JP2026511087A - Method and composition for producing ovarian support cell cocultures - Google Patents

Abstract

This disclosure features methods and compositions for the in vitro maturation of oocytes. In particular, this disclosure features methods and compositions relating to the manipulation of multiple ovarian supporting cells (OSCs) to promote the maturation of oocytes in a culture medium. Such methods and compositions are particularly useful in assisted reproductive technology (ART) procedures.
[Selection Diagram] None

Description

This disclosure relates to the field of in vitro oocyte maturation and assisted reproductive technology (ART).

One in ten women suffers from infertility and requires assisted reproductive technology (ART), such as in vitro fertilization (IVF). Maintaining the health of oocytes during culture remains a challenge, resulting in reduced oocyte quality and consequently, reduced embryo quality. Furthermore, developmentally immature oocytes are conventionally discarded, narrowing the pool of oocytes available for IVF. In vitro maturation culture (IVM) offers the possibility of maturing oocytes outside the body after egg retrieval, making all retrieved eggs usable. Current IVM methods using follicle-stimulating hormone (FSH) addition to the culture medium (spiking) are inefficient, with only 5-40% of immature eggs showing maturation. Even worse, this method results in many unhealthy eggs, and embryo survival rates are much lower than standard IVF (less than 17%). Therefore, there is still a need in this field to promote oocyte maturation for women undergoing ART.

In one embodiment, the present disclosure features an in vitro composition comprising one or more ovarian supporting cells (OSCs) and one or more diluents or excipients. In some embodiments, the composition promotes the maturation of one or more oocytes.

In some embodiments, one or more OSCs comprise one or more granulosa cells. In some embodiments, one or more OSCs express FOXL2, AMHR2, CD82, or any combination thereof. In some embodiments, one or more OSCs express one or more genes selected from GJA1, MDK, BBX, HES4, PBX3, YBX3, BMPR2, CD46, COL4A1, COL4A2, LAMC1, ITGAV, and ITGB. In some embodiments, one or more OSCs further express one or more genes selected from FOXO1, CDH1, CYP19A1, RARRES2, NOTCH2, NRG1, BMPR1B, EGFR (ERBB1), and ERBB4. In some embodiments, one or more OSCs further express one or more genes selected from RARRES2, NOTCH2, NOTCH3, ID3, and BMPR2. In some embodiments, one or more OSCs further express the gene CDH2 and/or NOTCH2. In some embodiments, one or more OSCs do not show significant expression of RARRES2.

In some embodiments, one or more OSCs express NR2F2. In some embodiments, one or more OSCs include ovarian stromal cells. In some embodiments, one or more OSCs include granulosa cells and ovarian stromal cells.

In some embodiments, one or more OSCs contain more than 60% granulosa cells, more than 70% granulosa cells, more than 80% granulosa cells, more than 90% granulosa cells, or more than 95% granulosa cells.

In some embodiments, one or more OSCs are obtained by differentiation of a population of iPSCs. In some embodiments, the iPSCs are human iPSCs (hiPSCs). In some embodiments, the hiPSCs express or overexpress the transcription factor RUNX2. In some embodiments, the hiPSCs express or overexpress the transcription factor NR5A1. In some embodiments, the hiPSCs express or overexpress the transcription factor GATA4. In some embodiments, the hiPSCs express or overexpress the transcription factor FOXL2.

In some embodiments, hiPSCs express or overexpress the transcription factors RUNX2 and NR5A1. In some embodiments, hiPSCs express or overexpress the transcription factors RUNX2 and GATA4. In some embodiments, hiPSCs express or overexpress the transcription factors RUNX2 and FOXL2. In some embodiments, hiPSCs express or overexpress the transcription factors NR5A1 and GATA4. In some embodiments, hiPSCs express or overexpress the transcription factors NR5A1 and FOXL2. In some embodiments, hiPSCs express or overexpress the transcription factors GATA4 and FOXL2.

In some embodiments, hiPSCs express or overexpress the transcription factors RUNX2, NR5A1, and GATA4. In some embodiments, hiPSCs express or overexpress the transcription factors RUNX2, GATA4, and FOXL2. In some embodiments, hiPSCs express or overexpress the transcription factors RUNX2, NR5A1, and FOXL2. In some embodiments, hiPSCs express or overexpress the transcription factors NR5A1, GATA4, and FOXL2. In some embodiments, hiPSCs express or overexpress the transcription factors RUNX2, NR5A1, GATA4, and FOXL2.

In some embodiments, the expression or overexpression of transcription factors is induced by doxycycline-responsive transcriptional regulators, such as doxycycline-responsive promoters or enhancers.

In some embodiments, hiPSCs come into contact with a Wnt/β-catenin pathway activator. In some embodiments, the Wnt/β-catenin pathway activator is a Rho-related protein kinase (ROCK) inhibitor, a glycogen synthase kinase-3 (GSK3) inhibitor, or a combination thereof.

In some embodiments, at least one of one or more OSCs is encapsulated. In some embodiments, one or more OSCs are encapsulated in alginate, laminin, collagen, vitronectin, chitosan, hyaluronic acid, poly-D-lactone, or any mixture thereof. In some embodiments, one or more OSCs are encapsulated in laminin, for example, laminin-521. In some embodiments, one or more OSCs are encapsulated in vitronectin.

In some embodiments, one or more OSCs have reduced or undetectable expression of one or more genes associated with pluripotency compared to unmodified iPSCs. In some embodiments, the one or more genes associated with pluripotency include NANOG. In some embodiments, the one or more genes associated with pluripotency include POU5F1.

In some embodiments, at least one of the one or more OSCs produces one or more growth factors. In some embodiments, the one or more growth factors include insulin-like growth factor (IGF), stem cell factor (SCF), epidermal growth factor (EGF), leukemia suppressor factor (LIF), vascular endothelial growth factor (VEGF), bone morphogenetic protein (BMP), C-type natriuretic peptide (CNP), or any combination thereof. In some embodiments, at least a portion of the one or more growth factors are secreted.

In some embodiments, one or more OSCs produce one or more steroids. In some embodiments, the one or more steroids include estradiol, progesterone, or a combination thereof. In some embodiments, one or more steroids are produced in response to hormonal stimulation. In some embodiments, the hormonal stimulation includes FSH, androstenedione treatment, or a combination thereof. In some embodiments, at least some of the one or more steroids are secreted.

In some embodiments, one or more OSCs are cryopreserved. In some embodiments, the composition further comprises an in vitro maturation (IVM) medium. In some embodiments, the IVM medium comprises a cell culture medium. In some embodiments, the IVM medium comprises Medicult-IVM medium. In some embodiments, the IVM medium comprises one or more supplements. In some embodiments, one or more supplements are
(i) Human serum albumin (HSA) at a concentration of approximately 5 to 15 mg/mL (optional), and at a concentration of 10 mg/mL (optional).
(ii) Recombinant follicle-stimulating hormone (rFSH) at a concentration of approximately 70 mIU/mL to approximately 80 mIU/mL, and at an optional concentration of 75 mIU/mL.
(iii)Optionally, human chorionic gonadotropin (hCG) at concentrations of approximately 95 mIU/mL to approximately 105 mIU/mL, and optionally at a concentration of 100 mIU/mL.
(iv)Optionally, androstenedione at concentrations of approximately 495 ng/mL to approximately 505 ng/mL, and further optionally, at a concentration of 500 ng/mL.
(v) Optionally, doxycycline at concentrations of approximately 0.5 μg/mL to approximately 1.5 μg/mL, and optionally at a concentration of 1 μg/mL.
or any combination of one or more supplements.

In some embodiments, one or more oocytes are collected from a donor subject. In some embodiments, the donor subject is approximately 19 to 45 years old. In some embodiments, the subject undergoes ovarian stimulation. In some embodiments, ovarian stimulation includes treatment with gonadotropin-releasing hormone (GnRH). In some embodiments, ovarian stimulation includes treatment with one or more GnRH analogs. In some embodiments, one or more GnRH analogs are GnRH agonists or antagonists. In some embodiments, ovarian stimulation includes one or more ovulation-inducing agents. In some embodiments, one or more ovulation-inducing agents include human chorionic gonadotropin (hCG). In some embodiments, one or more ovulation-inducing agents include a GnRH agonist, optionally, the GnRH agonist being leuprolide. In some embodiments, ovarian stimulation includes FSH treatment. In some embodiments, ovarian stimulation does not include FSH treatment. In some embodiments, this FSH treatment comprises 300 to 700 IU of FSH. In some embodiments, this FSH treatment comprises 400 to 600 IU of FSH. In some embodiments, this FSH treatment comprises one, two, three, or more FSH injections, and optionally, this FSH treatment comprises multiple injections, each injection comprising a dose of approximately 100 to 200 IU of FSH. In some embodiments, ovarian stimulation comprises the administration of clomiphene citrate, optionally administered in one or more doses for up to eight days, optionally each dose ranging from 50 mg to 150 mg (e.g., 50–75 mg, 60–80 mg, 75–100 mg, 90–115 mg, 110–130 mg, 125–150 mg, e.g., 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg). In some embodiments, ovarian stimulation further comprises one or more hCG inducers. In some embodiments, one or more hCG inducers comprise 2,500 IU to 10,000 IU of hCG or about 200 μg to about 700 μg of hCG, and optionally, the hCG is administered to the subject in doses of about 400 μg to about 600 μg, and optionally, the hCG is administered to the subject in doses of about 500 μg per dose.

In some embodiments, one or more oocytes are located within a cumulus-oocyte complex (COC). In some embodiments, one or more oocytes include one or more developed immature oocytes. In some embodiments, one or more oocytes are all developed immature oocytes. In some embodiments, one or more oocytes are not developed.

In some embodiments, one or more oocytes comprise one or more oocytes containing a nucleus vesicle (GV). In some embodiments, one or more of the oocytes comprise one or more oocytes in metaphase I (MI). In some embodiments, one or more of the oocytes comprise one or more oocytes in metaphase II (MII). In some embodiments, at least a portion of one or more oocytes comprises one or more previously vitrified oocytes. In some embodiments, at least a portion of one or more oocytes comprises one or more previously cryopreserved oocytes.

In some embodiments, one or more oocytes are co-cultured with one or more OSCs.

In some embodiments, before and/or after co-culture, one or more oocytes are evaluated for parameters selected from the group consisting of total oocyte score, oocyte maturation rate from GV to MII, oocyte maturation rate from GV to MI, oocyte maturation rate from MI to MII, mean oocyte shape, mean oocyte size, mean oocyte quality, mean pericoelous space (PVS) quality, mean zona pellucida (ZP) quality, and mean polar body quality. In some embodiments, one or more co-cultured oocytes have substantially the same morphological quality as oocytes matured in vivo, and morphological quality includes oocyte size, zona pellucida size, oocyte color, oocyte shape, oocyte cytoplasmic grain size, oocyte polar body quality, and oocyte PVS quality.

In some embodiments, one or more co-cultured oocytes have an improved maturation rate compared to oocytes in a culture without one or more OSCs. In some embodiments, one or more co-cultured oocytes have metaphase II spindles at substantially the same position as those in oocytes matured in vivo. In some embodiments, one or more co-cultured oocytes have substantially the same transcriptome profile as oocytes matured in vivo.

In some embodiments, one or more oocytes are co-cultured with one or more OSCs for approximately 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 hours. In some embodiments, one or more oocytes are co-cultured with one or more OSCs for approximately 24 to 28 hours.

In some embodiments, one or more oocytes co-cultured with one or more OSCs form one or more undifferentiated germ cells after contact with one or more mature spermatids.

In some embodiments, one or more oocytes are co-cultured in direct contact with one or more OSCs. In some embodiments, one or more oocytes are not in direct contact with OSCs. In some embodiments, the culture system is suspension culture. In some embodiments, the culture system is adherent culture.

In another embodiment, the present disclosure is characterized by a method for culturing oocytes, wherein one or more immature oocytes are co-cultured with one or more OSCs.

In another embodiment, the present disclosure features a method for preparing one or more oocytes previously collected from a human subject for use in an assisted reproductive technology (ART) procedure, the method comprising co-culturing one or more oocytes with one or more OSCs.

In another embodiment, the present disclosure features a method for generating mature oocytes for use in ART treatment, the method comprising co-culturing one or more oocytes previously collected from a human subject with a population of ovarian supporting cells differentiated from one or more iPSCs.

In another embodiment, the present disclosure features a method for inducing oocyte maturation in vitro, the method comprising co-culturing one or more oocytes with a population of ovarian supporting cells differentiated from one or more iPSCs, the co-culturing being optionally performed for approximately 6 to 120 hours.

In another embodiment, the method is characterized by a method for generating mature oocytes for use in ART treatment, and this method is
(a) Differentiating one or more iPSCs to generate one or more OSCs,
(b) Collect one or more immature oocytes from the subject,
(c) Co-culturing one or more oocytes with one or more OSCs to generate one or more mature oocytes.

In another aspect, the present disclosure features a method for promoting the maturation of oocytes in subjects who have undergone ART treatment and have previously been administered one or more follicle-inducing agents during the follicle-inducing period, wherein the method is
(a) Collect one or more immature oocytes from the subject,
(b) Co-culturing one or more oocytes with one or more OSCs differentiated from iPSCs, thereby generating one or more mature oocytes,
(c) comprising isolating one or more mature oocytes.

In some embodiments of any of the prior art, before and/or after co-culture, one or more oocytes are evaluated for parameters selected from the group consisting of total oocyte score, oocyte maturation rate from GV to MII, oocyte maturation rate from GV to MI, oocyte maturation rate from MI to MII, mean oocyte shape, mean oocyte size, mean oocyte quality, mean pericoelous space (PVS) quality, mean zona pellucida (ZP) quality, and mean polar body quality. In some embodiments, one or more co-cultured oocytes have substantially the same morphological quality as oocytes matured in vivo, and morphological quality includes oocyte size, zona pellucida size, oocyte color, oocyte shape, oocyte cytoplasmic grain size, oocyte polar body quality, and oocyte PVS quality.

In some embodiments, one or more co-cultured oocytes have an improved maturation rate compared to oocytes in a culture without one or more OSCs. In some embodiments, one or more co-cultured oocytes have an improved maturation rate compared to oocytes matured in vivo. In some embodiments, one or more co-cultured oocytes have metaphase II spindles at substantially the same position as those in oocytes matured in vivo. In some embodiments, one or more co-cultured oocytes have substantially the same transcriptome profile as oocytes matured in vivo.

In some embodiments, one or more OSCs express FOXL2, AMHR2, CD82, or any combination thereof. In some embodiments, one or more OSCs express one or more genes selected from GJA1, MDK, BBX, HES4, PBX3, YBX3, BMPR2, CD46, COL4A1, COL4A2, LAMC1, ITGAV, and ITGB. In some embodiments, one or more OSCs further express one or more genes selected from FOXO1, CDH1, CYP19A1, RARRES2, NOTCH2, NRG1, BMPR1B, EGFR (ERBB1), and ERBB4. In some embodiments, one or more OSCs further express one or more genes selected from RARRES2, NOTCH2, NOTCH3, ID3, and BMPR2. In some embodiments, one or more OSCs further express the CDH2 gene and/or the NOTCH2 gene, but do not exhibit significant expression of the RARRES2 gene.

In some embodiments, one or more OSCs include granulosa cells.

In some embodiments, one or more OSCs express NR2F2. In some embodiments, one or more OSCs include ovarian stromal cells.

In some embodiments, one or more OSCs comprise granulosa cells and ovarian stromal cells. In some embodiments, one or more OSCs comprise more than 60% granulosa cells, more than 70% granulosa cells, more than 80% granulosa cells, more than 90% granulosa cells, or more than 95% granulosa cells.

In some embodiments, one or more OSCs are obtained by differentiation of a population of iPSCs. In some embodiments, the iPSCs are hiPSCs. In some embodiments, the hiPSCs express or overexpress the transcription factor RUNX2. In some embodiments, the hiPSCs express or overexpress the transcription factor NR5A1. In some embodiments, the hiPSCs express or overexpress the transcription factor GATA4. In some embodiments, the hiPSCs express or overexpress the transcription factor FOXL2.

In some embodiments, hiPSCs express or overexpress the transcription factors RUNX2 and NR5A1. In some embodiments, hiPSCs express or overexpress the transcription factors RUNX2 and GATA4. In some embodiments, hiPSCs express or overexpress the transcription factors RUNX2 and FOXL2. In some embodiments, hiPSCs express or overexpress the transcription factors NR5A1 and GATA4. In some embodiments, hiPSCs express or overexpress the transcription factors NR5A1 and FOXL2. In some embodiments, hiPSCs express or overexpress the transcription factors GATA4 and FOXL2.

In some embodiments, hiPSCs express or overexpress the transcription factors RUNX2, NR5A1, and GATA4. In some embodiments, hiPSCs express or overexpress the transcription factors RUNX2, GATA4, and FOXL2. In some embodiments, hiPSCs express or overexpress the transcription factors RUNX2, NR5A1, and FOXL2. In some embodiments, hiPSCs express or overexpress the transcription factors NR5A1, GATA4, and FOXL2. In some embodiments, hiPSCs express or overexpress the transcription factors RUNX2, NR5A1, GATA4, and FOXL2.

In some embodiments, the expression or overexpression of any of the aforementioned transcription factors is induced by doxycycline-responsive transcriptional regulators, such as doxycycline-responsive promoters or enhancers.

In some embodiments, hiPSCs come into contact with a Wnt/β-catenin pathway activator. In some embodiments, the Wnt/β-catenin pathway activator is a Rho-related protein kinase (ROCK) inhibitor, a glycogen synthase kinase-3 (GSK3) inhibitor, or a combination thereof.

In some embodiments, one or more OSCs are encapsulated. In some embodiments, one or more OSCs are encapsulated in alginate, laminin, collagen, vitronectin, chitosan, hyaluronic acid, poly-D-lactone, or a mixture thereof. In some embodiments, one or more OSCs are encapsulated in laminin, optionally being laminin-521. In some embodiments, one or more OSCs are encapsulated in vitronectin.

In some embodiments, one or more OSCs have the expression of one or more genes associated with pluripotency that are low or undetectable compared to iPSCs. In some embodiments, the one or more genes associated with pluripotency include NANOG. In some embodiments, the one or more genes associated with pluripotency include POU5F1.

In some embodiments, one or more OSCs produce one or more growth factors. In some embodiments, the growth factors include IGF, SCF, EGF, LIF, VEGF, BMP, CNP, or any combination thereof. In some embodiments, at least some of the one or more growth factors are secreted.

In some embodiments, one or more OSCs produce one or more steroids. In some embodiments, one or more steroids include estradiol, progesterone, or a combination thereof. In some embodiments, one or more steroids are produced in response to hormonal stimulation of the OSC. In some embodiments, hormonal stimulation includes exposure to FSH, androstenedione, or a combination thereof. In some embodiments, at least some of the one or more steroids are secreted.

In some embodiments, one or more OSCs are cryopreserved. In some embodiments, the composition further comprises an in vitro maturation (IVM) medium. In some embodiments, the IVM medium comprises a cell culture medium. In some embodiments, the IVM medium comprises Medicult-IVM medium. In some embodiments, the IVM medium comprises one or more supplements. In some embodiments, one or more supplements are
(i) Human serum albumin (HSA) at a concentration of approximately 5 to 15 mg/mL (optional), and at a concentration of 10 mg/mL (optional).
(ii) Recombinant follicle-stimulating hormone (rFSH) at a concentration of approximately 70 mIU/mL to approximately 80 mIU/mL, and at an optional concentration of 75 mIU/mL.
(iii)Optionally, human chorionic gonadotropin (hCG) at concentrations of approximately 95 mIU/mL to approximately 105 mIU/mL, and optionally at a concentration of 100 mIU/mL.
(iv)Optionally, androstenedione at concentrations of approximately 495 ng/mL to approximately 505 ng/mL, and further optionally, at a concentration of 500 ng/mL.
(v) Optionally, doxycycline at concentrations of approximately 0.5 μg/mL to approximately 1.5 μg/mL, and optionally at a concentration of 1 μg/mL.
or any combination of one or more supplements.

In some embodiments, one or more oocytes are collected from a donor subject. In some embodiments, the donor subject is approximately 19 to 45 years old. In some embodiments, the subject undergoes ovarian stimulation. In some embodiments, ovarian stimulation includes treatment with gonadotropin-releasing hormone (GnRH). In some embodiments, ovarian stimulation includes treatment with one or more GnRH analogs. In some embodiments, one or more GnRH analogs are GnRH agonists or antagonists. In some embodiments, ovarian stimulation includes one or more ovulation-inducing agents. In some embodiments, one or more ovulation-inducing agents include human chorionic gonadotropin (hCG). In some embodiments, one or more ovulation-inducing agents include a GnRH agonist, optionally, the GnRH agonist being leuprolide. In some embodiments, ovarian stimulation includes FSH treatment. In some embodiments, ovarian stimulation does not include FSH treatment. In some embodiments, the FSH treatment comprises 300 to 700 IU of FSH. In some embodiments, the FSH treatment comprises 400 to 600 IU of FSH. In some embodiments, the FSH treatment comprises one, two, three, or more FSH injections, and optionally, the FSH treatment comprises multiple injections, each injection comprising a dose of approximately 100 to 200 IU of FSH. In some embodiments, ovarian stimulation comprises the administration of clomiphene citrate, optionally administered in one or more doses for up to eight days, optionally each dose ranging from 50 mg to 150 mg (e.g., 50–75 mg, 60–80 mg, 75–100 mg, 90–115 mg, 110–130 mg, 125–150 mg, e.g., 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg). In some embodiments, ovarian stimulation further comprises one or more hCG inducers. In some embodiments, one or more hCG inducers comprise 2,500 IU to 10,000 IU of hCG or about 200 μg to about 700 μg of hCG, and optionally, the hCG is administered to the subject in doses of about 400 μg to about 600 μg, and optionally, the hCG is administered to the subject in doses of about 500 μg per dose.

In some embodiments, one or more oocytes are located within a cumulus-oocyte complex (COC). In some embodiments, one or more oocytes include one or more veiled immature oocytes. In some embodiments, one or more oocytes are all veiled immature oocytes. In some embodiments, one or more oocytes are not veiled.

In some embodiments, one or more oocytes comprise one or more oocytes containing a nucleus vesicle (GV). In some embodiments, one or more of the oocytes comprise one or more oocytes in metaphase I (MI). In some embodiments, one or more of the oocytes comprise one or more oocytes in metaphase II (MII). In some embodiments, at least a portion of one or more oocytes comprises one or more previously vitrified oocytes. In some embodiments, at least a portion of one or more oocytes comprises one or more previously cryopreserved oocytes.

In some embodiments, one or more oocytes are co-cultured with one or more OSCs.

In some embodiments, before and/or after co-culture, one or more oocytes are evaluated for parameters selected from the group consisting of total oocyte score, oocyte maturation rate from GV to MII, oocyte maturation rate from GV to MI, oocyte maturation rate from MI to MII, mean oocyte shape, mean oocyte size, mean oocyte quality, mean pericoelous space (PVS) quality, mean zona pellucida (ZP) quality, and mean polar body quality. In some embodiments, one or more co-cultured oocytes have substantially the same morphological quality as oocytes matured in vivo, and morphological quality includes oocyte size, zona pellucida size, oocyte color, oocyte shape, oocyte cytoplasmic grain size, oocyte polar body quality, and oocyte PVS quality.

In some embodiments, one or more co-cultured oocytes have an improved maturation rate compared to oocytes in a culture without one or more OSCs. In some embodiments, one or more co-cultured oocytes have metaphase II spindles at substantially the same position as those in oocytes matured in vivo. In some embodiments, one or more co-cultured oocytes have substantially the same transcriptome profile as oocytes matured in vivo.

In some embodiments, one or more oocytes are co-cultured with one or more OSCs for approximately 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 hours. In some embodiments, one or more oocytes are co-cultured with one or more OSCs for approximately 24 to 28 hours.

In some embodiments, one or more oocytes co-cultured with one or more OSCs form one or more undifferentiated germ cells after contact with one or more mature spermatids.

In some embodiments, one or more oocytes are co-cultured in direct contact with one or more OSCs. In some embodiments, one or more oocytes are not in direct contact with OSCs. In some embodiments, the culture system is suspension culture. In some embodiments, the culture system is adherent culture.

In another aspect, the disclosure features a method for promoting the differentiation of one or more induced pluripotent stem cells (iPSCs) into one or more ovarian supporting cells (OSCs), wherein the method is
(a) Culturing one or more iPSCs in vitro,
(b) Inducing the expression or overexpression of one or more transcription factors, including FOXL2, NR5A1, RUNX2, GATA4, or any combination thereof, in one or more iPSCs, thereby generating differentiated cells,
(c) Determine that the differentiated cells obtained from (b) exhibit a gene expression profile similar to the gene expression profile of one or more OSCs,
(d) Co-culturing one or more OSCs with one or more oocytes previously collected from the subject, thereby maturing one or more oocytes.

In another aspect, the disclosure features a method for generating one or more ovarian supporting cells (OSCs) from one or more induced pluripotent stem cells (iPSCs), wherein the method is
(a) Culturing one or more iPSCs in vitro,
(b) Inducing the expression or overexpression of one or more transcription factors, including FOXL2, NR5A1, RUNX2, GATA4, or any combination thereof, in one or more iPSCs, thereby generating differentiated cells,
(c) Determine that the differentiated cells obtained from (b) exhibit a gene expression profile similar to the gene expression profile of one or more OSCs,
(d) Co-culturing one or more identified OSCs with one or more oocytes previously collected from the subject, thereby maturing one or more oocytes.

In another aspect, the present disclosure is characterized by a method for preparing a composition comprising one or more ovarian supporting cells (OSCs), the method comprising:
(a) Culturing one or more iPSCs in vitro,
(b) Inducing the expression or overexpression of one or more transcription factors, including FOXL2, NR5A1, RUNX2, GATA4, or any combination thereof, in one or more iPSCs, thereby generating differentiated cells,
(c) Determine that the differentiated cells obtained from (b) exhibit a gene expression profile similar to the gene expression profile of one or more OSCs,
(d) Co-culturing one or more identified OSCs with one or more oocytes previously collected from the subject, thereby maturing one or more oocytes.

In some embodiments of the prior art, the iPSCs are human iPSCs (hiPSCs). In some embodiments, the iPSCs have been previously cryopreserved. In some embodiments, co-culture is performed in in vitro maturation (IVM) medium. In some embodiments, the IVM medium comprises cell culture medium. In some embodiments, the IVM medium comprises Medicult-IVM medium. In some embodiments, the IVM medium comprises one or more supplements. In some embodiments, one or more supplements are
(i) Human serum albumin (HSA) at a concentration of approximately 5 to 15 mg/mL (optional), and at a concentration of 10 mg/mL (optional).
(ii) Recombinant follicle-stimulating hormone (rFSH) at a concentration of approximately 70 mIU/mL to approximately 80 mIU/mL, and at an optional concentration of 75 mIU/mL.
(iii)Optionally, human chorionic gonadotropin (hCG) at concentrations of approximately 95 mIU/mL to approximately 105 mIU/mL, and optionally at a concentration of 100 mIU/mL.
(iv)Optionally, androstenedione at concentrations of approximately 495 ng/mL to approximately 505 ng/mL, and further optionally, at a concentration of 500 ng/mL.
(v) Optionally, doxycycline at concentrations of approximately 0.5 μg/mL to approximately 1.5 μg/mL, and optionally at a concentration of 1 μg/mL.
or any combination of one or more supplements.

In some embodiments, induction from iPSC to OSC takes approximately 1 to 10 days, and optionally, induction takes approximately 5 days.

In some embodiments, iPSCs are cultured in a medium containing a matrix. In some embodiments, the matrix comprises alginate, laminin, collagen, vitronectin, chitosan, hyaluronic acid, poly-D-lactone, or a mixture thereof. In some embodiments, the matrix comprises laminin, and optionally, the laminin is laminin-521. In some embodiments, the matrix comprises vitronectin.

In some embodiments, iPSCs are reprogrammed using transposases to carry one or more inducible transcription factors. In some embodiments, iPSCs are transformed via electroporation, liposome-mediated transformation, or virus-mediated gene transfer. In some embodiments, the expression or overexpression of one or more transcription factors is induced in the presence of doxycycline. In some embodiments, iPSCs are contacted with a Wnt/β-catenin pathway activator. In some embodiments, the Wnt/β-catenin pathway activator is a Rho-related protein kinase (ROCK) inhibitor, a glycogen synthase kinase-3 (GSK3) inhibitor, or a combination thereof.

In some embodiments, one or more OSCs express FOXL2, AMHR2, CD82, or any combination thereof. In some embodiments, one or more OSCs express one or more genes selected from GJA1, MDK, BBX, HES4, PBX3, YBX3, BMPR2, CD46, COL4A1, COL4A2, LAMC1, ITGAV, and ITGB. In some embodiments, one or more OSCs further express one or more genes selected from FOXO1, CDH1, CYP19A1, RARRES2, NOTCH2, NRG1, BMPR1B, EGFR (ERBB1), and ERBB4. In some embodiments, one or more OSCs further express one or more genes selected from RARRES2, NOTCH2, NOTCH3, ID3, and BMPR2. In some embodiments, one or more OSCs further express the CDH2 gene and/or the NOTCH2 gene, and optionally lack significant expression of the RARRES2 gene.

In some embodiments, one or more OSCs include one or more granulosa cells.

In some embodiments, one or more OSCs express NR2F2. In some embodiments, one or more OSCs contain one or more ovarian stromal cells.

In some embodiments, one or more OSCs comprise granulosa cells and ovarian stromal cells. In some embodiments, one or more OSCs comprise more than 60% granulosa cells, more than 70% granulosa cells, more than 80% granulosa cells, more than 90% granulosa cells, or more than 95% granulosa cells.

In some embodiments, one or more OSCs have the expression of one or more genes associated with pluripotency that are low or undetectable compared to iPSCs. In some embodiments, the one or more genes associated with pluripotency include NANOG. In some embodiments, the one or more genes associated with pluripotency include POU5F1.

In some embodiments, one or more OSCs produce one or more growth factors. In some embodiments, the growth factors include IGF, SCF, EGF, LIF, VEGF, BMP, CNP, or any combination thereof. In some embodiments, at least some of the one or more growth factors are secreted.

In some embodiments, one or more OSCs produce one or more steroids. In some embodiments, one or more steroids include estradiol, progesterone, or a combination thereof. In some embodiments, one or more steroids are produced in the presence of one or more hormones. In some embodiments, one or more hormones include exposure to FSH, androstenedione, or a combination thereof. In some embodiments, at least some of the one or more steroids are secreted.

In some embodiments, one or more oocytes collected from a subject are immature oocytes. In some embodiments, co-culturing one or more OSCs with one or more oocytes promotes the maturation of one or more oocytes. In some embodiments, this method further includes collecting one or more oocytes for assisted reproductive technology procedures.

In some embodiments, the subjects undergo ovarian stimulation before harvesting one or more oocytes. In some embodiments, ovarian stimulation includes treatment with gonadotropin-releasing hormone (GnRH). In some embodiments, ovarian stimulation includes treatment with one or more GnRH analogs. In some embodiments, one or more GnRH analogs are GnRH agonists or antagonists. In some embodiments, ovarian stimulation includes one or more ovulation-inducing agents. In some embodiments, one or more ovulation-inducing agents include hCG. In some embodiments, one or more ovulation-inducing agents include a GnRH agonist, optionally, the GnRH agonist being leuprolide. In some embodiments, ovarian stimulation includes FSH treatment. In some embodiments, ovarian stimulation does not include FSH treatment. In some embodiments, FSH treatment includes 300 IU to 700 IU of FSH. In some embodiments, FSH treatment includes 400 IU to 600 IU of FSH. In some embodiments, FSH treatment comprises one, two, three, or more FSH injections, and optionally, FSH treatment comprises multiple injections, each injection comprising a dose of FSH of about 100 IU to about 200 IU. In some embodiments, ovarian stimulation comprises the administration of clomiphene citrate, and optionally, clomiphene citrate is administered in one or more doses for up to 8 days, each dose being 50 mg to 150 mg (e.g., 50-75 mg, 60-80 mg, 75-100 mg, 90-115 mg, 110-130 mg, 125-150 mg, e.g., 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg). In some embodiments, ovarian stimulation further comprises one or more hCG inducers. In some embodiments, one or more hCG inducers comprise 2,500 IU to 10,000 IU of hCG or about 200 μg to about 700 μg of hCG, and optionally, the hCG is administered to the subject in doses of about 400 μg to about 600 μg, and optionally, the hCG is administered to the subject in doses of about 500 μg per dose.

In some embodiments, one or more oocytes are located within a cumulus-oocyte complex (COC). In some embodiments, one or more oocytes include one or more developed immature oocytes. In some embodiments, one or more oocytes are all developed immature oocytes. In some embodiments, one or more oocytes are not developed before or after co-culture.

In some embodiments, one or more oocytes comprise one or more oocytes containing a nucleus vesicle (GV). In some embodiments, one or more oocytes comprise one or more oocytes in metaphase I (MI). In some embodiments, one or more oocytes comprise one or more oocytes in metaphase II (MII).

In some embodiments, at least a portion of one or more oocytes includes one or more previously vitrified oocytes. In some embodiments, at least a portion of one or more oocytes includes one or more previously cryopreserved oocytes.

In some embodiments, before and/or after co-culture, one or more oocytes are evaluated for parameters selected from the group consisting of total oocyte score, oocyte maturation rate from GV to MII, oocyte maturation rate from GV to MI, oocyte maturation rate from MI to MII, mean oocyte shape, mean oocyte size, mean oocyte quality, mean pericoelous space (PVS) quality, mean zona pellucida (ZP) quality, and mean polar body quality. In some embodiments, one or more co-cultured oocytes have substantially the same morphological quality as oocytes matured in vivo, and morphological quality includes oocyte size, zona pellucida size, oocyte color, oocyte shape, oocyte cytoplasmic grain size, oocyte polar body quality, and oocyte PVS quality. In some embodiments, one or more co-cultured oocytes have an improved maturation rate compared to oocytes in a culture that does not contain one or more OSCs. In some embodiments, one or more co-cultured oocytes have metaphase II spindles located substantially in the same position as those of mature oocytes in vivo. In some embodiments, one or more co-cultured oocytes have substantially the same transcriptome profile as mature oocytes in vivo.

In some embodiments, one or more oocytes are co-cultured with one or more OSCs for approximately 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 hours. In some embodiments, one or more oocytes are cultured with one or more OSCs for approximately 24 to 28 hours.

In some embodiments, the method further includes isolating one or more MII-stage oocytes from a co-culture comprising one or more oocytes and one or more OSCs collected from a subject.

In some embodiments, one or more oocytes co-cultured with one or more OSCs form one or more undifferentiated germ cells after contact with one or more mature spermatids.

In some embodiments, one or more oocytes are cultured in direct contact with one or more OSCs. In some embodiments, one or more oocytes are not in direct contact with one or more OSCs. In some embodiments, the co-culture is suspension co-culture. In some embodiments, the co-culture is adherent co-culture.

In another embodiment, the present disclosure features a cell culture system comprising one or more ovarian supporting cells (OSCs), the system promoting the maturation of one or more oocytes.

In some embodiments, one or more OSCs comprise one or more granulosa cells. In some embodiments, one or more OSCs express FOXL2, AMHR2, CD82, or any combination thereof. In some embodiments, one or more OSCs express one or more genes selected from GJA1, MDK, BBX, HES4, PBX3, YBX3, BMPR2, CD46, COL4A1, COL4A2, LAMC1, ITGAV, and ITGB. In some embodiments, one or more OSCs further express one or more genes selected from FOXO1, CDH1, CYP19A1, RARRES2, NOTCH2, NRG1, BMPR1B, EGFR (ERBB1), and ERBB4. In some embodiments, one or more OSCs further express one or more genes selected from RARRES2, NOTCH2, NOTCH3, ID3, and BMPR2. In some embodiments, one or more OSCs further express the gene CDH2 and/or NOTCH2. In some embodiments, one or more OSCs do not show significant expression of RARRES2.

In some embodiments, one or more OSCs express NR2F2. In some embodiments, one or more OSCs include ovarian stromal cells. In some embodiments, one or more OSCs include granulosa cells and ovarian stromal cells.

In some embodiments, one or more OSCs contain more than 60% granulosa cells, more than 70% granulosa cells, more than 80% granulosa cells, more than 90% granulosa cells, or more than 95% granulosa cells.

In some embodiments, one or more OSCs are obtained by differentiation of a population of iPSCs. In some embodiments, the iPSCs are human iPSCs (hiPSCs). In some embodiments, the hiPSCs express or overexpress the transcription factor RUNX2. In some embodiments, the hiPSCs express or overexpress the transcription factor NR5A1. In some embodiments, the hiPSCs express or overexpress the transcription factor GATA4. In some embodiments, the hiPSCs express or overexpress the transcription factor FOXL2.

In some embodiments, hiPSCs express or overexpress the transcription factors RUNX2 and NR5A1. In some embodiments, hiPSCs express or overexpress the transcription factors RUNX2 and GATA4. In some embodiments, hiPSCs express or overexpress the transcription factors RUNX2 and FOXL2. In some embodiments, hiPSCs express or overexpress the transcription factors NR5A1 and GATA4. In some embodiments, hiPSCs express or overexpress the transcription factors NR5A1 and FOXL2. In some embodiments, hiPSCs express or overexpress the transcription factors GATA4 and FOXL2.

In some embodiments, hiPSCs express or overexpress the transcription factors RUNX2, NR5A1, and GATA4. In some embodiments, hiPSCs express or overexpress the transcription factors RUNX2, GATA4, and FOXL2. In some embodiments, hiPSCs express or overexpress the transcription factors RUNX2, NR5A1, and FOXL2. In some embodiments, hiPSCs express or overexpress the transcription factors NR5A1, GATA4, and FOXL2. In some embodiments, hiPSCs express or overexpress the transcription factors RUNX2, NR5A1, GATA4, and FOXL2.

In some embodiments, the expression or overexpression of transcription factors is under the control of doxycycline.

In some embodiments, hiPSCs come into contact with a Wnt/β-catenin pathway activator. In some embodiments, the Wnt/β-catenin pathway activator is a Rho-related protein kinase (ROCK) inhibitor, a glycogen synthase kinase-3 (GSK3) inhibitor, or a combination thereof.

In some embodiments, at least one of one or more OSCs is encapsulated. In some embodiments, one or more OSCs are encapsulated in alginate, laminin, collagen, vitronectin, chitosan, hyaluronic acid, poly-D-lactone, or any mixture thereof. In some embodiments, one or more OSCs are encapsulated in laminin, optionally being laminin-521. In some embodiments, one or more OSCs are encapsulated in vitronectin.

In some embodiments, one or more OSCs have lower or undetectable expression of one or more genes associated with pluripotency compared to iPSCs. In some embodiments, the one or more genes associated with pluripotency include NANOG. In some embodiments, the one or more genes associated with pluripotency include POU5F1.

In some embodiments, at least a portion of the OSCs produce one or more growth factors. In some embodiments, the one or more growth factors include insulin-like growth factor (IGF), stem cell factor (SCF), epidermal growth factor (EGF), leukemia suppressor factor (LIF), vascular endothelial growth factor (VEGF), bone morphogenetic protein (BMP), C-type natriuretic peptide (CNP), or any combination thereof. In some embodiments, at least a portion of the one or more growth factors are secreted.

In some embodiments, one or more OSCs produce one or more steroids. In some embodiments, the one or more steroids include estradiol, progesterone, or a combination thereof. In some embodiments, one or more steroids are produced in response to hormonal stimulation. In some embodiments, the hormonal stimulation includes FSH, androstenedione treatment, or a combination thereof. In some embodiments, at least some of the one or more steroids are secreted.

In some embodiments, one or more OSCs are cryopreserved. In some embodiments, the composition further comprises an in vitro maturation (IVM) medium. In some embodiments, the IVM medium comprises a cell culture medium. In some embodiments, the IVM medium comprises Medicult-IVM medium. In some embodiments, the IVM medium comprises one or more supplements. In some embodiments, one or more supplements are
(i) Human serum albumin (HSA) at a concentration of approximately 5 to 15 mg/mL (optional), and at a concentration of 10 mg/mL (optional).
(ii) Recombinant follicle-stimulating hormone (rFSH) at a concentration of approximately 70 mIU/mL to approximately 80 mIU/mL, and at an optional concentration of 75 mIU/mL.
(iii)Optionally, human chorionic gonadotropin (hCG) at concentrations of approximately 95 mIU/mL to approximately 105 mIU/mL, and optionally at a concentration of 100 mIU/mL.
(iv)Optionally, androstenedione at concentrations of approximately 495 ng/mL to approximately 505 ng/mL, and further optionally, at a concentration of 500 ng/mL.
(v) Optionally, doxycycline at concentrations of approximately 0.5 μg/mL to approximately 1.5 μg/mL, and optionally at a concentration of 1 μg/mL.
or any combination of one or more supplements.

In some embodiments, one or more oocytes are collected from a donor subject. In some embodiments, the donor subject is approximately 19 to 45 years old. In some embodiments, the subject undergoes ovarian stimulation. In some embodiments, ovarian stimulation includes treatment with gonadotropin-releasing hormone (GnRH). In some embodiments, ovarian stimulation includes treatment with one or more GnRH analogs. In some embodiments, one or more GnRH analogs are GnRH agonists or antagonists. In some embodiments, ovarian stimulation includes one or more ovulation-inducing agents. In some embodiments, one or more ovulation-inducing agents include human chorionic gonadotropin (hCG). In some embodiments, one or more ovulation-inducing agents include a GnRH agonist, optionally, the GnRH agonist being leuprolide. In some embodiments, ovarian stimulation includes FSH treatment. In some embodiments, ovarian stimulation does not include FSH treatment. In some embodiments, the FSH treatment comprises 300 to 700 IU of FSH. In some embodiments, the FSH treatment comprises 400 to 600 IU of FSH. In some embodiments, the FSH treatment comprises one, two, three, or more FSH injections, and optionally, the FSH treatment comprises multiple injections, each injection comprising a dose of approximately 100 to 200 IU of FSH. In some embodiments, ovarian stimulation comprises the administration of clomiphene citrate, optionally administered in one or more doses for up to eight days, with each dose ranging from 50 mg to 150 mg (e.g., 50–75 mg, 60–80 mg, 75–100 mg, 90–115 mg, 110–130 mg, 125–150 mg, e.g., 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg). In some embodiments, ovarian stimulation further comprises one or more hCG inducers. In some embodiments, one or more hCG inducers comprise 2,500 IU to 10,000 IU of hCG or about 200 μg to about 700 μg of hCG. Optionally, hCG is administered to subjects in doses of about 400 μg to about 600 μg, and further optionally, hCG is administered to subjects in doses of about 500 μg per dose. In some embodiments, one or more oocytes are located within a cumulus-oocyte complex (COC). In some embodiments, one or more oocytes comprise one or more vellused immature oocytes. In some embodiments, one or more oocytes are all vellused immature oocytes. In some embodiments, one or more oocytes are not vellused.

In some embodiments, one or more oocytes comprise one or more oocytes containing a nucleus vesicle (GV). In some embodiments, one or more of the oocytes comprise one or more oocytes in metaphase I (MI). In some embodiments, one or more of the oocytes comprise one or more oocytes in metaphase II (MII). In some embodiments, at least a portion of one or more oocytes comprises one or more previously vitrified oocytes. In some embodiments, at least a portion of one or more oocytes comprises one or more previously cryopreserved oocytes.

In some embodiments, one or more oocytes are co-cultured with one or more OSCs.

In some embodiments, before and/or after co-culture, one or more oocytes are evaluated for parameters selected from the group consisting of total oocyte score, oocyte maturation rate from GV to MII, oocyte maturation rate from GV to MI, oocyte maturation rate from MI to MII, mean oocyte shape, mean oocyte size, mean oocyte quality, mean pericoelous space (PVS) quality, mean zona pellucida (ZP) quality, and mean polar body quality. In some embodiments, one or more co-cultured oocytes have substantially the same morphological quality as oocytes matured in vivo, and morphological quality includes oocyte size, zona pellucida size, oocyte color, oocyte shape, oocyte cytoplasmic grain size, oocyte polar body quality, and oocyte PVS quality.

In some embodiments, one or more co-cultured oocytes have an improved maturation rate compared to oocytes in a culture without one or more OSCs. In some embodiments, one or more co-cultured oocytes have metaphase II spindles at substantially the same position as those in oocytes matured in vivo. In some embodiments, one or more co-cultured oocytes have substantially the same transcriptome profile as oocytes matured in vivo.

In some embodiments, one or more oocytes are co-cultured with one or more OSCs for approximately 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 hours. In some embodiments, one or more oocytes are co-cultured with one or more OSCs for approximately 24 to 28 hours.

In some embodiments, one or more oocytes co-cultured with one or more OSCs form one or more undifferentiated germ cells after contact with one or more mature spermatids.

In some embodiments, one or more oocytes are co-cultured in direct contact with one or more OSCs. In some embodiments, one or more oocytes are not in direct contact with OSCs. In some embodiments, the culture system is suspension culture. In some embodiments, the culture system is adherent culture.

In another embodiment, the present disclosure features a kit comprising an in vitro composition and accompanying documentation from any one of the prior embodiments, the accompanying documentation instructing the user of the kit to co-culture a population of ovarian supporting cells with one or more oocytes according to any one of the prior methods.

In another embodiment, the present disclosure features a kit comprising a vial containing a population of iPSCs and a package insert, the package insert instructing the user of the kit to differentiate the iPSC population into one or more ovarian supporting cells according to one of the prior art methods.

In another embodiment, the present disclosure features a kit comprising a vial containing one or more OSCs and a document accompanying the kit, the document instructing the user of the kit to culture one of the prior embodiments of the cell culture system.

The attached drawings illustrate embodiments of this disclosure and are included for further understanding of those embodiments.

These are a series of UMAP projections and bar graphs showing single-cell RNA sequencing (scRNA-seq) data obtained from six independent batches of hiPSCs after differentiation for 5 days using three inducible transcription factors (NR5A1, RUNX2, and GATA4). The gene expression profiles of the obtained batches were divided into clusters (early GC, GC, occlusion/luteolysis, mitochondrial gene enrichment, ribosome gene enrichment) and subclusters (early GCI, early GCII, and early GCIII, as well as GCI, GCII, and GCIII). Figure 1A is a collection of gene expression enrichment data obtained from scRNA-seq data, shown as bubble plots, violin plots, and UMAP projections, each representing the relative expression of genes within each cluster or subcluster. This is a series of UMAP projections showing gene expression enrichment data of differentiated hiPSCs against gene expression data corresponding to the gene signatures of primary GC, secondary GC, hydatidiform GC, and pre-ovulatory GC. A series of UMAP projections and bar graphs showing scRNA-seq clustering data of differentiated hiPSCs cultured on either a laminin or a vitronectin matrix. This is an image of a gel showing genotype data from PCR reactions to evaluate the relative expression levels of transcription factors NR5A1, GATA4, and RUNX2 from individual clones after hiPSC reprogramming. This is a series of representative microscopic images showing the morphology of clones after hiPSC differentiation. This graph shows the relative expression levels of ovarian support cell biomarkers and hiPSC biomarkers expressed in each indicated clone, based on the cutoff (dotted line) measured by flow cytometry. This bar graph shows the levels of estradiol (E2) secreted by selected clones in response to the application of follicle-stimulating hormone (+F), androstenedione (+A), or a combination of follicle-stimulating hormone and androstenedione (+F+A) to cell culture medium (DK10). This table characterizes the clones based on the OSC:hiPSC ratio, cell viability, relative expression levels of the indicated biomarkers, and expression levels of progesterone (P4) and estradiol (E2). This image shows a series of fluorescence micrographs illustrating the relative expression of specified hiPSC biomarkers, along with a karyotype graph showing the cellular identity of the selected clones before reprogramming. These are UMAP projections and bar graphs showing scRNA-seq clustering data for two independently reprogrammed batches of hiPSC populations obtained from the same clone. This is an example table of metabolite preparations. An exemplary flowchart for preparing a granulosa co-culture is shown. This is a schematic diagram of a device that assists in the maturation of human oocytes in vitro. This is a schematic diagram of an experimental co-culture IVM approach. hiPSCs are differentiated using overexpression of inducible transcription factors to form ovarian supporting cells (OSCs). Immature human cumulus oocyte complexes (COCs) are harvested from donors in the clinic after being stimulated with shortened gonadotropins. In the laboratory, developmental dishes are prepared, including OSC seeding as needed, and COCs are introduced for IVM co-culture. Oocyte maturation and morphological quality are assessed after 24–28 hours of IVM co-culture, and samples are stored for analysis or used for embryogenesis. This is a typical image of a co-culture setup during plating, containing human COCs (n=5) and 100,000 OSCs. The scale bar is 100 μm. In suspension culture, COCs with both swollen and non-swollen cumulus oophores are observed along with surrounding OSCs. This table shows the maturation rates of oocytes after 24–28 hours of IVM experiment in Experiment 1 or the media control (including oocyte co-culture with OSC). n represents the number of individual oocytes under each culture condition. Error bars indicate the mean ± SEM. The p-value is derived from an asymmetric t-test comparing OSC-IVM with the media control condition. The total oocyte score (TOS) generated from image analysis of MII oocytes after 24–28 hours of IVM experiment is shown. n represents the number of individual MII oocytes analyzed. The median (dashed line) and quartiles (dotted lines) are shown. Unpaired t-tests showed no significant difference between means (p = 0.2909). Due to the small number of oocytes collected per donor, it was not possible to consistently separate oocytes between the two analyzed conditions. Groups mainly consist of oocytes from non-overlapping donor cohorts, and pairwise comparisons were not used. Experiment 2 (including co-culture of oocytes with OSCs or culture with a commercially available IVM control) shows the maturation rate of oocytes after 28 hours of IVM experimentation. n represents the number of individual oocytes under each culture condition. Error bars represent the mean ± SEM. The p-value was derived from paired t-tests comparing experimental OSC-IVM with the control condition (commercially available IVM control). The total oocyte score (TOS) generated from image analysis of MII oocytes after a 28-hour IVM experiment is shown. n represents the number of individual MII oocytes analyzed. The median (dashed line) and quartiles (dotted line) are shown. Unpaired t-tests showed no significant differences between means (p = 0.9420). COCs from each donor were randomly and fairly distributed between control and intervention to allow for paired statistical comparisons. The embryogenesis results after 28 hours of IVM experiment are shown for a subset of oocytes used for embryogenesis in Experiment 2 (including oocyte co-culture with OSCs or commercially available IVM controls). Error bars indicate mean ± SEM. Results are shown as a percentage of total COCs treated in that group. For both IVM conditions, fertilization, cleavage, blastocyst formation, high-quality blastocyst formation, and euploid blastocyst formation are evaluated. Representative images of embryo development under OSC-IVM conditions and commercially available IVM conditions are shown for cleavage day 3, and blastocyst formation days 5, 6, and 7. Embryos with appropriate vitrification quality were classified as "usable quality embryos" and used for trophectoderm biopsy. This is a schematic diagram of an experimental co-culture IVM approach. hiPSCs were differentiated using overexpression of inducible transcription factors to form OSCs. Human oocytes were obtained from donors in the clinic after standard gonadotropin stimulation, and immature oocytes (GV and MI) identified after denucleation were assigned to this study. In the developmental biology laboratory, dishes containing OSC seeding were prepared as needed, and immature oocytes were introduced for IVM co-culture. Oocyte maturation and health were assessed 24–28 hours after IVM co-culture, and oocyte samples were stored for further analysis. This is a typical image of a co-culture setup during plating, containing immature human oocytes (n=3) and OSCs. The scale bar is 200 μm. In suspension culture, deprived GV oocytes are observed together with surrounding OSCs. This table shows the maturation rate of oocytes after 24–28 hours of IVM experimentation (including oocyte co-culture with OSC or oocyte culture in a control medium (medium-IVM)). n represents the number of individual oocytes under each culture condition. Error bars indicate the mean ± SEM. The p-value was derived from an asymmetric t-test comparing experimental OSC-IVM with control medium-IVM. Due to the small number of oocytes collected per donor, each group mainly contains oocytes from non-overlapping donor groups, and pairwise comparisons were not used. The total oocyte score (TOS) generated from image analysis of MII oocytes after 24–28 hours of IVM experiment is shown. n represents the number of individual MII oocytes analyzed. The median (dashed line) and quartiles (dotted line) are shown. Asymmetric t-tests showed no significant difference between means (ns, p = 0.5725). Representative images showing the meiotic spindle of MII oocytes after 28 hours of IVM co-culture with OSCs, stained with fluorescent α-tubulin dye. The blue line crossing the center of the PB1 and spindle assembly from the center of the oocyte was used to derive the PB1 spindle angle. The range of the PB1-spindle angle is shown above. An example of MII with a spindle deficiency is provided under medium-IVM conditions. This figure shows the quantification of the angle between PB1 and the spindle, derived from oocyte fluorescence imaging analysis (similar to Figure 14A). 'n' represents the number of individual oocytes analyzed under each condition. The number of MII oocytes in which spindle formation was not observed is also shown below the axis label. The median (dashed line) and quartiles (dotted line) are shown. ANOVA statistical analysis showed no significant difference in the mean values across the conditions (ns, p = 0.1155). The UMAP projection of the oocyte transcriptome is shown, using symbols colored by the experimental batch, experimental conditions (OSC-IVM, medium-IVM, IVF-MII), oocyte maturation state, and Leiden clusters. Each symbol represents one oocyte. n = 81 oocytes. The UMAP projection is shown colored according to the scores of each gene marker set (GV and IVF MII). The UMAP projections generated from the scores of cells in two different signature marker sets (GV vs. IVF MII) are shown, colored according to experimental conditions, oocyte maturation status, and Leiden clusters. This chart shows the quantification of oocytes in each maturation outcome (GV, MI, MII) under experimental conditions (IVM or IVF). The color distribution indicates the proportion of the population within each Leiden cluster. Striped bars are primarily used to indicate clusters with characteristics similar to IVF. The images show immunofluorescence images of human ovaloid (F66/N.R1.G.F #4 granulosa-like cells + hPGCLC) sections stained with FOXL2 (granulosa), OCT4 (germ cells/pluripotent), and DAZL (mature germ cells) at culture days 2, 4, 14, and 32. The scale bar is 40 μm. A section of mouse ovaloid (mouse fetal ovarian somatic cells + hPGCLCs) stained in the same manner as in Figure 18A is shown. The scale bar is 40 μm. The proportion of OCT4+ cells and DAZL+ cells in human and mouse xenoplasmic obaroids is shown over time compared to total cells (DAPI+). Counts were performed at 11 time points for images from two human obaroid replicas (F66/N.R1.G.F #4 and F66/N.R2 #1 granulosa-like cells + hPGCLCs) and one mouse xenoplasmic obaroid replica. The images show immunofluorescence stains for SOX17 (germ cells), TFAP2C (early germ cells), and AMHR2 (granulosa) in sections of human ovaloid (F66/N.R2 #1 granulosa-like cells + hPGCLC) cultured on days 4 and 8. The scale bar is 40 μm. This image shows the expression of DAZL and OCT4 observed by immunofluorescence in obaroids on day 16. Several DAZL+OCT4- cells (arrows) and DAZL+OCT4+ cells (arrows) are visible. The obaroids are also beginning to form follicle-like morphology (arrows). The scale bar is 40 μm. Sections of human ovaloid (F66/N.R1.G #7+hPGCLC) stained for FOXL2, OCT4, and AMHR2 at day 35 are shown. The scale bar is 40 μm. Follicle-like structures are marked with triangles. This image shows the overall obaroid of the follicle-like structure in human obaroid (F66/N.R1.G #7). The scale bar represents 1 mm. This image shows a section of human ovaloid (F66/N.R1.G.F #4+ hPGCLC) cultured 70 days prior, stained with FOXL2, NR2F2, and AMHR2, revealing multiple small follicles (triangular) consisting of a monolayer of FOXL2+AMHR2+ cells. NR2F2+ cells are scattered among them. The scale bar is 100 μm. This image shows a section of human ovaloid (F66/N.R2 #1+hPGCLC) cultured 70 days prior, stained for FOXL2, NR2F2, and AMHR2, revealing an antral follicle composed of FOXL2+AMHR2+granulosa-like cells arranged in several layers around a central cavity. NR2F2 staining is observed on the outside of the hair follicle (marked "stroma"). The scale bar is 100 μm. The selected granulosa (FOXL2), stromal/capsular (NR2F2), and germ cell (PRDM1) markers (log2 CPM) are shown. Expression was obtained from scRNA-seq analysis of obaroids (F66/N.R1.G.F #4 granulosa-like cells + hPGCLC). Data from all samples (days 2, 4, 8, and 14) were combined and subjected to join dimensionality reduction and clustering. The Leiden clustering of the four main clusters is shown, and the expression of marker genes (log2 CPM) is plotted for each cluster from scRNA-seq analysis of the ovaloid (similar to Figure 18A). This shows the mapping of cells to the Human Fetal Ovarian Reference Atlas (Garcia-Alonso et al., 2022) and the assignment of cell types based on scRNA-seq analysis as described in (Figure 18A). Based on the scRNA-seq analysis shown in Figure 18A, the proportions of somatic cells, germ cells, DAZL+ cells, and DDX4+ cells in the ovaloid for each day are shown. This shows oocytes that have been deprived of their cells by standard treatment. This demonstrates COC (Critical Occlusion) with minimal stimulation. This study demonstrates that OSC-IVM statistically significantly improves the maturation rate of oocytes. This shows the morphological quality of oocytes cultured and grown in OSCs-IVM. This shows the angle between PB1 and the spindle of oocytes cultured and grown in OSC-IVM. Oocytes cultured and grown in OSC-IVM show a high degree of similarity to MII oocytes in the body. Oocytes cultured and grown in OSC-IVM show a high degree of similarity to MII oocytes in the body. This shows the oocyte degradation rate from toxicity assessments of OSC-IVM products. This shows the fertilization and blastocyst formation processes of the OSC-IVM product.

Definitions Unless otherwise defined herein, scientific and technical terms used herein have the meanings generally understood by those skilled in the art. Where there may be any ambiguity, the definitions provided herein shall prevail over any dictionary or external definitions. Unless specifically required by context, singular terms shall include plural forms, and plural terms shall include singular forms. The use of “or” shall mean “and/or” unless otherwise specified. The use of the term “including,” and its variations such as “include” and “included,” shall mean “not limiting.”

As used herein, the term "approximately" refers to a value within plus or minus 10% of the stated value (10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less). For example, the expression "approximately 50 mg" refers to a value between 45 mg and 55 mg (including 45 mg and 55 mg).

As used herein, the terms “assisted reproductive technology” or “ART” refer to infertility treatments in which one or more female reproductive cells (oocytes) or gametes (oocytes) are manipulated in vitro to promote embryo formation, and the embryos are implanted in a subject seeking pregnancy. For example, in some embodiments, oocytes collected from a subject undergoing ART treatment can be matured in vitro using, for example, the co-culture method described herein. In some embodiments, once mature oocytes (oocytes) are formed, they may be treated with one or more spermatids to promote zygote and ultimately embryo formation. The embryos may then be implanted in the uterus of a female subject using, for example, the compositions and methods of the art. Exemplary ART treatments include in vitro fertilization (IVF) and intracytoplasmic sperm injection (ICSI) techniques described herein and known in the art.

As used herein, the term "subject" refers to a living organism receiving treatment for a specific disease or condition described herein. Examples of subjects include mammals such as humans (e.g., females) receiving treatment for diseases or conditions related to reduced ovarian reserve or the release of immature oocytes.

As used herein, the terms “modified ovarian stimulation” or more concisely “ovarian stimulation” refer to procedures that induce ovulation in subjects such as humans before the collection of oocytes or eggs for use in embryogenesis, such as in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI). Modified ovarian stimulation may include administering follicle-stimulating hormone (FSH), human chorionic gonadotropin (hCG), and/or gonadotropin-releasing hormone (GnRH) antagonists to subjects to promote follicular maturation. While methods of modified ovarian stimulation are known in the art, they are also described herein because they are methods for inducing follicular maturation and ovulation in combination with assisted reproductive technology.

As used herein, the term "derived from" with respect to cells derived from a subject means that a cell (e.g., a mammalian oocyte) is isolated from the subject, or obtained from the growth, division, maturation, or manipulation (e.g., in vitro growth, division, maturation, or manipulation) of one or more cells isolated from the subject. For example, when an oocyte is "derived from" a subject or an oocyte described herein, the oocyte is either directly isolated from the subject or obtained from the maturation of an oocyte isolated from the subject (e.g., an oocyte isolated from a subject approximately 1 to 5 days after ovarian stimulation (e.g., an oocyte isolated from a subject approximately 2 to 4 days after ovarian stimulation)).

As used herein, the term “dose” refers to the amount of a therapeutic agent (e.g., a follicle-stimulating agent as described herein) administered to a subject for the treatment of a disease or condition (e.g., to promote oocyte maturation and/or release of live oocytes, and to promote the collection and in vitro maturation of live oocytes). The therapeutic agents described herein may be administered once or multiple times. In each case, the therapeutic agent may be administered using one or more unit dosage forms. For example, one dose of 100 mg of the therapeutic agent may be administered using, for example, two 50 mg unit dosage forms of the therapeutic agent. Similarly, one dose of 300 mg of the therapeutic agent may be administered using, for example, six 50 mg unit dosage forms of the therapeutic agent, or two 50 mg unit dosage forms of the therapeutic agent and one 200 mg unit dosage form of the therapeutic agent. Similarly, a single dose of 900 mg of the therapeutic agent may be administered, for example, using six 50 mg units of the therapeutic agent and three 200 mg units of the therapeutic agent, or using ten 50 mg units of the therapeutic agent and two 200 mg units of the therapeutic agent.

As used herein, the term “follicle induction period” refers to the time for administering the follicle induction agent. The time for administering the follicle induction agent to a female subject (i.e., the follicle induction period) is day 1, day 2, or day 3 of the subject’s menstrual cycle, with day 2 being preferred. However, if the female subject is taking hormonal contraceptives, the time for administering the follicle induction agent is 4 to 6 days after the last oral contraceptive dose (e.g., day 4, day 5, or day 6), with day 5 being preferred.

As used herein, the term “follicle-stimulating hormone” (FSH) refers to a biologically active heterodimer human reproductive hormone capable of inducing ovulation in a subject. FSH may be purified from the urine of postmenopausal women or produced as a recombinant protein product. Exemplary recombinant FSH products include follitropin alpha (GONAL-F, Merck Serono/EMD Serono) and follitropin beta (PUREGON/FOLLISTIM, MSD/Scherig-Plough).

As used herein, the term “human chorionic gonadotropin” (hCG) refers to a polypeptide hormone that interacts with the luteinizing hormone chorionic gonadotropin receptor (LHCGR) to induce follicular maturation and ovulation. hCG can be purified from the urine of pregnant women or produced as a recombinant protein product. An example of a recombinant hCG product is chorionic gonadotropin α (Ovidrel®, Merck Serono/EMD Serono).

As used herein, the term “in vitro fertilization” (IVF) refers to the process of bringing an egg, such as a human egg, into contact with one or more spermatids outside the body to promote fertilization of the egg and formation of a zygote. The egg may originate from a subject, such as a human subject undergoing various ARTs known in the art. For example, one or more oocytes can be obtained from a subject after injection of a follicular maturation stimulant for controlled ovarian stimulation, for example, about 1 to 5 days after the injection of the drug (for example, about 4 days after the injection of the follicular maturation stimulant into the subject). The egg may also be collected directly from the subject, for example, by transvaginal oocyte retrieval methods known in the art.

As used herein, the term “intracytoplasmic sperm injection” (ICSI) refers to the process of directly injecting spermatids into an egg, such as a human egg, to facilitate fertilization and zygote formation. For example, spermatids can be injected into an egg by perforating the egg cell membrane with a microinjector to deliver them directly into the egg cell cytoplasm. ICSI methods useful in combination with the compositions and methods described herein are known in the art, for example, described in WO2013/158658, WO2008/051620, and WO2000/009674, and these disclosures, in particular, relating to compositions and methods for carrying out intracytoplasmic sperm injection, are incorporated herein by reference.

As used herein, the terms “oocyte” and “oocyte” refer to haploid female germ cells or gametes. In the context of assisted reproductive technology as described herein, oocytes may be produced in vitro by maturing one or more oocytes isolated from a subject undergoing ART. Oocytes can also be isolated directly from a subject, for example, by transvaginal oocyte retrieval methods described herein or known in the art. As used in this disclosure, oocyte or oocyte may refer to multiple oocytes. Oocytes may form complexes with surrounding cells (e.g., cumulus-oocyte complexes (COCs)).

As used herein, the terms “mature oocyte” and “mature oocyte” refer to one or more oocytes or oocytes in metaphase II (MII) of meiosis, typically having morphological or structural features consistent with metaphase II, such as the polar body and other features described herein.

As used herein, the terms “immature oocyte” and “immature oocyte” refer to one or more oocytes or oocytes that have not reached meiotic phase MII. In some embodiments, immature oocytes may include oocytes in the vesicle (GV) phase and/or metaphase I (MI) phase, as determined by morphological features and/or other indicators known in the art.

As used herein, the term “oocyte maturation” refers to the developmental process by which immature oocytes transition to mature oocytes. Oocyte maturation occurs as immature oocytes receive cellular signaling events triggered by external and internal stimuli. External stimuli may be generated by adjacent or supporting cells as described herein. Oocyte maturation may occur before oocyte release and oocyte retrieval from the subject. Oocyte maturation may occur in vitro as a result of the culture methods and culture compositions described herein.

As used herein, “induced pluripotent stem cells (iPSCs),” such as human iPSCs (hiPSCs), refer to one or more cells that self-replicate in an undifferentiated state and can differentiate into any one type of differentiated cell present in an organism. iPSCs can be induced from non-embryonic cell sources, such as somatic cells, and can proliferate indefinitely. Depending on the induction of transcription factors and/or the use of gene editing methods known in the art, iPSCs can differentiate into each of the three germ layers (i.e., endoderm, mesoderm, and ectoderm) and further into cell types within them. In one example, iPSCs can differentiate into a population of ovarian supporting cells, as described herein. The pluripotency or "stem-like" nature of iPSCs can be verified by the expression of one or more pluripotency-specific markers, including OCT4, SSEA3, SSEA4, TRA1-60, TRA1-81, NANOG, SOX2, and/or POU5F1, among others known in the art and described herein.

As used herein, “Ovarian supporting cells” (OSCs) or “supporting cells” refers to one or more cells that promote the maturation of one or more oocytes. OSCs may be ovarian granulosa cells (e.g., a type of granulosa cell as described herein). Additionally or alternatively, OSCs may be ovarian stromal cells (e.g., a type of stromal cell as described herein). OSCs may form cumulus-oocyte complexes (COCs) with oocytes. OSCs may be generated from exogenous sources such as induced pluripotent stem cells (iPSCs), e.g., human induced pluripotent stem cells (hiPSCs), as described herein. OSCs can be applied to harvested oocytes using the in vitro cell culture methods and compositions described herein. OSCs may be a mixture of two or more cell types. OSCs may be a mixture of stromal cells and granulosa cells such that the stromal cells and granulosa cells are aggregated in a ratio of approximately 1:1. OSC may be a mixture of stromal cells and granulosa cells in which one cell type is more abundant than one or more cell types, and in possible population distributions, for example, a population of about 2:1, about 3:1, about 4:1, or about 5:1. OSC may be a mixture of stromal cells and granulosa cells in which one cell type is more abundant (for example, in possible distributions, 90% stromal cells and 10% granulosa cells, 80% stromal cells and 20% granulosa cells, 70% stromal cells and 30% granulosa cells, 60% stromal cells and 40% granulosa cells, 40% stromal cells and 60% granulosa cells, 30% stromal cells and 70% granulosa cells, 20% stromal cells and 80% granulosa cells, or 10% stromal cells and 90% granulosa cells). In some embodiments, OSCs may be a mixture of stromal cells and granulosa cells combined with one or more further cell types.

As used herein, “ovarian stromal cells” or “stromal cells” are cumulus cells that surround an oocyte to ensure healthy oocyte development and subsequent embryonic development. Ovarian stromal cells may form a cross-occipital cell (COC) with the oocyte. Ovarian stromal cells may express markers consistent with the stromal subtype, such as nuclear receptor subfamily 2 group F member 2 (NR2F2). NR2F2 can be detected by methods known in the art. Ovarian stromal cells may be steroid-producing stromal cells. Ovarian stromal cells can be generated from differentiated hiPSCs as described herein.

As used herein, “steroid-producing stromal cells” are stromal cells capable of producing one or more steroids, such as estradiol, progesterone, or a combination thereof. One or more steroids may be produced in response to hormonal stimulation, such as FSH, androstenedione, or a combination thereof. One or more steroids may be secreted.

As used herein, the terms "EGFR" and "ERBB1" are interchangeable terms referring to genes or biomarkers expressed by cells (e.g., ovarian supporting cells, (e.g., differentiated iPSCs)). EGFR and ERBB1 are gene names for the epidermal growth factor receptor (e.g., human epidermal growth factor receptor, NCBI gene ID: 1956). Other names for this gene are known in the art and include ERRP, HER1, mENA, PIG51, and NISBD2.

As used herein, “ovarian granulosa cells” or “granulosa cells” are cumulus cells surrounding the oocyte, ensuring healthy oocyte development and subsequent embryonic development. Ovarian granulosa cells can form COCs with the oocyte. Ovarian granulosa cells may express markers consistent with the granulosa subtype, such as FOXL2, CD82, and/or follicle-stimulating hormone receptor (FSHR). Such markers can be detected by methods known in the art. Ovarian granulosa cells may be steroid-producing granulosa cells. Ovarian granulosa cells can be generated from differentiated hiPSCs as described herein.

As used herein, “steroid-producing granulosa cells” are granulosa cells capable of producing one or more steroids, such as estradiol, progesterone, or a combination thereof. One or more steroids may be produced in response to hormonal stimulation, such as FSH, androstenedione, or a combination thereof. One or more steroids may be secreted.

As used herein, the terms “biological sample” or “sample” may refer to one or more isolated cells, an entire or partial population of cells, and/or components of an in vitro cell culture system such as cell culture medium. Furthermore, a biological sample or sample may refer to a specimen (e.g., blood, blood components (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissue (e.g., placenta or dermis), pancreatic juice, chorionic villi samples, hair, oocytes, oocytes, and/or cells isolated from the subject).

As used herein, the term “expression” refers to one or more of the following events: (1) the generation of an RNA template from a DNA sequence (e.g., by transcription); (2) the processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and/or 3' end processing); (3) the translation of RNA into a polypeptide or protein; and (4) post-translational modification of a polypeptide or protein. The expression of a target gene or biomarker in a sample (e.g., a biological sample, e.g., a biological sample comprising one or more iPSCs, OSCs, oocytes, or combinations thereof) can be revealed, for example, by detecting the amount or concentration of mRNA encoding the corresponding protein in the sample (e.g., assessed using RNA detection procedures such as quantitative polymerase chain reaction (qPCR), reverse transcription PCR (RT-PCR), and RNA sequencing (RNA-seq) techniques, as well as other RNA detection methods known in the art), the amount or concentration of the corresponding protein (e.g., assessed using protein detection methods described herein or known in the art, e.g., enzyme-linked immunosorbent assay (ELISA), immunofluorescence, Western blotting, or mass spectrometry), and/or the activity of the corresponding protein (e.g., in the case of an enzyme, assessed using enzyme activity assays known in the art). Any one of the preceding processes may result in increased or decreased expression compared to a control sample. In some embodiments, the control sample is a cell or cell population that has not undergone one or more of the procedures that the target sample has undergone. Exemplary procedures include reprogramming or differentiation methods, such as any one or more types of reprogramming or differentiation methods for iPSCs described herein; in vitro maturation methods, such as in vitro maturation methods for one or more oocytes described herein; or in vitro fertilization methods, such as in vitro fertilization methods described herein. In some embodiments, the control sample is a cell or cell population representative of a particular cell type, such as an established cell line or other characterized cell type obtained from a manufacturer (e.g., iPSC or OSC). In some embodiments, if one or more cells are determined to express a gene or biomarker, the expression level is within an acceptable range compared to the expression level of the control sample (e.g., within 20%, 15%, 10%, or 5% of the expression level of the control sample). In some embodiments, significant expression refers to an expression level relative to a cutoff value or threshold, such as a cutoff value or threshold for RNA sequencing or flow cytometry. In some embodiments, a cell population may be determined to express a gene or biomarker of interest if a significant portion of the population (e.g., about 50%, 60%, 70%, 80%, 90%, 95%, or 99%) exhibits significant expression of the gene or biomarker of interest by meeting or exceeding a specific cutoff or threshold.

In some cases, significant expression of one or more target genes or biomarkers may not be observed. In some embodiments, “no significant expression” or “no detectable expression” refers to an expression level below the detection limit of a particular detection method (e.g., RT-PCR or ELISA) and/or an expression level less than approximately 95% (e.g., less than 95%, 96%, 97%, 98%, 99%, or less than 99%) compared to a suitable control such as a particular cell type (e.g., a typical OSC such as an undifferentiated iPSC or an in vivo OSC). In some embodiments, “no significant expression” or “no detectable expression” refers to an expression level relative to a cutoff value or threshold, such as a cutoff value or threshold for RNA sequencing or flow cytometry. No significant expression or no detectable expression refers to the relative expression level of a cell population, where a significant portion of that population (e.g., approximately 50%, 60%, 70%, 80%, 90%, 95%, or 99%) has expression levels below a specific cutoff or threshold, resulting in no significant expression of the gene or biomarker of interest.

As used herein, the term “overexpression” refers to the increased expression of a gene or biomarker compared to the baseline level of expression in a particular cell type (e.g., iPSC). The expression of a gene or biomarker may be greater than 100%, such as 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, or, in some cases, greater than 125%, 150%, 175%, 200%, 225%, 250%, 275%, 300%, 325%, 350%, 375%, 400%, or greater than 400%. Overexpression of a gene or biomarker can be detected or measured by any suitable method for detecting or measuring gene or biomarker expression, such as genotyping methods, mRNA detection methods, or protein detection methods, as described herein and known in the art.

As used herein, the term “doxycycline-responsive transcriptional regulator” refers to a nucleotide sequence, such as a promoter, that initiates gene transcription in the presence of doxycycline (e.g., an effective amount of doxycycline).

As used herein, the terms “oral contraceptive therapy,” “oral contraception,” “contraception,” or “oral contraceptives” typically refer to hormonal therapies used to prevent pregnancy. Oral contraceptive therapy may block the release of oocytes from the ovaries and may contain hormones including estrogen and progestin.

As used herein, the term “ovarian reserve” refers to the number and quality of oocytes in the ovary of a subject. Ovarian reserve naturally decreases with age and/or the medical conditions described herein. Subjects with reduced ovarian reserve may seek IVF or other ART to achieve successful pregnancy. As described herein, levels of anti-Müllerian hormone (AMH) may indicate a subject's ovarian reserve.

As used herein, the term "stimulation protocol" refers to the process of administering one or more follicle-inducing agents to a target during a follicle-inducing period.

As used herein, the terms “follicle-inducing agent” or “inducer” refer to a chemical or biological composition that stimulates the release of oocytes from the ovary during ovulation. Follicle-inducing agents may contain hormones such as human chorionic gonadotropin and follicle-stimulating hormone. As used herein, the term “induced pluripotent stem cell” (iPSC) refers to an induced stem cell obtained by reprogramming and/or otherwise manipulating harvested somatic cells. iPSCs can differentiate into ovarian supporting cells or other cell types, including granulosa cells, by methods known in the art and those described herein. iPSCs may be human iPSCs (hiPSCs) or, for example, iPSCs derived from other mammals.

As used herein, the terms “Clomid” or “clomifene citrate” are used interchangeably and refer chemically to a nonsteroidal ovulatory agent having the molecular formula _C26H28ClNO・_C6H8O7 and a molecular weight of 598.01 g/mol. Clomifene citrate is a mixture of two geometric isomers, cis (zuclomifene) and trans (enclomifene), the mixture containing 30% _{to 50} % (e.g., about 25%, about 30%, _about 35%, about 40%, about 45%, about 50%, or about 55%) of the cis isomer (zuclomifene).

As used herein, the term "cell culture" refers to laboratory methods that enable the extracorporeal proliferation and/or culture of prokaryotic or eukaryotic cell types.

As used herein, the term “matrix” in the context of cell culture or in vitro cell culture methods refers to a coating such as a substrate or membrane on the surface of a cell culture vessel (e.g., a well plate or Petri dish) that facilitates the fixation or adhesion of cells to the surface. A suitable matrix may be a protein such as glycoproteins or proteoglycans, or a combination thereof. A suitable matrix may be derived from plant or animal products, or may be a synthetic product (e.g., a recombinant protein or polymer). A suitable matrix may be an extracellular matrix protein, its specific isoform, or a purified or partially purified fraction concentrated based on its molecular weight. A suitable matrix may include a mixture of components. An exemplary matrix includes alginates, laminins (e.g., laminin-111, laminin-211, laminin-121, laminin-221, laminin-332, laminin-311, laminin-321, laminin-411, laminin-421, laminin-511, laminin-521, and/or laminin-213), vitronectins (e.g., type I, type II, and/or type III), fibronectins (e.g., The matrix includes type I, type II, and/or type III collagen (e.g., type I, type II, type III, type IV, type V, type VI, type VII, type VIII, type IX, type X, type XI, type XII, type XIII, type XIV, type XVI, type XVII, type XVIII, type XVIIII, type XIX, type XX, type XXI, type XXII, type XIII, and/or type XXIV), chitosan, hyaluronic acid, poly-D-lactone, and hydrogel. The choice of matrix depends on the cell type or relative cellular composition (e.g., lipid content, surface protein expression, e.g., relative abundance of one or more adhesion receptors).

As used herein, the term “mounting” or its variations refers to the coating of one or more cells in an in vitro culture system, which, optionally, are delivered (e.g., embedded, distributed, or deposited) into a cellular niche (e.g., a tissue or organ of an organism). Mounting one or more cells may improve the efficiency of delivery. Suitable mounting reagents may be proteins such as glycoproteins and proteoglycans, or combinations thereof. Reagents for mounting may be derived from plant or animal products, or synthetic products (e.g., recombinant proteins or polymers). Suitable mounting reagents may be extracellular matrix proteins, their specific isoforms, or purified or partially purified fractions concentrated based on molecular weight. Suitable mounting reagents may include mixtures of components. Exemplary mounting media include alginates, laminins (e.g., laminin-111, laminin-211, laminin-121, laminin-221, laminin-332, laminin-311, laminin-321, laminin-411, laminin-421, laminin-511, laminin-521, and/or laminin-213), vitronectin (e.g., type I, type II, and/or type III), fibronectin (e.g., type I) The reagents include type I, type II, and/or type III), collagen (e.g., type I, type II, type III, type IV, type V, type VI, type VII, type VIII, type IX, type X, type XI, type XII, type XIII, type XIV, type XVI, type XVII, type XVIII, type XVIIII, type XIX, type XX, type XXI, type XXII, type XIII, and/or type XXIV), chitosan, hyaluronic acid, poly-D-lactone, and hydrogel. The selection of reagents for inclusion may vary depending on the cell type or relative cellular composition (e.g., lipid content, surface protein expression, relative abundance of one or more adhesion receptors) of the cells to be inclusion or the cells in the niche receiving one or more inclusiond cells.

As used herein, the term “Wnt/β-catenin activator” refers to any agent (e.g., nucleic acids, proteins, lipids, small molecules, or combinations thereof) that ultimately upregulates, stimulates, initiates, drives, or induces the activation of the Wnt/β-catenin signaling pathway. A Wnt/β-catenin activator may upregulate the pathway by directly contacting a positive regulator of the pathway. A Wnt/β-catenin activator may upregulate the pathway by directly contacting a negative regulator of the pathway (i.e., acting as an inhibitor of an inhibitor of the Wnt/β-catenin pathway). Regulators of the Wnt/β-catenin signaling pathway are known in the art and are described in other literature, such as Nusse and Clevers, Cell. 169(6):985–999, 2017, which are incorporated herein by reference.

In some embodiments, the Wnt/β-catenin activator is a Rho-related protein kinase (ROCK) inhibitor, such as Y-27642 or an equivalent salt or derivative thereof, whose structure is shown below.

In some embodiments, the Wnt/β-catenin activator is a glycogen synthase kinase 3 inhibitor such as CHIR099021, or an equivalent salt or derivative thereof, the structure of which is shown below.

As used herein, the term "co-culture" refers to a type of cell culture method in which multiple cell types or cell populations are cultured in some degree of contact. In a typical co-culture system, two or more cell types may share an artificial growth medium.

As used herein, the terms “adherent co-culture system” or “adherent cell culture” refer to a cell culture configuration in which cells are attached to a surface for proper growth and proliferation.

As used herein, the terms “suspension co-culture system” or “suspension cell culture” refer to a cell culture configuration in which cells are cultured by dispersion in a liquid medium for appropriate growth and proliferation.

Detailed Description This specification describes compositions and methods for use in assisted reproductive technology (ART). For example, the compositions and methods described herein relate to producing, manipulating, and culturing one or more ovarian supporting cells (OSCs) (e.g., ovarian granulosa cells, ovarian stromal cells, or a combination thereof), and to in vitro maturating oocytes.

Advantageously, the compositions and methods described herein facilitate the collection and use of oocytes previously discarded for in vitro fertilization (IVF) purposes by performing in vitro maturation of immature oocytes via co-culture with ovarian support cells (e.g., reprogrammed iPSC-derived ovarian support cells). The described IVF maturation methods improve the ability to use immature oocytes that would normally be discarded in IVF procedures, potentially leading to more cost-effective treatment strategies and reduced risks for patients undergoing treatment. For example, the method can reduce the risk of in vivo ovarian hyperstimulation by requiring fewer hormonal injections and/or lower doses of hormonal injections for subjects seeking IVF treatment than current IVF treatment options. Aspects of this disclosure can increase the overall pool of available, healthy oocytes from women for use in IVF. Furthermore, aspects of this disclosure can significantly reduce the amount of hormonal administration to subjects during oocyte retrieval and improve the quality of oocytes in culture. This could significantly expand access to reproductive technologies, drastically shorten the cycle duration, and reduce the overall number of cycles required to achieve pregnancy.

I. In Vitro Compositions and Cell Culture Media In some embodiments, the Disclosure provides engineered cell culture systems. In some embodiments, the engineered cell culture system includes a population of artificial ovarian supporting cells (OSCs). In some embodiments, the subject matter described herein relates to a method for differentiating a population of induced pluripotent stem cells (iPSCs), such as human iPSCs (hiPSCs), into a population of OSCs. In some embodiments, the subject matter described herein relates to an in vitro maturation (IVM) method. In some embodiments, the engineered cell culture system promotes the maturation of one or more oocytes. In some embodiments, the subject matter described herein relates to an in vitro fertilization (IVF) method. In some embodiments, one or more mature oocytes are utilized in an ART or IVF method. In some embodiments, the engineered cell culture system is an engineered cell co-culture system. In some embodiments, the co-cultured cells are in suspension culture. In some embodiments, the co-cultured cells are in adherent culture.

A. Ovarian Supporting Cells Derived from Human Induced Pluripotent Stem Cells Any one or more OSCs used in the methods described herein may be created from iPSCs using a transcription factor (TF)-directed protocol. In some embodiments, the iPSCs are mammalian iPSCs. In preferred embodiments, the iPSCs are human iPSCs (hiPSCs). In some embodiments, the hiPSCs may be transformed with one or more plasmids encoding any one or more transcription factors. In some embodiments, differentiation from hiPSCs to OSCs is facilitated by the overexpression of one or more transcription factors. In some embodiments, the one or more TFs include FOXL2, NR5A1, RUNX2, GATA4, or any combination thereof. In some embodiments, undifferentiated hiPSCs are reprogrammed using a transposase method (e.g., piggyBac transposase method) to carry specific inducible transcription factors (e.g., FOXL2, NR5A1, RUNX2, and/or GATA4). In some embodiments, hiPSCs can be transformed by electroporation, liposome-mediated transformation, or virus-mediated gene transfer, among other cell transformation methods known in the art. In some embodiments, gene expression of a desired transcription factor can be induced in a doxycycline-dependent manner. In some embodiments, the plasmid or expression vector used to reprogram hiPSCs may include a reporter gene such as a fluorescent protein. In some embodiments, hiPSCs can differentiate into stromal cells by the induced expression of transcription factors including GATA4, FOXL2, or a combination thereof. In some embodiments, hiPSCs can differentiate into granulosa cells by the induced expression of transcription factors including FOXL2, NR5A1, GATA4, RUNX1, RUNX2, or a combination thereof. In addition to a combination of one or more transcription factors such as FOXL2, NR5A1, GATA4, RUNX1, and/or RUNX2, hiPSCs can differentiate into granulosa via the expression of KLF2, TCF21, NR2F2, or a combination thereof.

OSCs used in the methods described herein may be generated using a CRISPR (Critical Repeating Sequence) technique, which involves the aggregation and regularly spaced arrangement of short palindromic sequences. CRISPR is a programmable technique for editing DNA at precise locations by targeting specific genetic codes. CRISPR techniques may include CRISPR-CAS 9. Cas9 (or "CRISPR-associated protein 9") is an enzyme that uses a CRISPR sequence as a guide to recognize and cleave specific strands of DNA complementary to the CRISPR sequence, thereby enabling the insertion of exogenous nucleic acids into the cell's genome. For example, CRISPR-based gene editing techniques can be used to introduce one or more genes encoding factors that induce differentiation into OSCs (e.g., granulosa cells or stromal cells) into an iPSC genome. These factors include, for example, FOXL2, NR5A1, GATA4, RUNX1, and RUNX2.

Exemplary CRISPR systems include those utilizing the Cas9 enzyme. The Cas9 enzyme, along with the CRISPR sequence, forms the basis of a technology known as CRISPR-Cas9, which can be used to edit genes in living organisms. The CRISPR method may include a Class 1 CRISPR system comprising Type I (cas3), Type III (cas10), and Type IV and 12 subtypes. The CRISPR method may also include a Class 2 CRISPR system comprising Type II (cas9), Type V (cas12), Type VI (cas13), and nine subtypes. In some embodiments, the CRISPR method may include a CRISPR-Cas design tool, which is a computer software platform, and bioinformatics tools used to facilitate the design of guide RNA (gRNA) for use with the CRISPR/Cas gene editing system. For example, CRISPR-Cas design tools include CRISPRon, CRISPRoff, Invitrogen TrueDesign Genome Editor, Breaking-Cas, Cas-OFFinder, CASTING, CRISPy, CCTop, CHOPCHOP, CRISPR, sgRNA Designer, and Synthego Design Tool. The CRISPR method can also be used as a diagnostic tool. For example, CRISPR-based diagnostics can be linked to enzymatic processes such as SHERLOCK-based in vitro transcription profiling (SPRINT). SPRINT can be used to detect various substances, such as metabolites in target samples or contaminants in environmental samples, using high-throughput or portable point-of-care devices.

In some embodiments, the overexpression of one or more TFs is driven by any suitable induction agent known in the art. In some embodiments, the induction period lasts for one, two, three, four, five, or more than five days. In some embodiments, the transcription factors are constitutively expressed. In some embodiments, the plasmid or expression vector used for reprogramming hiPSCs may include a reporter gene such as a fluorescent protein. In some embodiments, cells (e.g., hiPSCs) are treated with a drug that prepares the cells for mesoderm induction. In some embodiments, during the initial induction of one or more transcription factors, cells (e.g., hiPSCs) are treated with a drug that activates Wnt/β-catenin signaling to prepare the cells for the fate of mesoderm cells. In some embodiments, during the initial induction of one or more transcription factors, cells (e.g., hiPSCs) are treated with agents that inhibit serine-threonine protein kinases, such as Rho-related protein kinase (ROCK) (e.g., small molecule ROCK inhibitors, e.g., Y-27642) or glycogen synthase kinase-3 (GSK3) (e.g., GSK3 inhibitors, e.g., CHIR099021).

In some embodiments, the resulting OSCs comprise a cell population functionally and biologically similar to human granulosa cells (cells expressing FOXL2 and AMHR2, among other granulosa biomarkers known in the art and described herein). In some embodiments, the resulting OSCs comprise a cell population functionally and biologically similar to ovarian stromal cells (cells expressing R2F2, among other stromal cell biomarkers known in the art and described herein). In some embodiments, the resulting OSCs comprise a cell population containing both granulosa cells and stromal cells. In some embodiments, the OSC population comprises primarily granulosa cells, resulting in the OSC population containing 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, or more than 95% granulosa cells. The reprogramming of hiPSCs into granulosa can be determined by the genotyping methods described herein.

Differentiation from hiPSCs to OSCs (such as granulosa cells) can be determined by the relative expression of biomarkers typical of granulosa cell types, including FOXL2, AMHR2, CD82, FSHR, IGFBP7, KRT19, STAR, WNT4, or combinations thereof, among other granulosa cell biomarkers known in the art. hiPSCs differentiated into OSCs (such as granulosa cells) can be classified into one or more clusters based on transcriptome profiling. In some embodiments, differentiation from hiPSCs to OSCs (such as granulosa cells) can be determined by the relative expression of biomarkers or genes, such as genes related to cell adhesion, chemotaxis, growth factors and/or growth factor receptors, steroids and/or steroid receptors, or combinations thereof, among other genes or biomarkers associated with one or more types of OSCs. Differentiation from hiPSCs to OSCs may be determined by the relative expression of biomarkers typical of OSCs (e.g., granulosa cells), including GJA1, MDK, BBX, HES4, PBX3, YBX3, BMPR2, CD46, COL4A1, COL4A2, LAMC1, ITGAV, and/or ITGB. In some embodiments, biomarkers typical of OSCs may further include FOXO1, CDH1, CYP19A1, RARRES2, NOTCH2, NRG1, BMPR1B, EGFR (ERBB1), and/or ERBB4. In some embodiments, biomarkers typical of OSCs may further include RARRES2, NOTCH2, NOTCH3, ID3, and/or BMPR2. In some embodiments, typical biomarkers for OSC may further include CDH2 and/or NOTCH2 without marked expression of RARRES2. In some embodiments, functional screening of individual clones identifies stable lines that maintain an optimal balance of expression for each transcription factor or biomarker.

In some embodiments, application of doxycycline to cells or cell cultures maintains the activation of one or more TFs within the OSC (e.g., in the case of doxycycline-dependent induction).

The expression of one or more genes, transcription factors, and/or biomarkers can be detected or measured by methods of protein and/or mRNA expression routinely used in the art. Exemplary methods for detecting or measuring the relative expression of one or more transcription factors or biomarkers include flow cytometry, RNA sequencing (RNA-seq) (e.g., single-cell RNA-seq), real-time reverse transcription polymerase chain reaction (RT-PCR), quantitative PCR (qPCR), RT-qPCR, Northern blotting, mass spectrometry and proteomic methods, Western blotting, enzyme-linked immunosorbent assay (ELISA), immunofluorescence or immunodetection, and other detection methods known in the art.

In some embodiments, reprogramming hiPSCs into OSCs yields one or more OSCs that share strong gene expression similarity with in vivo granulosa cells (e.g., cells expressing FOXL2 and AMHR2). In some embodiments, the OSCs share strong gene expression similarity with in vivo stromal cells (e.g., cells expressing NR2F2). In some embodiments, the OSCs replicate the progression of follicular formation in vitro through follicular development. In some embodiments, reprogramming from hiPSCs into one or more OSCs may be determined by the production of growth factors and/or hormones that can adequately support the in vitro maturation of oocytes recovered via paracrine and juxtacrine cell signaling. In some embodiments, the OSCs produce one or more growth factors, including insulin-like growth factor (IGF), stem cell factor (SCF), epidermal growth factor (EGF), leukemia suppressor factor (LIF), vascular endothelial growth factor (VEGF), bone morphogenetic protein (BMP), C-type natriuretic peptide (CNP), or any combination thereof. In some embodiments, one or more growth factors are secreted from OSCs. In some embodiments, OSCs are steroid-producing and produce hormones including estradiol and/or progesterone. In some embodiments, OSCs are steroid-producing in the presence of exogenously supplied reagents. In some embodiments, OSCs such as granulosa cells produce estradiol and/or progesterone upon stimulation with androstenedione and FSH or forskolin. In some embodiments, the secretion of estradiol and/or progesterone can be detected or measured by one or more protein detection methods known in the art. In some embodiments, doxycycline application to OSCs maintains cellular identity and drives steroid-producing activity. In some embodiments, doxycycline is applied to OSCs or OSC medium when thawing and seeding OSCs obtained from iPSCs that have been cryopreserved and reprogrammed after cryopreservation (e.g., in the case of doxycycline-dependent induction).

B. Cell Culture Medium OSCs derived from iPSCs (e.g., hiPSCs) or transgenic OSCs may be provided as a composition further comprising a cell culture medium. The OSCs or progenitor cells (e.g., hiPSCs before reprogramming) may be cultured in the cell culture medium. In some embodiments, the manipulated OSCs may be added to commercially available reproductive media (e.g., IVF, IVM, (e.g., MEDICULT IVM® medium), or LAG medium). In some embodiments, the cell culture medium comprises DMEM/F12 supplemented with knockout serum replacement (KSR). In some embodiments, the cell culture medium comprises an L-glutamine analog such as GLUTAMAX® (GIBCO®, Thermo Fisher Scientific, Waltham, MA), and optionally, GLUTAMAX® is adapted to use reagents that do not contain animal-derived components. In some embodiments, the developmental biology laboratory procures appropriate IVF cell culture medium. In some embodiments, the cell culture medium includes Medicult IVM medium. In some embodiments, the developmental biology laboratory procures appropriate cell culture plates. In some embodiments, the cell culture plates are GPS Universal dishes. In some embodiments, the developmental biology laboratory procures ART-grade mineral oil. In some embodiments, co-culture is achieved by preparing IVM medium. In some embodiments, the IVM medium includes a basic medium formulation. In some embodiments, the basic medium formulation includes MEDICULT IVM® medium.

In some embodiments, the hiPSCs and/or obtained OSCs described herein are cultured on a matrix or encapsulated in a matrix during induction, culture, or co-culture with oocytes. In some embodiments, the hiPSCs and/or OSCs are cultured on a matrix or encapsulated in a matrix containing alginate. In some embodiments, the hiPSCs and/or OSCs are cultured on a matrix or encapsulated in a matrix containing laminin. In some embodiments, the hiPSCs and/or OSCs are cultured on a matrix or encapsulated in a matrix containing laminin-521. In some embodiments, the hiPSCs and/or OSCs are cultured on a matrix or encapsulated in a matrix containing vitronectin. In some embodiments, the hiPSCs and/or OSCs are cultured on a matrix or encapsulated in a matrix containing collagen. In some embodiments, the hiPSCs and/or OSCs are cultured on a matrix or encapsulated in a matrix containing chitosan. In some embodiments, hiPSCs and/or OSCs are cultured on a matrix or encapsulated in a matrix containing hyaluronic acid. In some embodiments, hiPSCs and/or OSCs are cultured on a matrix or encapsulated in a matrix containing dextran hydrogel. In some embodiments, hiPSCs and/or OSCs are cultured on a matrix or encapsulated in a matrix containing MATRIGEL® matrix.

In some embodiments, the cell culture medium is supplemented. The cell culture medium may be supplemented with human serum albumin (HSA) (e.g., about 5–15 mg/mL, e.g., 10 mg/mL), follicle-stimulating hormone (FSH) (e.g., about 70–80 mIU/mL, e.g., 75 mIU/mL), human chorionic gonadotropin (hCG) (e.g., about 95–105 mIU/mL, e.g., 100 mIU/mL), androstenedione (e.g., about 495–505 ng/mL, e.g., 500 ng/mL), doxycycline (e.g., 0.5–1.5 μg/mL, e.g., 1 μg/mL), and other compounds such as hyaluronidase and/or dPBS. In some embodiments, one or more supplemented proteins are recombinant proteins. In some embodiments, the cell culture medium is supplemented with a drug that activates Wnt/β-catenin signaling, such as a ROCK inhibitor or a GSK3 inhibitor.

In some embodiments, the supplemented cell culture medium forms an in vitro maturation (IVM) medium. In some embodiments, the IVM medium is prepared the day before oocytes are collected from the subject by placing approximately 100 μL of the medium in a suitable cell culture dish and topping it with mineral oil. In some embodiments, the manipulated OSCs are thawed approximately 2 to 4 hours before IVM culture. In some embodiments, the thawed manipulated OSCs are centrifuged. In some embodiments, the manipulated OSCs are washed with IVM medium. In some embodiments, the manipulated OSCs are seeded into cell culture droplets. In some embodiments, the manipulated OSCs are seeded at a final concentration of approximately 1,000 cells per μL (e.g., approximately 500–1,000 cells/μL, approximately 700–1,000 cells/μL, approximately 1,000–1,200 cells/μL, approximately 1,000–1,500 cells/μL, or approximately 1,000–2,000 cells/μL).

C. Production and Preservation of Ovarian Supporting Cells In some embodiments, one or more OSCs described herein may be produced in multiple batches. In some embodiments, OSCs may be frozen and thawed before the co-culture method. In some embodiments, OSCs are newly reprogrammed from a population of iPSCs before the in vitro maturation method. In some embodiments, OSCs may be seeded before adding oocytes for in vitro maturation and equilibrated over 2 to 8 hours (e.g., 2 to 3 hours, 2 to 4 hours, 3 to 4 hours, 4 to 6 hours, 5 to 7 hours, 6 to 8 hours, e.g., 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours). In some embodiments, OSCs may be seeded and equilibrated for about 25 to 90 minutes (for example, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes, about 65 minutes, about 70 minutes, about 75 minutes, about 80 minutes, about 85 minutes, or about 90 minutes).

In some embodiments, hiPSCs may be provided depending on the subject. The provision of hiPSCs can be carried out according to the oocyte harvesting process described herein. The subjects participating in the provision of hiPSCs may be different from or the same as the subjects from which oocytes were harvested. In some embodiments, hiPSC donors may undergo the stimulation protocols disclosed herein. Selected hiPSC clones may be expanded to generate an intermediate cell bank. Selected hiPSC clones may be expanded and fully differentiated for immediate use.

In some embodiments, the hiPSCs (e.g., undifferentiated hiPSCs, e.g., intermediate cell banks) or engineered OSCs described herein are provided in cryovials. In some embodiments, the cryovials consist of approximately 125,000 cells (e.g., hiPSCs or OSCs). In some embodiments, the hiPSCs or engineered OSCs are suspended in antifreeze. In some embodiments, the antifreeze comprises CryoStor CS10. In some embodiments, the cryovials are plastic vials. In some embodiments, the cryovials are plastic vials with female threads suitable for liquid nitrogen. In some embodiments, the hiPSCs or engineered OSCs are provided in one or more aggregates. In some embodiments, the hiPSCs or engineered OSCs are provided in one or more single-cell suspensions.

In some embodiments, hiPSCs or engineered OSCs (e.g., OSCs produced from reprogrammed hiPSCs) are prepared and frozen or cryopreserved for later use. Cryopreservation or freezing methods include the use of cryoprotectants such as dimethyl sulfoxide and/or other freezing methods known in the art. In some embodiments, hiPSCs or engineered OSCs are stored in liquid nitrogen. In some embodiments, hiPSCs or engineered OSCs are stored in liquid nitrogen until use. In some embodiments, cryopreserved undifferentiated hiPSCs (e.g., an intermediate cell bank) are stored in liquid nitrogen. Undifferentiated hiPSCs (e.g., an intermediate cell bank) can be thawed for further differentiation steps. In some embodiments, engineered OSCs are stored in liquid nitrogen until use in IVM methods and applications.

In some embodiments, frozen cells are sent to various batch release assays in which one or more tests are performed to determine the quality of the cells. In some embodiments, one or more aliquots of frozen cells are subjected to a series of tests to measure cell number and/or viability upon thawing, confirm genetic stability, verify genotype, confirm sterility, or a combination thereof. One or more aliquots of undifferentiated cells (e.g., undifferentiated hiPSCs, e.g., an intermediate cell bank) can be further subjected to a series of tests to measure the presence and/or relative abundance of one or more pluripotency markers (e.g., NANOG, POU5F1, SOX2, OCT4). One or more aliquots of one or more OSC populations (e.g., a final target cell bank) can be subjected to a series of tests to measure the presence and/or relative abundance of markers associated with one or more types of OSCs (e.g., granulosa cells or stromal cells), or the potency of secreted proteins or steroids (estradiol and/or progesterone production). In further embodiments, one or more aliquots of frozen cells are tested to determine the risk of embryotoxicity (e.g., the presence of one or more chemicals or factors that may interfere with the normal growth, development, or genetic differentiation of the embryo).

D. Ovarian Support Cells and Oocytes for In Vitro Maturation In some embodiments, one or more oocytes are human oocytes. In some embodiments, one or more oocytes are mammalian oocytes. In some embodiments, one or more oocytes are mouse oocytes. In some embodiments, one or more oocytes are rat oocytes. In some embodiments, one or more oocytes are monkey oocytes. In some embodiments, one or more oocytes are rhesus macaque oocytes. In some embodiments, one or more oocytes are obtained from conventional stimulation.

In some embodiments, the manipulated OSC promotes the in vitro maturation (i.e., in vitro maturation (IVM)) of one or more oocytes by forming a cumulus-oocyte complex (COC) with one or more oocytes. In some embodiments, one or more oocytes may be evaluated at any point during IVM based on an oocyte score, as described in Section II(C)(i). In some embodiments, the manipulated OSC expands access to in vitro fertilization (IVF) and oocyte freezing by providing a more cost-effective, more efficient, and less invasive method for individuals seeking infertility treatment or ART.

In some embodiments, the manipulated OSC promotes IVM of one or more oocytes, as described in more detail in Section II. In some embodiments, the manipulated OSC does not have any continuing effects (e.g., biological or developmental effects) on one or more mature oocytes after maturation. In some embodiments, the manipulated OSC is physically separated from one or more mature oocytes after IVM and is not present in the sample used for further steps of the IVF procedure (e.g., embryogenesis and/or implantation). In some embodiments, one or more mature oocytes are utilized for subsequent in vitro fertilization procedures, including but not limited to embryogenesis and implantation. In some embodiments, the manipulated OSC is not separated from one or more mature oocytes and does not have any biological or developmental effects on the developing embryo.

II. Methods for Stimulating Oocyte Release The methods described herein may be adapted for subjects who wish to increase the number of usable oocytes from any standard ART utilizing controlled ovarian hyperstimulation (COH). The increase in the number of usable oocytes is achieved by co-culturing immature oocytes with one or more OSCs (e.g., one or more reprogrammed OSCs as described herein) to promote the maturation of immature oocytes commonly obtained in typical COH and oocyte harvesting procedures. The current standard treatment is to discard the harvested immature oocytes. The OSCs described herein can promote the maturation of immature oocytes and thus increase the number of mature and usable oocytes from the harvested oocyte population.

Furthermore, while the methods described herein may be applicable to subjects seeking assisted reproductive technology (ART) treatment, access may be limited due to the exorbitantly high costs and/or risks associated with conventional methods of ovarian stimulation for oocyte retrieval (e.g., the risk of ovarian hyperstimulation syndrome (OHSS)). Conventional methods typically require administering gonadotropins to subjects to stimulate the ovaries and retrieve mature oocytes. The delivery of administered gonadotropins is typically inefficient, requiring high concentrations of gonadotropins to ensure that sufficient levels of gonadotropins are delivered to the follicles for oocyte maturation and release after systemic injection. By performing the IVM method described herein, the amount of gonadotropin administered for oocyte retrieval can be reduced, thereby providing a method to avoid the costly and potentially dangerous side effects associated with the systemic administration of high levels of gonadotropins. When immature oocytes are collected and exposed to conditions optimized for maturation outside the body, the resulting mature oocytes can be used for subsequent fertilization, embryo development, blastocyst formation, implantation, and pregnancy, ultimately producing healthy offspring.

A. Subject Selection The methods for stimulating oocyte release described herein are intended for subjects seeking IVF treatment options. Typically, subjects are women with a low number of oocytes retrieved or a high number of immature oocytes. Subjects may be between 20 and 45 years of age, and are typically 35 years of age or older. Subjects may have age-related decline in ovarian reserve and/or genetic or medical conditions that lead to decline in ovarian reserve (e.g., polycystic ovary syndrome (PCOS)). The subjects may have ovarian reserve with 20 or fewer oocytes, such as having 1 to 5 oocytes, 4 to 10 oocytes, 8 to 16 oocytes, or 15 to 20 oocytes (for example, 1 oocyte, 2 oocytes, 3 oocytes, 4 oocytes, 5 oocytes, 6 oocytes, 7 oocytes, 8 oocytes, 9 oocytes, 10 oocytes, 11 oocytes, 12 oocytes, 13 oocytes, 14 oocytes, 15 oocytes, 16 oocytes, 17 oocytes, 18 oocytes, 19 oocytes, or 20 oocytes). The subjects may have anti-Müllerian hormone (AMH) levels consistent with reduced ovarian reserve. The subjects may have AMH levels measured by blood tests and other methods known in the art. The subjects may have AMH levels of 1–6 ng/mL (e.g., 1–2 ng/mL, 2–4 ng/mL, or 4–6 ng/mL, e.g., 1 ng/mL, 2 ng/mL, 3 ng/mL, 4 ng/mL, 5 ng/mL, or 6 ng/mL). The subjects may have estradiol levels of 20–50 pg/mL (e.g., 20–30 pg/mL, 25–35 pg/mL, 30–40 pg/mL, 35–45 pg/mL, or 40–50 pg/mL, e.g., 20 pg/mL, 21 pg/mL, 22 pg/mL, 23 pg/mL, 24 pg/mL, 25 pg/mL, 30 pg/mL, 35 pg/mL, 40 pg/mL, 45 pg/mL, or 50 pg/mL).

A physician or expert may assess a subject for methods of stimulating oocyte release by taking a biological sample from the subject. Biological samples may include laboratory specimens held in a biorepository for examination. In some embodiments, biological samples may include bodily fluids such as blood, saliva, urine, semen (seminal plasma), vaginal secretions, cerebrospinal fluid (CSF), synovial fluid, pleural fluid (pleural lavage), pericardial fluid, ascites, amniotic fluid, saliva, nasal secretions, ocular fluid, gastric juice, breast milk, and cell culture supernatant. Biological samples may include medical diagnoses, user input describing the user's mood and/or symptom complaints, and information collected from wearable devices related to the user. For example, biological samples may include information obtained from visits to healthcare professionals, such as medical history. In yet another non-limiting example, biological samples may include information such as data collected from wearable devices worn by the user. Wearable devices are designed to collect information about the user's sleep patterns, exercise patterns, etc. In one embodiment, the biological sample is collected on a specific day and/or time of the user's menstrual cycle. For example, without limitation, the biological sample may be collected on the second day of the user's menstrual cycle to evaluate one or more hormone levels. The biological sample can be used to determine the subject's AMH level and/or other hormone levels, or markers of the subject's ovarian reserve that can be measured by other indicators. An AMH level of 1 ng/mL or less may be used to indicate low ovarian reserve. Subjects with low ovarian reserve may have measured AMH levels of 1.0 ng/mL, 0.9 ng/mL, 0.8 ng/mL, 0.7 ng/mL, 0.6 ng/mL, 0.5 ng/mL, 0.4 ng/mL, 0.3 ng, 0.2 ng/mL, or 0.1 ng/mL. Other biological samples that can be used to determine one or more markers of the subject's overall health status include, but are not limited to, the progression of the menstrual cycle and/or monitoring of circulating hormone levels such as estradiol (E2), luteinizing hormone (LH), follicle-stimulating hormone (FSH), progesterone (P4), estrone (E1), estriol (E3), testosterone, androgens, dehydroepiandrosterone (DHEA), triiodothyronine (T3), tetraiodothyronine (T4), calcitonin, melatonin, insulin, cortisol, human growth hormone (HGH), adrenaline levels, and other hormones.

Other biological sample data taken from the subject includes at least one oocyte. As used in this disclosure, “biological sample data” is data describing the biological, genetic, biochemical and/or physiological properties, composition, or activity characteristics of the biological sample. In some embodiments, the oocyte may be an immature oocyte. As used in this disclosure, “immature oocyte” is one or more immature germ cells that develop in the ovary. In some embodiments, the immature oocyte may be an oocyte containing a GV oocyte and/or an MI oocyte. In some embodiments, the immature oocyte may be multiple oocytes. The immature oocyte may be an immature cumulus-oocyte complex (COC) taken from the subject. As used in this disclosure, “cumulus-oocyte complex” is an oocyte surrounded by specific granulosa cells. As used in this disclosure, “specific granulosa cells” are cumulus cells that surround the oocyte to ensure healthy oocyte and embryonic development. In some embodiments, immature oocytes may include oocytes in which specific granulosa cells are added to a cell culture (e.g., a co-culture) to mature the oocytes and create COCs.

In some embodiments of this method, a biological sample may be collected from the user via a collection device. A "collection device" is a device and/or tool capable of acquiring, recording, and/or verifying measurements related to the sample. A collection device may include needles, syringes, vials, lancets, vacuum collection tubes (ECTs), tourniquets, vacuum collection tube systems, or any combination thereof. For example, a collection device may include a butterfly needle set. Data from the biological sample may include measurements such as serum calcium, phosphate, electrolytes, blood urea nitrogen and creatinine, and uric acid.

In one embodiment of this method, the biological sample information of the subject may be obtained from an ultrasound examination. As used in this disclosure, “ultrasound examination” is any method that uses sound waves to generate one or more images of the user’s body. For example, ultrasound examination may be used to obtain images of the subject’s reproductive organs and/or tissues. In one embodiment, the ultrasound examination may be performed at a specific time in the subject’s menstrual cycle. For example, the subject may undergo an ultrasound examination on the second day of the cycle, which can be used to determine the follicle size and/or number of follicles. The selection and/or adjustment of the stimulation protocol can be made using such information. For example, for an ultrasound-examined subject showing PCOS, the dosage of one or more drugs ingested and/or used during the stimulation protocol may be adjusted. Furthermore, the length of the stimulation protocol for the subject can be modified based on the subject’s PCOS diagnosis. In one embodiment, the ultrasound examination may be repeated one or more times throughout the subject’s stimulation protocol, and the information obtained can be used to adjust the subject’s stimulation protocol in real time.

B. Oocyte Stimulation Protocols A physician or expert may use the biological parameters described herein to determine a stimulation protocol for oocyte release directed at a subject. Such biological parameters include, among those known to those skilled in the art, hormone levels (e.g., baseline hormone levels and/or hormone levels due to the use of contraceptives) and the physical characteristics of the subject (e.g., follicle size, number of follicles, ovarian morphology, and/or uterine morphology). Those skilled in the art may implement a stimulation protocol using any one or a combination of the inducers or compositions intended to stimulate follicular maturation and oocyte release described herein.

Other relevant hormonal levels or concentrations of compounds in a biological sample may include estradiol (E2), luteinizing hormone (LH), follicle-stimulating hormone (FSH), progesterone (P4), estrone (E1), estriol (E3), testosterone, androgens, dehydroepiandrosterone (DHEA), triiodothyronine (T3), tetraiodothyronine (T4), calcitonin, melatonin, insulin, cortisol, human growth hormone (HGH), and adrenaline. In some embodiments, the measurement of hormone levels may be based on blood analysis of the biological sample. For example, blood analysis may include plasma hormone analysis. In some embodiments, the measurement of hormone levels may be based on salivary hormone testing. The measurement of hormone levels may also be based on other forms of analysis (e.g., hair, urine, and any other form of the biological sample described throughout this disclosure). Prior to the follicular induction period, the baseline serum level of the target estradiol may be approximately 30 pg/mL to approximately 60 pg/mL (e.g., approximately 30 pg/mL to approximately 45 pg/mL, approximately 40 pg/mL to approximately 55 pg/mL, or approximately 45 pg/mL to approximately 60 pg/mL, e.g., approximately 30 pg/mL, approximately 35 pg/mL, approximately 40 pg/mL, approximately 45 pg/mL, approximately 50 pg/mL, approximately 55 pg/mL, or approximately 60 pg/mL). Prior to the follicular induction period, the baseline serum level of the target progesterone may be approximately 0.5 ng/mL to approximately 2.5 ng/mL (e.g., approximately 0.5 ng/mL to approximately 1.0 ng/mL, approximately 1.0 ng/mL to approximately 1.5 ng/mL, approximately 1.5 ng/mL to approximately 2.0 ng/mL, or approximately 2.0 ng/mL to approximately 2.5 ng/mL, e.g., approximately 1.0 ng/mL, approximately 1.5 ng/mL, approximately 2.0 ng/mL, or approximately 2.5 ng/mL).

Furthermore, the subject's use of contraception (e.g., hormonal contraception) may influence the setting of the stimulation protocol. Consideration of contraception can help determine the timing of follicle induction in the woman's menstrual cycle. For example, without limitation, a subject not using any form of contraception may initiate a stimulation protocol with recombinant follicle-stimulating hormone (rFSH) between days 1 and 3 of the menstrual cycle, preferably on day 2. In yet another non-limiting example, a subject using contraception may initiate a stimulation protocol with rFSH 4 to 6 days after taking the last oral contraceptive (e.g., day 4, day 5, or day 6), preferably 5 days after the last oral contraceptive administration. In one embodiment, rFSH stimulation may be used for 2 to 3 days (e.g., 2 or 3 days), depending on the subject's tolerance, follicle size, and/or growth dynamics. Following this two- or three-day period, a one- to three-day (e.g., one, two, or three days) coasting period can be used to monitor follicular size and allow for further follicular maturation and development. As used in this disclosure, “coasting period” is any period during which the drugs used throughout the stimulation protocol are not administered and/or ingested. The coasting period may last, for example, one, two, three, or longer, if medically necessary. During the coasting period, the subject may continue to undergo one or more ultrasound examinations to monitor their progress.

When the follicular size reaches any of the following ranges (e.g., 7.5 mm, 8 mm, 8.5 mm, 9 mm, 9.5 mm, 10 mm, 10.5 mm, or greater): the subject may be triggered by the administration of an induction agent such as human chorionic gonadotropin (hCG). As used in this disclosure, “follicular measurement” is any measurement of a follicle. A follicle may include any sac present in the ovary containing an unfertilized egg. Follicular measurements can be obtained using any method described herein, such as ultrasound, manual measurement, or automated measurement. In one embodiment, a double hCG injection may be used to induce follicular maturation and prepare one or more follicles for collection. A double hCG injection may be two or three injections of hCG. On the day of commencement of the double hCG injection, blood tests for one or more hormone levels, such as E2, P4, and LH, may be performed to monitor hormone levels. After the two hCG administrations, one or more hormone levels may be measured, for example, by a blood test that determines and examines the levels of E2, P4, and LH.

A "fertilization inducer" is a chemical substance that causes cell development in the ovary. Fertilization inducers (e.g., follicle inducers) may include any substance, including any commercially available and/or prescription drugs. Inducement agents (e.g., follicle induction agents) include Lupron Depot® (Abbott Laboratories, North Chicago, IL), Ganirelix (Ferring Pharmaceuticals, Saint-Prex, Switzerland), Cetrotide (Merck Global, Readington Township, NJ), Gonal-F® (Merck Global), Follistim® (Merck Global), Bravelle® (Ferring Pharmaceuticals), and Clomid® (Patheon Pharmaceuticals). Inc., Waltham, mA), Seraphine (Teva, Tel Aviv-Yafo, Israel), Glucophage (Registered Trademark) (Merck Global), Fortamet (Registered Trademark) (Mylan, Canonsburg, PA), Pregnyl (Registered Trademark) (Schering Plough, Kenilworth, NJ), Novarel (Registered Trademark) (Ferring Laboratories, Parsippany, NJ), Repronex (Ferring Pharmaceuticals), Factorel (Registered Trademark) (Zoetic Canada) This may include any one or a combination of non-limiting examples such as Menopur® (Ferring Pharmaceuticals), Inc., Kirkland, Canada, and other agents that induce ovarian cell production as understood by those skilled in the art to be applicable. Inducing agents (e.g., follicle-inducing agents) may include, among other inducers known in the art, human serum albumin, FSH, hCG, androstenedione, and doxycycline.

In one embodiment, the subject may not be administered an induction agent that stimulates oocyte production (e.g., a follicle induction agent). In one embodiment, the subject may receive multiple injections of an induction agent over 1 to 4 days (e.g., day 1, day 2, day 3, or day 4), but not exceeding 5 days, in a preferred stimulation protocol. The subject may receive multiple injections over several days, such as receiving five injections of one or more induction agents. For example, the subject may receive 300 to 700 IU per injection (e.g., 300 to 500 IU, 400 to 600 IU, 500 to 700 IU, 300 to 350 IU, 350 to 400 IU, 400 to 450 IU, 450 to 500 IU, 500 to 550 IU, 550 to 600 IU, 600 to 650 IU, 650 to 700 IU, e.g., 30 Patients may receive a 3-day stimulation course (one or more injections per day) using rFSH in doses of 0 IU, 325 IU, 350 IU, 375 IU, 400 IU, 425 IU, 450 IU, 475 IU, 500 IU, 525 IU, 550 IU, 575 IU, 600 IU, 625 IU, 650 IU, 675 IU, or 700 IU. The subjects are administered 200-700 μg or 2,500-10,000 IU of hCG (e.g., 200-500 μg, 300-600 μg, 400-700 μg, 200-300 μg, 300-400 μg, 400-500 μg, 500-600 μg, or 600-700 μg) by injection as an induction agent (e.g., a follicle induction agent), with a preferred stimulation dose of 500 μg. The target population may receive clomiphene citrate in doses of 50–150 mg per injection (e.g., 50–75 mg, 60–80 mg, 75–100 mg, 90–115 mg, 110–130 mg, 125–150 mg, e.g., 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg) in combination with other inducers, one or more times over a maximum of eight days (e.g., one, two, three, four, five, six, seven, or eight days).

Before administering the inducer, the levels of the hormone or other related compounds in the target serum may be evaluated. Before administration of the inducer, the serum level of the target estradiol should be approximately 250 pg/mL to approximately 400 pg/mL (e.g., approximately 250 pg/mL to approximately 275 pg/mL, approximately 275 pg/mL to approximately 300 pg/mL, approximately 300 pg/mL to approximately 325 pg/mL, approximately 325 pg/mL to approximately 350 pg/mL, approximately 350 pg/mL to approximately 375 pg/mL, or approximately 375 pg/mL to approximately 400 pg/mL). For example, it could be about 250 pg/mL, about 260 pg/mL, about 270 pg/mL, about 280 pg/mL, about 290 pg/mL, about 300 pg/mL, about 310 pg/mL, about 320 pg/mL, about 330 pg/mL, about 340 pg/mL, about 350 pg/mL, about 360 pg/mL, about 370 pg/mL, about 380 pg/mL, about 390 pg/mL, or about 400 pg/mL). Prior to administration of the progesterone induction agent, the serum level of the target progesterone should be approximately 0.25 ng/mL to approximately 0.75 ng/mL (for example, approximately 0.25 ng/mL to approximately 0.35 ng/mL, approximately 0.35 ng/mL to approximately 0.45 ng/mL, approximately 0.45 ng/mL to approximately 0.55 ng/mL, approximately 0.55 ng/mL to approximately 0.65 ng/mL, or approximately 0.65 ng/mL). g/mL to approximately 0.75 ng/mL, for example, approximately 0.25 ng/mL, approximately 0.30 ng/mL, approximately 0.35 ng/mL, approximately 0.40 ng/mL, approximately 0.45 ng/mL, approximately 0.50 ng/mL, approximately 0.55 ng/mL, approximately 0.60 ng/mL, approximately 0.65 ng/mL, approximately 0.70 ng/mL, or approximately 0.75 ng/mL). Prior to administration of the induction agent, the serum level of the target LH may be approximately 1.0 mIU/mL to approximately 2.5 mIU/mL (e.g., approximately 1.0 mIU/mL to approximately 1.5 mIU/mL, approximately 1.5 mIU/mL to approximately 2.0 mIU/mL, or approximately 2.0 mIU/mL to approximately 2.5 mIU/mL, e.g., approximately 1.0 mIU/mL, approximately 1.25 mIU/mL, approximately 1.5 mIU/mL, approximately 1.75 mIU/mL, approximately 2 mIU/mL, approximately 2.25 mIU/mL, or approximately 2.5 mIU/mL). Prior to administration of the induction agent, the serum level of the target FSH may be approximately 11 mIU/mL to approximately 14 mIU/mL (e.g., approximately 11 mIU/mL to approximately 12 mIU/mL, approximately 12 mIU/mL to approximately 13 mIU/mL, or approximately 13 mIU/mL to approximately 14 mIU/mL, e.g., approximately 11 mIU/mL, approximately 12 mIU/mL, approximately 13 mIU/mL, or approximately 14 mIU/mL).

Inducement agents (e.g., follicle-inducing agents) may be administered over time to produce a minimal stimulation protocol, which is a follicle-stimulation protocol. A minimal stimulation protocol is configured by those skilled in the art to induce cell release over a span of about three days. A “minimally stimulating protocol” is a shorter stimulation process compared to an average in vitro fertilization (IVF) stimulation protocol and helps to induce the ovary to produce oocytes. Typically, the average duration for a stimulation protocol using standard IVF is about 8 to 14 days. A minimal stimulation protocol can induce cell release over a period of 8 days or less (e.g., 8 days or less, 7 days or less, 6 days or less, 5 days or less, 4 days or less, 3 days or less, 2 days or less, or 1 day, e.g., 1 to 3 days, 2 to 4 days, 3 to 5 days, 4 to 6 days, 5 to 7 days, or 6 to 8 days, e.g., 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, or 8 days). This is a shorter period compared to the 8 to 14 days of a standard IVF stimulation protocol. The average time required to perform the minimal stimulation protocol may be two days. The average time required to perform the minimal stimulation protocol may be three days. The average time required to perform the minimal stimulation protocol may be four days. The average time required to perform the minimal stimulation protocol may be five days. The average time required to perform the minimal stimulation protocol may be six days. In one embodiment, the minimal stimulation protocol may not require the administration of follicle-inducing agents for successful oocyte collection and subsequent maturation. In one embodiment, the minimal stimulation protocol may include selecting a first inducer (e.g., a follicle-inducing agent) and a second inducer (e.g., a follicle-inducing agent) depending on follicle measurement values and/or other biological sample data.

C. Oocyte Collection After follicular stimulation, oocytes (or a group of cells including oocytes) are collected from the subject. As used in this disclosure, “oocytes” are germ cells derived from the ovary. Approximately 24 to 48 hours after the last dose of the induction agent (e.g., a follicle induction agent) (e.g., 24 to 32 hours, 32 to 40 hours, 40 to 48 hours, e.g., approximately 24 hours, approximately 28 hours, approximately 32 hours, approximately 36 hours, approximately 40 hours, approximately 44 hours, approximately 48 hours), the subject may undergo oocyte collection. On the day of oocyte collection, blood tests may be performed to check the levels of one or more hormones, such as E2, LH, FSH and/or P4, to confirm indicators of quality, that hormone levels are within the range, and/or that the appropriate hCG dose has been taken. E2 hormone levels may range from approximately 300 pg/mL to approximately 450 pg/mL on the day of oocyte collection (e.g., approximately 300 pg/mL to approximately 350 pg/mL, approximately 350 pg/mL to approximately 400 pg/mL, or approximately 400 pg/mL to approximately 450 pg/mL, e.g., approximately 300 pg/mL, approximately 325 pg/mL, approximately 350 pg/mL, approximately 375 pg/mL, approximately 400 pg/mL, approximately 425 pg/mL, or approximately 450 pg/mL). LH hormone levels may range from approximately 3 mIU/mL to approximately 6 mIU/mL on the day of oocyte retrieval (for example, approximately 3 mIU/mL to approximately 4 mIU/mL, approximately 4 mIU/mL to approximately 5 mIU/mL, or 5 mIU/mL to approximately 6 mIU/mL, for example, approximately 3 mIU/mL, approximately 3.5 mIU/mL, approximately 4 mIU/mL, approximately 4.5 mIU/mL, approximately 5 mIU/mL, approximately 5.5 mIU/mL, or approximately 6 mIU/mL). FSH hormone levels may range from approximately 6 mIU/mL to approximately 9 mIU/mL on the day of oocyte collection (e.g., approximately 6 mIU/mL to approximately 7 mIU/mL, approximately 7 mIU/mL to approximately 8 mIU/mL, or approximately 8 mIU/mL to approximately 9 mIU/mL, e.g., approximately 6 mIU/mL, approximately 6.5 mIU/mL, approximately 7 mIU/mL, approximately 7.5 mIU/mL, approximately 8 mIU/mL, approximately 8.5 mIU/mL, or approximately 9 mIU/mL). The P4 hormone level on the day of oocyte collection may be approximately 0.5 ng/mL to approximately 1.5 ng/mL (for example, approximately 0.5 ng/mL to approximately 1.0 ng/mL, approximately 0.75 ng/mL to approximately 1.0 ng/mL, approximately 1.0 ng/mL to approximately 1.5 ng/mL, or approximately 1.25 ng/mL to approximately 1.5 ng/mL, for example, approximately 0.5 ng/mL, approximately 0.75 ng/mL, approximately 1.0 ng/mL, approximately 1.25 ng/mL, or approximately 1.5 ng/mL).

Oocytes (or cell populations containing oocytes) are collected from the subject using methods known in the art. For example, oocytes can be collected by using a transvaginal ultrasound device, attaching a needle guide to the probe, and aspirating the released follicular contents. The follicular aspirate is then examined under a dissecting microscope, washed with HEPES medium (G-MOPS Plus, VITROLIFE®), and filtered through a 70-micron cell strainer (FALCON®, Corning). Next, the oocytes and/or COCs are transferred to a culture dish and medium, and co-culture and appropriate control are initiated as described herein. Other collection methods may include collection devices such as needles, syringes, vials, lancets, vacuum collection tubes (ECTs), tourniquets, vacuum collection tube systems, or any combination thereof. For example, the collection device may include a butterfly needle set.

The collected oocytes may include, but are not limited to, immature oocytes, mature oocytes, groups of one or more oocytes, or groups of one or more cells, such as a cumulus-oocyte complex. As used in this disclosure, a "cumulus-oocyte complex" (COC) is an oocyte containing one or more surrounding cumulus cells. A COC may include immature oocytes. A COC may include mature oocytes.

As used in this disclosure, “immature oocyte” refers to one or more immature germ cells that develop in the ovary. In some embodiments, immature oocytes may include, but are not limited to, oocytes in the vesicular stage (GV) and metaphase I (MI), as further described below. In some embodiments, immature oocytes may be multiple oocytes. Immature oocytes may be an immature cumulus-oocyte complex (COC) collected from a subject. As used in this disclosure, “mature oocyte” may be one or more mature oocytes in metaphase II (MII). After collection, the COC may be allowed to stand for one, two, three hours or longer to equilibrate to in vitro conditions for in vitro maturation.

At the time of collection, one or more of the collected oocytes or cells described herein can be appropriately frozen and stored using methods known in the art for future use, analysis, or experimentation. Furthermore, one or more of the collected oocytes or cells described herein can be used fresh (i.e., suitable for immediate use, such as for in vitro maturation or for any one or more analyses or experiments described herein).

III. Methods for Rescuing Maternal Cells A. Oocyte Denucleation After the oocyte collection method described above, one or more COCs may require oocyte denucleation. In this disclosure, “oocyte denucleation” means removing cumulus cells or other cell types from oocytes by mechanical separation, chemical separation, or a combination thereof. Several methods of oocyte denucleation are known in the art. In some embodiments, denucleation may occur in an IVM well by gently mechanically dissociating the cells by pipetting to remove most cumulus cells and/or granulosa cells. If enzymatic dissociation is required, the cells may be transferred to a separate dish for hyaluronidase treatment. The COCs may be stripped with stripper tips and washed in IVM medium or MOPS plus medium to wash the oocytes for imaging and inactivate hyaluronidase as needed. Stripper tips may include those with a diameter of 200 microns and/or 400 microns for fine washing. In some embodiments, oocytes in the gestational vesicle (GV) and metaphase I (MI) stages can be prepared and used for culture after developing the COC. The developed COC may be transferred to a separate culture dish for imaging.

B. Co-culture of oocytes for in vitro maturation i. Content and timing of co-culture In the methods described herein, oocytes may be combined with one or more OSCs, such as one or more granulosa cells or stromal cells. OSCs are cumulus cells that surround the oocyte and ensure the health of the oocyte and subsequent embryonic development. In some embodiments, the granulosa and/or stromal co-culture cells are derived from differentiated induced pluripotent stem cells (iPSCs), such as human iPSCs (hiPSCs) as described herein (see Section I(A)). As used in this disclosure, “co-culture” is a cell culture apparatus that cultures two or more different cell populations in some degree of contact. In some embodiments, steroid-producing granulosa cells derived from human induced pluripotent stem cells (hiPSCs) can be co-cultured with immature oocytes to form a COC, thereby promoting rapid and efficient oocyte maturation in a way that reconstructs the follicular niche in vitro and enhances the health and developmental capacity of the oocyte. As used in this disclosure, “steroid-producing granulosa cells” are granulosa cells that express high levels of estradiol-producing steroid-producing enzymes. For example, steroid-producing granulosa cells may be wall granulosa cells collected from encapsulated follicles. Applying steroid-producing granulosa cells to co-culture of COCs promotes in vitro oocyte maturation after oocyte/oocyte retrieval, making all retrieved oocytes available by directly supplying nutrients, raw materials, and mechanical support to the oocytes through gamete formation and follicular formation. Steroid-producing granulosa cells can grow in standard IVF and IVM media, as further described below, and may induce oocyte maturation of immature oocytes. This may increase the overall pool of healthy oocytes available for in vitro fertilization and reduce the number of oocyte/oocyte retrieval procedures the user undergoes.

In some embodiments of this method, the cell culture is formed by combining a population of immature oocytes and manipulated OSCs, and after extracting one or more oocytes according to a minimal stimulation protocol, the oocytes are added in the cell culture to mature and create COCs. In one embodiment, one or more specific granulosa cells and/or specific stromal cells may be thawed during the resting period of one or more COCs. In one embodiment, during culture, 50,000 to 150,000 cells (e.g., 50,000 to 60,000 cells, 60,000 to 70,000 cells, 70,000 to 80,000 cells, 80,000 to 90,000 cells, 90,000 to 100,000 cells, 100,000 to 110,000 cells, 110,000 cells) are added. 00 to 120,000 cells, 120,000 to 130,000 cells, 130,000 to 140,000 cells, or 140,000 to 150,000 cells, for example, 50,000 cells, 55,000 cells, 60,000 cells, 65,000 cells, 70,000 cells, 75,000 cells, Certain granulosa cells (80,000, 85,000, 90,000, 95,000, 100,000, 105,000, 110,000, 115,000, 120,000, 125,000, 130,000, 135,000, 140,000, 145,000, or 150,000 cells) can be combined with COC. In one embodiment, thawed specific granulosa cells can be placed in culture medium at any time, such as before COC harvesting, about 24 to 120 hours before (e.g., about 24 to 48 hours, about 48 to 72 hours, about 72 to 96 hours, about 96 to 120 hours; for example, about 24 to 36 hours, about 30 to 40 hours, about 36 to 48 hours, about 48 to 56 hours, about 56 to 72 hours, about 72 to 84 hours, about 80 to 96 hours, about 90 to 100 hours, about 96 to 108 hours, about 108 to 120 hours; for example, about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 56 hours, about 60 hours, about 65 hours, about 72 hours, about 78 hours, about 86 hours, about 92 hours, about 96 hours, about 102 hours, about 110 hours, about 115 hours, about 120 hours). Co-cultures (COCs) can be transferred to a culture medium containing thawed specific granulosa cells to form group cultures, as will be described in more detail below. In one embodiment, group cultures can be cultured in an incubator for a range of 12 to 48 hours (e.g., 12 to 16 hours, 12 to 20 hours, 18 to 24 hours, 18 to 36 hours, 24 to 36 hours, 36 to 48 hours, e.g., 12 hours, 16 hours, 20 hours, 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, 44 hours, 48 hours). Co-cultures can be carried out at a biologically appropriate temperature, e.g., 37°C.

In some embodiments of this method, the harvested oocytes, including the immature cumulus oocyte complex, may be cultured in group culture. “Group culture” is the combination of the harvested COC with one or more additional cells. The additional cells may include any cells that grow together with the harvested COC. The additional cells may include specific stromal cells. The additional cells may include specific granulosa cells. In one embodiment, the group culture is cultured for a specific time, for example, 12 to 120 hours (for example, 12 to 24 hours, 12 to 36 hours, 24 to 48 hours, 36 to 60 hours, 54 to 72 hours, 68 to 96 hours, 96 to 120 hours, for example, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours, 46 hours, 48 hours, 50 hours, 52 hours, 54 hours). The cells can be cultured and/or incubated for 100 hours, 56 hours, 58 hours, 60 hours, 62 hours, 64 hours, 66 hours, 68 hours, 70 hours, 72 hours, 74 hours, 76 hours, 78 hours, 80 hours, 82 hours, 84 hours, 86 hours, 88 hours, 90 hours, 92 hours, 94 hours, 96 hours, 98 hours, 100 hours, 102 hours, 104 hours, 106 hours, 108 hours, 110 hours, 112 hours, 114 hours, 116 hours, 118 hours, or 120 hours. For example, group culture may include culturing COCs with a granulosa cell co-culture as further described below. In some embodiments, group culture may include culturing a control group of COCs without co-culture as further described below. In some embodiments, the user can provide immature oocytes such as GV and MI stage oocytes. Such immature oocytes can be used in culture medium as part of a group culture to aid in the growth of COCs. Oocyte provision can be carried out according to the oocyte harvesting process described above. The subjects participating in oocyte provision may be different from or the same as those involved in the second biological sample containing immature COCs. In some embodiments, the subjects providing oocytes may undergo a stimulation protocol as disclosed above.

In some embodiments, the maturity level of oocytes collected from the subject may determine the length of time the oocytes should be co-cultured with ovarian supporting cells (e.g., specific granulosa cells and/or specific stromal cells). For example, less mature oocytes (e.g., GV oocytes) may require a longer co-culture period than oocytes in a more advanced stage of meiosis (e.g., MI oocytes).

In some embodiments relating to oocyte culture, the cell culture medium may include LAG medium (Medicult, COOPERSURGICAL®). For example, LAG medium may be used for incubation of oocytes and/or COCs after collection from a minimal stimulation protocol. For example, modified Medicult IVM medium may be used as a baseline control during the culture process. In some embodiments, the cell culture medium may include metabolites, as illustrated in Figure 5. For example, modified Medicult IVM medium may include human serum albumin, FSH, hCG, androstenedione, doxycycline, or any combination thereof. The culture medium may be equilibrated for approximately 18–24 hours (e.g., approximately 18 hours, approximately 20 hours, approximately 22 hours, approximately 24 hours) before culturing in a standard sterile 37°C incubator under O2 (e.g., 1–10% O2 atmosphere, e.g., 4–8% O2 or 5–7% O2, e.g., 6% O2) and appropriate CO2 levels (these are known in the art). Co-culture and specific granulosa cell cultures may be adherent cell cultures in cell culture dishes or flasks. Co-culture and specific granulosa cell cultures may be suspension cell cultures in cell culture flasks. Cell culture materials and methods include standard sterile cell culture methods known in the art. Cell morphology and cell viability may be evaluated by one or more established methods known in the art.

In some embodiments, co-culture is carried out according to the steps outlined in Figure 6. For example, a population of ovarian supporting cells (e.g., ovarian granulosa cells) can be cryopreserved and thawed. In some embodiments, the ovarian supporting cells are centrifuged to form a cell pellet, which is then resuspended in a medium suitable for in vitro maturation. In some embodiments, the ovarian supporting cells are centrifuged one or more times, and each time they are resuspended in the in vitro maturation medium. The ovarian supporting cells are then co-cultured with oocytes obtained from subjects undergoing ART treatment, thereby inducing oocyte maturation.

ii. Granulosa cells derived from human iPSCs The specific granulosa cells used in the methods described herein may be prepared from hiPSCs using the transcription factor (TF)-directed protocol described herein.

In some embodiments, hiPSCs may be provided depending on the subject. The provision of hiPSCs can be carried out according to the oocyte harvesting process described above. The subjects participating in hiPSC provision may be different from or the same as the subjects from which the oocytes were harvested. In some embodiments, hiPSC donors can undergo the stimulation protocols disclosed above.

In some embodiments, hiPSCs, granulosa cells, cumulus cells, oocytes, GV-phase oocytes, MI-phase oocytes, MII-phase oocytes, and all other types of cells described throughout this disclosure may be lysed, their genomic material extracted, and rapidly frozen for further manipulation and/or analysis (e.g., analysis as part of the omics data acquisition techniques described in Section III(C)(iii) below). For example, cells may undergo enzymatic cell lysis using enzymes such as lysozyme, lysostafin, zymolase, cellulose, protease, or glycanase. Other lysis methods, e.g., chemical lysis, surfactant lysis, alkaline lysis, mechanical lysis, thermal lysis, sonication lysis, physical lysis, non-mechanical lysis, and other lysis methods known in the art may be applied. In some embodiments, the culture medium may be rapidly frozen. Freezing methods include the use of cryoprotectants such as dimethyl sulfoxide and/or other freezing methods known in the art.

C. Oocyte Rescue i. Oocyte Scoring Oocytes and/or granulosa cells can be appropriately frozen and stored at any stage of in vitro maturation, or immediately after in vitro maturation, for future analysis, experimentation, or use in oocyte maturation. Oocytes can be scored by a scoring metric based on their morphology, determined by image analysis. In some embodiments, setting the scoring metric may include taking images of group cultures and analyzing images of one or both of the co-culture group and the control group without co-culture growth medium. In some embodiments, oocytes are scored and comparatively analyzed during any such stage of in vitro maturation. For example, images of group cultures may include pre-culture group COC images, post-culture group COC images, and post-culture naked oocyte images. In some embodiments, the oocytes used for scoring are those that have never been frozen. In some embodiments, the oocytes used for scoring by image analysis may be those that have been thawed after storage by freezing. In some embodiments, the oocytes used for scoring may be those collected without in vitro maturation as described. In some embodiments, the oocytes used for scoring may be cultured without the granulosa cells described. In some embodiments, the images may be sent to a qualified third party (e.g., an embryologist, developmental biologist, or other relevant expert) for scoring purposes.

In some embodiments of the methods described herein, oocytes can be evaluated and then classified according to their maturity state according to the following criteria.

GV – Typically, an oocyte contains a single nucleolus within the oocyte.

MI – The oocyte lacks a nuclear vesicle, and the polar body is absent in the perivitelline space between the oocyte and the zona pellucida.

MII – The oocyte lacks a nuclear vesicle, and the polar body is present in the perivitelline space between the oocyte and the zona pellucida.

In some embodiments of this method, the scoring metric may include a total oocyte score (TOS) based on the analysis of group cultures imaged by appropriate microscopy or image analysis software. Methods and approaches for TOS have been described in the Art (Lazzaroni-Tealdi et al., PLOS One 10:e0143632, 2015). The oocyte score may include metrics (indicators) such as shape, size, cytoplasmic properties, perivitelline space structure (PVS), zona pellucida (ZP), and polar body (PB) morphology, among other possible qualitative indicators. The total oocyte score for both pre- and post-culture oocyte images for generating the TOS metric may be based on a scale system from -6 to +6.

Regarding oocyte morphology, a value of -1 can be assigned if the oocyte morphology is poor (the oocyte is generally dark and/or oval-shaped), a value of 0 can be assigned if it is nearly normal (the overall coloration of the oocyte is not very dark and it is not very oval-shaped), and a value of +1 can be assigned if it is judged to be normal. Regarding oocyte size, if the oocyte size is judged to be abnormally small or large, a value of -1 can be assigned if the size is less than 120 μm or greater than 160 μm. If the size is nearly normal, i.e., does not deviate from normal by more than 10 μm, a value of 0 can be assigned, and if the oocyte size is within the normal range of more than 130 μm but less than 150 μm, a value of +1 can be assigned. Regarding the characteristics of the oocyte cytoplasm, a value of -1 can be assigned if the oocyte cytoplasm is very granular and/or very vacuolated and/or shows some inclusions. If it is only slightly granular and/or shows almost no inclusions, a value of 0 can be assigned. If there is no granularity or inclusions, the value may be +1. Regarding the structure of the perivitelline space (PVS), if the PVS is abnormally large, absent, or very granular, the PVS may be defined as -1. For moderately large and/or moderately small and/or less granular PVS, a value of 0 may be assigned. A value of +1 may be assigned to a normal-sized PVS that is not granular. Regarding the zona pellucida (ZP), if the ZP is very thin or thick (<10 μm or >20 μm), the oocyte may be assigned -1. If the ZP does not deviate from normal by more than 2 μm, the ZP may be assigned 0. A normal zona pellucida (>12 μm and <18 μm) may be assigned +1. Regarding the morphology of the polar body (PB), the morphology of the PB is defined as follows: -1 is assigned to flat and/or multiple PBs, no PBs, granular and/or abnormally small or large PBs. PBs that are normal but not judged as superior may be assigned 0, and PBs of normal size and shape may be assigned +1. In some embodiments, the MII oocyte PB score may not aggregate with the TOS.

In some embodiments of this method, the scoring metric may include performing the results analysis as a function of TOS, as defined and illustrated in Figure 7. Parametric or non-parametric tests may be applied to determine the significance of the results obtained during the analysis. The results analysis may be used to determine the maturation rate of oocytes from the GV to MII stage, the maturation rate of oocytes from the GV to MI stage, the maturation rate of oocytes from the MI to MII stage, the mean total oocyte score, the mean oocyte shape, the mean oocyte size, the mean egg quality, the mean PVS quality, the mean ZP quality, the mean polar body quality, and so on. In some embodiments, these results may be reported as mean, median, and deviation.

ii. In Vitro Fertilization and Embryo Culture In some embodiments of this method, one or more eggs or oocytes as described herein can be evaluated for quality or maturity using the scoring metrics described herein, etc., to determine whether they can be used for in vitro fertilization and embryo development.

In some embodiments of this method, oocytes or oocytes are matured through in vitro maturation and then used in IVF and/or ART as described herein. Any one or more oocytes can be used for intracytoplasmic sperm injection (ICSI). Following fertilization of the oocyte by contact with one or more spermatids, the subsequently formed zygote can be matured in vitro to produce an embryo such as a morula or blastula (e.g., mammalian blastocyst), which can then be transferred to the uterus of a subject (e.g., the subject from which the oocytes were initially collected) for implantation into the endometrium. Embryo transfers that can be carried out using the methods described herein include fresh embryo transfer, in which oocytes or oocytes to be used for embryonic development are collected from a subject, and the subsequent embryos are implanted in the subject during the same menstrual cycle. Alternatively, embryos can be generated and cryopreserved for long-term storage prior to implantation in the subject.

iii. Collection of Omics Data and Analysis of Oocytes, Cells, and Culture Mediums In some embodiments of this method, the scoring metrics may include omics-based analyses. For example, frozen cell lysates and cell culture media can be analyzed for bulk RNA sequencing, whole-genome bisulfite sequencing (WGBS), mass spectrometry-based proteomics, and metabolomics. Cell culture media can be used for metabolomics analysis to determine changes in the molecular content of the medium after co-culture compared to a pre-culture medium control. This can be used to profile dynamic changes in paracrine signaling between granulosa cells and oocytes. The collected data can then be aggregated for subsequent analysis to identify changes in epigenetic state, the presence of metabolites, and gene expression between various co-culture conditions and controls.

In some embodiments of this method, omics-based analyses may include genomics, proteomics, transcriptomics, pharmacogenomics, epigenomics, microbiomics, lipidomics, glycomics, transcriptomics-culturomics, and/or any other omics that are apparent to those skilled in the art as applicable. In some embodiments, immature oocytes exhibiting GV or MI characteristics after culture can be collected along with associated granulosa cells from the culture for single-cell RNA sequencing analysis. For this purpose, the oocytes and granulosa cells can be rapidly frozen for library preparation. Half of the oocytes exhibiting MII oocyte development can be collected and, along with their associated granulosa cells, subjected to single-cell RNA sequencing analysis using the rapid freezing method described throughout this disclosure. The remaining half of the MII oocytes can be used for proteomics analysis. The culture media for all conditions can be further rapid-frozen and used for metabolomics and proteomics to identify cholesterol metabolite levels and paracrine protein production. For example, frozen cell lysates and cell culture media can be analyzed using bulk RNA sequencing, whole-genome bisulfite sequencing (WGBS), mass spectrometry-based proteomics, and metabolomics. Cell culture media can be used for metabolomics analysis to determine changes in molecular content of the co-culture medium compared to a pre-culture medium control, in order to profile the dynamic changes in paracrine signaling between granulosa cells and oocytes. Rapid freezing of the medium components effectively quenches the samples, making them suitable for metabolic assessment. The collected data can then be aggregated for downstream analysis to determine changes in epigenetic state, metabolite presence, and gene expression between different co-culture conditions and controls.

IV. Kits or Products The compositions or methods described herein may be provided as kits for use in reprogramming iPSCs (e.g., hiPSCs) into populations of OSCs (e.g., granulosa cells and/or stromal cells). In some embodiments, the compositions and methods described herein may be provided as kits for use in co-culturing one or more oocytes with OSCs to produce one or more mature oocytes, which are optionally further fertilized by ART or IVF treatment to form embryos. In some embodiments, the kit may include instructions for the user of the kit to perform iPSC differentiation and/or in vitro maturation. In further embodiments, the kit may include instructions for the user of the kit to perform one of the ovarian stimulation and/or oocyte harvesting methods described herein. The kit optionally includes a syringe or device for administering the compositions of the Disclosure or for harvesting one or more oocytes. In some embodiments, the kit may include one or more additional cell media or agents used for cell culture. In some embodiments, the kit comprises one or more antibodies or binding molecules for detecting the expression of one or more genes or biomarkers described herein.

Example 1. Method for Producing Ovarian Supporting Cells by Reprogramming Induced Pluripotent Stem Cells Induced pluripotent stem cells (iPSCs) are widely used in biomedicine because they are obtained from adult cells that have been reprogrammed to have the potential to differentiate into any cell type in the body. We applied our established combinatorial and technical platform to manipulate iPSCs that possess inducible transcription factors that promote differentiation into ovarian cell types. These cells, called ovarian supporting cells (OSCs), express protein markers similar to granulosa cells, which are important functional cells of the ovary, and exhibit similar transcriptome and steroid production profiles. Our OSCs provide the IVM with a physiologically more appropriate and ovarian-like dynamic environment than culture medium alone, leading to improved maturation outcomes and, consequently, creating great potential for our artificial cells to improve infertility treatment in clinics as ART. This example details our efforts to develop and optimize an approach to large-scale, consistent manufacturing under Good Manufacturing Practice conditions using animal-free materials without affecting product purity, efficacy, or safety. We provide case studies and methodologies on the clinical applications of both iPSCs in cell therapy and ART for infertility treatment, demonstrating that a strategic plan for scalable and controlled manufacturing ultimately leads to far more consistent and functional products.

i. Method of Example 1 Raw Materials First, an allogeneic hiPSC strain (VCT-37-F35) was procured from Reprocell USA (9000 Virginiamanor Rd #207, Beltsville, mD 20705) as the starting material for a clinical-grade cell line. The human dermal fibroblast-derived stem cell line was generated under Good Manufacturing Practice (GMP) conditions using a non-integrated mRNA-based reprogramming technique with controlled conditions and GMP-compliant reagents used throughout the manufacturing process. Appropriate controls for fibroblast induction were implemented for specific stages of the process in accordance with established guidelines, and reprogramming and cell proliferation were carried out under full GMP conditions in compliance with FDA, EMA, and PMDA regulatory standards and guidelines. Based on supporting documentation to assess compliance with regulatory requirements, we conducted a risk-based assessment of the safety and suitability of the VCT-37-F35 parent cell line, confirming that the donor eligibility complied with 21 CFR 1271, subpart C, and that donor consent for broad use and indications had been obtained. The procured cloned hiPSC line was grown in a research cell bank (RCB) prior to in-house cell engineering. The research cell bank was created by growing the parent cell line for one passage and cryopreserving multiple samples to ensure sufficient material for engineering and to secure the stock solution of the parent cell line under ideal culture conditions for backup or future use. Reprocell, Inc., the supplier of the original hiPSC line, conducted a detailed characterization to ensure compliance with established commercial release standards, while we conducted further testing at downstream steps in the process to confirm the pluripotency, identity, genetic stability, and potency of the cells.

Plasmids used in plasmid manufacturing engineering were verified for identity, integrity, and purity by sequencing the entire plasmid using nanopore technology and stored at -20°C, while the glycerol stock solution of the transformed bacteria was stored at -80°C. Plasmids encoding transcription factors and piggyBac translocations were introduced into hiPSCs using a Lonza NUCLEOFECTOR® device.

Cell Engineering: Engineering of hiPSCs with specific transcription factors was performed using the piggyBac transposase strategy, which allows for the integration of multiple copies of the trans gene into the host genome. To enhance the efficiency of the engineering process, puromycin selection was used to remove cells that did not incorporate the transcription factors. The engineered stem cells were exposed to the Rho-related protein kinase inhibitor Y-27632, WNT activator, and glycogen synthase kinase 3 inhibitor CHIR99021, which together prepare the cells for mesoderm fate. Exposure to doxycycline throughout the process induced overexpression of transcription factors NR5A1, RUNX2, and GATA4, promoting differentiation from hiPSCs to OSCs. This potent Dox-induced TF-oriented differentiation requires 5 days of culture.

Cell screening, selection, and preliminary characterization: Following preliminary testing of a pooled population of transfected hiPSCs, clones were established by limiting dilution in a multi-well plate format. All wells were closely monitored daily until a single clone was identified in each well. Wells in which multiple clones were identified were discarded. Each identified clone was further expanded and cryopreserved to generate 43 seed clones. Each clone was initially assigned a unique code based on its position on the plate. Genotyping PCR was performed on the 43 seed clones to identify the presence of three transcription factors. This initial screening identified nine clones containing all transcription factors, and a more detailed screening process was then performed on each clone, including identity, potency, and safety testing to identify candidate lead cell lines. To identify the most promising candidate, each of the nine clones was individually differentiated and subjected to a series of assays to confirm clonal identity (pluripotency markers, donor identity, and genotype), compatibility (cell number and viability), potency (OSC generation and function), and, most importantly, safety (genomic integrity, vector copy number, mycoplasma, sterility, and accidental pathogens). The most promising candidate clone selected for use as a starting material for clinical-grade cell lines was named GTO-101.

ii. Transcription factor-mediated differentiation consistently generates ovarian supporting cells at various stages of ovarian development and follicular formation.
To evaluate the feasibility of using gene-modified hiPSC strains containing three inducible transcription factors (NR5A1, RUNX2, GATA4) as a source for generating consistent and functional OSCs, six independent batches of hiPSC-derived OSCs differentiated over eight months by multiple operators following standard operating procedures were compared. Differentiation of OSCs mediated by the overexpression of inducible transcription factors is a rapid and straightforward process compared to standard protocols that rely on small molecules to replicate the developmental trajectory. Five days after induction, hiPSCs proliferated 3.12 ± 1.77 times and acquired morphological features similar to human granulosa cells, including clusters of cells with pointed edges and granules within the cell body. Differentiated OSCs also expressed FOXL2 and CD82, two well-characterized markers indicating granulosa cell fate, demonstrating successful differentiation into the desired cell type.

To further characterize the molecular phenotypes of differentiated OSCs and gain a deeper understanding of the differences and similarities between independent batches, single-cell RNA sequencing (scRNA-seq) was performed on cryopreserved samples taken from six differentiated batches (Figure 1A). Initially, 15 Leiden clusters were identified, and these were combined based on molecular similarities to ultimately create nine clusters. Twelve of the fifteen identified early Leiden clusters expressed markers (GJA1, MDK, BBX, HES4, PBX3, YBX3, BMPR2, CD46, COL4A1, COL4A2, LAMC1, ITGAV, ITGB1) that have been shown to exhibit different expression in human granulosa cells compared to other cell types in developing ovaries (Garcia-Alonso et al.). Since all of these clusters were identified as granulosa cells, they were classified into two major classes: early GC or GC. The remaining three clusters were included in a third major class, identified as "other," because the expression of the major markers was not evident in these cell groups (Figure 1A).

Despite the expression of all granulosa markers, the class classified as early GC also shares transcriptional similarities with preGC-I and -IIa/IIb, including the expression of FOXO1 and CDH1 genes (Figure 1B). These cell subclusters, named early GC I, express the aromatase gene CYP19A1, which is upregulated in preGC-I cells in the ovarian medulla, and the gene for the chemotactic protein RARRES2, which has been shown to suppress steroid production and inhibit the progression of oocyte meiosis in bovine models. The early GC II subcluster expresses the RARRES2 gene in addition to the receptor NOTCH2, similar to the aforementioned subclusters (Figure 1B). The NOTCH signaling pathway is involved in oocyte-GC crosstalk during follicular formation, and high levels of NOTCH2 and NOTCH3 expression in cumulus cells have been shown to be positively correlated with the IVF response. Finally, in the subcluster, early GC III expression of RARRES2 was not observed, as in the previous subcluster, but NOTCH2 expression continued to be detected at significant levels. Overall, these expression patterns suggest that the cluster of early GC classes shares transcriptional signatures with granulosa cells and both preGC-I and -IIa/IIb, and the differential expression of CYP19A1, RARRES2, and NOTCH2 suggests a stepwise development and functional progression from early GC I to early GC III in the subcluster.

The GC class is marked by the expression of CDH2, in addition to all other granulosa markers described so far, including the NOTCH2/3 receptor (Figure 1B). Subcluster GC I is enriched with the NRG1 gene, BMPR1B gene, and ERBB family receptor genes (Figure 1B). NRG1 has been shown to exhibit different expression in preGC-IIa/IIb and is known to be expressed and secreted by granulosa cells in response to a surge in ovulation. BMPR1B, EGFR (ERBB1), and ERBB4 are all receptors identified in granulosa cells, with corresponding ligands expressed in oocytes (BMP6, TGFA, and NRG4, respectively). These interactions have been proposed to mediate follicular aggregation. The subcluster labeled GC II is enhanced by the expression of gene ID3, a target of the receptor BMPR2, which is also expressed by these cells. Interestingly, while BMPR2 is expressed by all early GC and GC clusters, the CG II subcluster is the subcluster with the highest enrichment of this target gene (Figure 1B), suggesting that the receptor BMPR2 is activated in these cells. The final subcluster of the GC class, GC III, consists of cells expressing both CDH2 and NOTCH2, but this subcluster does not enrich any of the other genes previously described in this class of subclusters. In summary, the three subclusters of GC appear to represent ovarian supporting cells in slightly different cellular states, mediated by distinct combinations of active signaling pathways.

The last three identified subclusters (obstructed/luteal, ribosome enriched, and mitochondrial enriched) were incorporated into a third class labeled "Other." In these clusters, the expression of most markers, including GJA1 and CDH2, was generally low (Figure 1B). Low expression levels of GJA1 and CDH2 have been reported in GCs in the early stages of obstruction. Interestingly, cells in the obstructed/luteal subcluster express not only CGA, the estrogen receptor α-response gene in human breast cancer cells, but also genes involved in steroid production, such as CYP11A1, CYP19A1, and HSD17B1. The other two clusters also showed enrichment of GCA genes; however, the most expressed genes within each cluster were mitochondrial genes in the mitochondrial enriched subcluster and ribosomal genes in the ribosome enriched subcluster. Generally, enrichment of mitochondrial and/or ribosomal genes in scRNA-seq analysis is associated with decreased cell quality, and further suggests that these clusters consist of dying cells. It is important to emphasize that it is unclear whether this observation is a biological consequence of ovarian supporting cells or a result of sample processing and handling.

After confirming that the majority of the analyzed cells were classified as granulosa cells (early GC and GC), we sought to understand whether OSCs were generated at various stages of follicular development by our protocol, or whether cells were overexpressed at specific follicular stages. To this end, we generated gene signature scores referencing a publicly available transcriptome landscape of human follicular development and applied them to our samples. In our samples, neither the primary nor secondary GC phase was clearly expressed, and most of the genes associated with these signature scores were not abundant in the analyzed cells. In contrast, the signature scores for pyloric GC and pre-ovulatory GC were more clearly expressed within clusters identified in our analysis, and it appeared that multiple genes driving these signatures were reinforced by multiple clusters (Figure 1C).

Following the characterization of cellular outcomes obtained from the differentiation process, the degree of reproducibility and consistency between independent batches of hiPSC-derived OSCs was investigated. Overall, all analyzed batches consistently generated clusters from the three major classes mentioned above (early GC, GC, and others), five of which were remarkably similar in terms of batch-by-batch cluster distribution. These results demonstrate a consistent methodology for generating OSCs from reprogrammed hiPSCs for clinical use in ART or infertility treatment.

iii. Applying the protocol to clinical manufacturing can lead to more reproducible cell results.
As part of a strategy to align research manufacturing protocols with clinical standards, a risk assessment of the bill of materials was conducted, and key components of the protocol were replaced with higher-quality alternatives, such as reagents free of animal-derived components, GMP-manufactured components, and cell therapy-grade raw materials. Among all the raw materials used to generate OSCs for research purposes, one of the most complex reagents is Matrigel, which is extracted from Engelblesse-Holm swarm mouse sarcoma cells and contains multiple extracellular matrix components of the tissue basement membrane. Due to the nature of this reagent, Matrigel exhibits significant lot-to-lot variability, which can affect the overall reproducibility of the final product. The most commonly used alternative substrates for hiPSC culture are human recombinant laminin-521 and vitronectin. Laminin, in particular, is one of the main components of Matrigel, while vitronectin is present in trace amounts.

The differentiation process was conducted by directly comparing differentiation induced by either human recombinant laminin-521 or vitronectin. All other raw materials remained identical under both conditions. Initial assessments of cell morphology during the differentiation process revealed subtle differences between the two groups. Laminin-OSCs produced smaller cells that organized into compact cell clusters. In contrast, vitronectin-OSCs exhibited larger cell bodies and organized as more sparse cell clusters. FOXL2 and CD82 expression also differed slightly between these two groups.

To gain a deeper understanding of the molecular profiles of OSCs generated under each condition and to assess the reproducibility of their cellular outcomes, we ran scRNA-seq on two batches of laminin-OSCs and two batches of vitronectin-OSCs and compared them to previous datasets (Figure 2). Laminin-OSCs were distributed primarily across subclusters of GC classes (GC I, GC II, GC III) and possessed a molecular profile similar to the cellular outcomes observed in the GC3_GC_batch_II_2D batch, which stood out in the previous analysis. In contrast, vitronectin-OSCs were primarily represented by early GC II, early GC III, and GC III (Figure 2). Interestingly, OSCs differentiated on vitronectin also showed a higher proportion of cells in clusters enriched with both ribosomal and mitochondrial genes (Figure 2). Overall, these data indicate that differentiation on laminin-521 and vitronectin generated cell fates similar to those characterized in the previous section; therefore, these alterations are unlikely to affect cell function. Interestingly, these results suggest that the final OSC fate is not only regulated by the overexpression of three transcription factors, but may also be significantly influenced by the properties of the matrix utilized as a substrate (Figure 2).

Although differences were observed in the cell results generated under each of these two conditions, there were no significant differences in cluster distribution between the two different independent batches under the same conditions (Figure 2). This indicates that by modifying the bill of materials to include higher-quality reagents, consistent and reproducible cell results were obtained regardless of the matrix used. It is also important to emphasize that each independent differentiation batch was performed by a different operator, which further strengthens the evidence for reproducibility.

iv. Differentiation mediated by laminin-521 leads to a scalable, pure, and functional population of ovarian supporting cells.
After confirming that the transition to a high-quality bill of materials overall would not negatively impact reproducibility or the final cellular outcome, we investigated which of the two conditions (laminin-521 and vitronectin) yielded better clinical outcomes and should be carried over to clinical manufacturing. As measures of successful clinical outcomes, we considered several parameters that directly indicate throughput, safety, and efficacy for each condition. For throughput, we compared the OSC:hiPSC ratio of each batch analyzed for each condition. Conditions that can increase the yield of viable cells harvested without changing the initial cell number or surface area for culturing cells would justify a more scalable alternative. Laminin-OSCs were harvested with a viability of 94.63 ± 0.01% and proliferated with an OSC:hiPSC ratio of 14.83 ± 4.48 during differentiation. Vitronectin-OSCs were harvested with a viability of 85.25 ± 0.11% and proliferated with an OSC:hiPSC ratio of 5.86 ± 1.30 during differentiation.

Regarding the safety of hiPSC-derived products, it is essential to confirm whether residual hiPSCs are present in the final cell composition. If a large amount of residual hiPSCs is found in the final cell composition without further purification steps, safety concerns would impair the product's use in assisted reproductive technology or in vitro fertilization procedures. To identify the presence of residual hiPSCs in terminal differentiation, RT-qPCR was used to identify the presence of two major regulators of pluripotency, POU5F1 and NANOG. To determine the sensitivity of the assay, concentration curves were generated by adding various concentrations of hiPSCs (0%, 0.0001%, 0.001%, 0.01%, 0.1%, 1%, 10%, 100%) to fully differentiated OSCs. This assay confirmed that hiPSCs in the final product could be identified down to 0.01%. Next, the presence of residual hiPSCs in different differentiation batches was characterized. The obtained data demonstrated that the final product was free of residual hiPSCs. However, it is noteworthy that while POU5F1 mRNA was detected in both the vitronectin-OSC batches using scRNA-Seq, it was not detected in the laminin-OSC batch. NANOG was not detected under either condition. POU5F1 is an important regulator and marker of hiPSC, but without NANOG and SOX2, this gene cannot maintain pluripotency. Furthermore, there is evidence that POU5F1 may be expressed by a subset of human ovarian granulosa cells.

Next, we investigated whether the final cell composition was functional and could promote human oocyte maturation. Following an approach similar to that discussed herein, we co-cultured both laminin-OSCs and vitronectin-OSCs independently with immature human oocytes and evaluated the oocyte maturation yield of each group (see, for example, Example 5). Co-culture with laminin-OSCs resulted in a 1.50-fold (64.91 ± 6.64%) increase in MII formation compared to the control group, while co-culture with vitronectin-OSCs resulted in a 1.26-fold (54.35 ± 8.10%) increase in MII formation compared to the control group. Overall, although the MII formation rate was slightly higher in the laminin-OSC group, both approaches successfully produced functional OSCs and improved oocyte maturation rates compared to control conditions.

v. Generation of a clinical-grade hiPSC strain capable of producing functional ovarian supporting cells The prior experiments for this example were performed using hiPSC strain GC3, a cell line designated for research use only (RUO) and not clinical-grade. Next, the goal was to generate a clinical-grade hiPSC strain from allogeneic female donors for commercial-grade material. To minimize discrepancies between the results of the original RUO hiPSC strain, which formed the basis of the initial preclinical studies and efficacy trials, and the clinical-grade hiPSC strain, the same manufacturing strategy as the RUO hiPSC strain was applied to the generation of the clinical-grade hiPSC strain, ultimately demonstrating that the same strategy could be utilized.

Clinical-grade hiPSC strains were engineered to retain inducible versions of three transcription factors (NR5A1, RUNX2, GATA4) that promote differentiation into OSCs. Individual clones were generated by limiting dilution of a pooled artificial population and expanded into a seed bank. All clones that grew successfully underwent initial screening by genotyping PCR to confirm the integration of the three transcription factors (Figure 3A). Nine seed clones containing all transcription factors were selected, and a more thorough screening process was carried out, including evaluation of their identity, potency, and safety. For this purpose, each clone was differentiated individually into OSCs (Figure 3B), and several characteristics were recorded for the purpose of screening and to identify the most promising candidates. To specifically assess clone identity, the expression of OSC markers, FOXL2 and CD82, was examined 5 days after differentiation, and it was confirmed that all clones were positive for both markers, despite differences in expression levels between clones, suggesting that overall OSC generation was successful (Figure 3C). Furthermore, we confirmed that the levels of OCT4 (hiPSC marker) in all clones were very low or zero, confirming efficient and pure OSC results.

As a functional readout of individual clones, cells were differentiated for 5 days and exposed to follicle-stimulating hormone (FSH or F), androstenedione (A4 or A), or a combination of both hormones (F + A) for 48 hours. Functional OSCs respond to FSH to produce estradiol (E2), and A4 is used as a substrate by the OSC to complete the reaction. Individual clones showed different responses to treatment with FSH + A4, and it was found that more responsive clones were more likely to perform better in maturing immature human oocytes (Figure 3D). In addition to the aforementioned attributes, three clones, including clone GTO-101, were identified as the most promising candidates based on the OSC:hiPSC ratio and viability at harvesting (Figure 3E). This decision was based primarily on the expression levels of FOXL2 and CD82, and the responsiveness of the clones to FSH and A4 regarding estradiol production. In clone 3-E3 (GTO-103), a low level of E2 was observed; however, this sample was selected for further analysis using an IVM assay with immature oocytes due to its high levels of identity markers (FOXL2 and CD82) (Figure 3E). The GTO-101 clone showed a higher oocyte maturation rate (MII formation) compared to the other clones. Therefore, clone GTO-101 not only presented high levels of OSC markers during differentiation but also demonstrated high performance in OSC generation and oocyte maturation.

vi. Generation of clinical-grade hiPSC strains capable of producing functional ovarian supporting cells To further characterize clone GTO-101 for clinical application, the presence of hiPSC markers was evaluated and confirmed, and cell identity and normal karyotype were verified (Figure 4A). Next, two independent batches of differentiated GTO-101 were generated using a protocol previously identified as most suitable for transition to clinical manufacturing. Specifically, GTO-101 hiPSCs were differentiated into laminin-521 coated dishes using the highest quality raw materials. As expected, the differentiated cell morphology was characterized by small cells with granules within the cell body, densely clustered in pointed-edge clusters. Furthermore, the survival rate at the time of collection remained high at an average of 96.90 ± 0.00%, and the OSC:hiPSC ratio was confirmed to be similar to that achieved in the RUO hiPSC strain when differentiated beyond laminin-521 (14.83 ± 4.48), averaging 11.41 ± 2.19. OSC identity was confirmed by the expression of markers FOXL2 and CD82, and since the hiPSC markers POU5F1 and NANOG were not detected after performing the differentiation protocol, cell identity was confirmed, and it was further shown that there was no contamination by residual hiPSCs (Figure 4B).

To further characterize the transcriptional signatures of differentiated OSCs and assess reproducibility between independent lots, scRNA-seq was performed on two batches of differentiated GTO-101 (Figure 4B). Surprisingly, the two batches were nearly identical in terms of cluster distribution compared to previously analyzed samples, and these consisted mainly of GC class clusters, particularly subclusters GC I and GC III. Interestingly, the transcriptome profiles of GTO-101-derived OSCs were similar to the first batch of RUO hiPSC strain produced (GC3_GC_batch_II_2D) and the two batches of laminin-OSCs produced after the raw material optimization described in the previous section. These results strongly suggest that GTO-101-derived OSCs lead to successful functional outcomes. Therefore, these observations further demonstrate the reproducibility of successful reprogramming from hiPSCs to OSCs, even when performed across independent cell batches, independent genetic backgrounds (RUO and clinical-grade hiPSC lines), and by different operators. These data collectively demonstrate the promising clinical utility of these cells and this methodology for hiPSC differentiation in ART and IVF applications.

Example 2. Method for Producing Ovarian Supporting Cells from iPSCs This example demonstrates how iPSC differentiation (e.g., reprogramming or engineering) into one or more types of ovarian supporting cells can be achieved. It should be understood that this example is a non-limiting embodiment of the present disclosure and is intended to illustrate a potential protocol for producing OSCs from iPSC precursors.

iPSCs (e.g., hiPSCs) from previously cryopreserved stock cells or freshly harvested from donor subjects are cultured in vitro in appropriate culture dishes containing cell medium (e.g., in vitro maturation (IVM) cell medium) and a matrix such as a laminin-containing matrix. Undifferentiated hiPSCs are reprogrammed using transposase expression plasmids (e.g., piggyBac transposase method) to carry specific inducible transcription factors (e.g., FOXL2, NR5A1, RUNX2, and/or GATA4). Electroporation of the transposase expression plasmid hiPSCs was performed. Transcription factors were induced upon coating the medium with doxycycline. Wnt/β-catenin pathway activators, such as ROCK inhibitors (e.g., Y-27642) and GSK3 inhibitors (e.g., CHIR099021), were also added to the medium to prepare the cellular environment for the fate of mesodermal cells.

The cells are induced for approximately 5 days (e.g., approximately 2, 3, 4, 5, 6, and 7 days). During the reprogramming process, the expression of genes or biomarkers corresponding to one or more types of OSCs is evaluated. mRNA and protein expression levels, assessed by RT-PCR and flow cytometry, confirmed that, compared to the undifferentiated hiPSC population, a portion of the cell population expressed FOXL2 and AMHR2, while another portion expressed NR2F2, thus confirming that the hiPSCs differentiated into a mixed population of granulosa cells and ovarian stromal cells. Furthermore, the resulting OSCs, stimulated by androstenedione and FSH or forskolin, produce steroids such as estradiol or progesterone. Steroid production is confirmed by ELISA, which measures the level of steroids secreted into the cell medium using antibodies that detect one or more steroids and compares it to the medium of a sample containing undifferentiated hiPSCs as a negative control.

Furthermore, the cellular identity and relative purity of the obtained OSCs were confirmed by RT-PCR, and no significant detection of one or more pluripotency markers (e.g., POU5F1, NANOG, SOX2, and/or OCT4) was detected compared to the expression levels detected in undifferentiated hiPSCs, which served as a positive control. When the detected expression levels of pluripotency markers were less than approximately 5% of the expression levels of hiPSCs (e.g., <5%, <4%, <3%, <2%, <1%, or <0.10%), it was confirmed that the obtained OSC population was pure and that the hiPSCs had successfully differentiated and been reprogrammed into OSCs. These OSCs may be further clonally expanded and/or cryopreserved as stock solutions for use in IVM methods or ART applications, such as any of the methods or applications described herein.

Example 3. Method for the release of oocytes from the ovary by follicular stimulation and in vitro maturation of oocytes This example demonstrates a method for performing minimal follicular stimulation on subjects with low ovarian reserve, followed by the collection of oocytes and in vitro maturation.

i. Follicular stimulation for ovarian release of oocytes: A 30-year-old woman undergoes a blood test, which detects an anti-Müllerian hormone (AMH) level of 6 ng/mL or less (e.g., 1 ng/mL, 2 ng/mL, 3 ng/mL, 4 ng/mL, 5 ng/mL, or 6 ng/mL). Therefore, the subject is determined to have low ovarian reserve. Further blood tests revealed that the target estradiol level was between 20 and 50 pg/mL (for example, 20-30 pg/mL, 25-35 pg/mL, 30-40 pg/mL, 35-45 pg/mL, or 40-50 pg/mL, e.g., 20 pg/mL, 21 pg/mL, 22 pg/mL, 23 pg/mL, 24 pg/mL, 25 pg/mL, 30 pg/mL, 35 pg/mL, 40 pg/mL, 45 pg/mL, or 50 pg/mL), reaffirming the diagnosis of low ovarian reserve.

To stimulate follicular maturation and oocyte release, the subject is administered an induction agent (e.g., clomiphene citrate 50 mg). Since the subject is taking hormonal contraceptives, administration of the induction agent is started around 5 ± 1 days (e.g., day 4, day 5, or day 6) after the last dose of contraceptives, and continued daily for 1 to 4 days (e.g., day 1, day 2, day 3, or day 4). The subject's follicular size is monitored by ultrasound until the average follicular size reaches approximately 8–10 mm (e.g., 7.5 mm, 8 mm, 8.5 mm, 9 mm, 9.5 mm, 10 mm, 10.5 mm, or larger), after which oocytes (or cell groups containing oocytes, e.g., COCs) are collected from the subject by aspiration. For example, oocyte collection can be performed using transvaginal ultrasound with a needle guide on the probe, by aspirating and removing the follicular contents. The follicular contents containing oocytes (e.g., follicular aspirate) are washed with HEPES medium (G-MOPS Plus, VITROLIFE®), filtered through a 70-micron cell strainer (FALCON®, Corning), and examined under a dissecting microscope. The oocytes (or cell groups containing oocytes, e.g., COC) are transferred to a culture dish containing cell culture medium (e.g., IVM medium, IVF medium, or LAG medium) and cultured for approximately 1-3 hours (e.g., 1, 2, or 3 hours), after which granulosa cells are introduced for co-culture.

ii. In vitro maturation of oocytes If present, cultured COCs can be separated from their cumulus cells (and any other non-oocytes) in a process referred herein as oocyte denucleation. Oocyte denucleation is performed by mechanically dissociating the cells from the COCs in the IVM well using a pipette, thereby removing the cumulus cells and/or granulosa cells. Further oocyte denucleation may be performed by enzymatic dissociation (e.g., hyaluronidase treatment). The COCs can be stripped with a stripper tip and washed in IVM medium or MOPS plus medium to wash the oocytes for imaging, and hyaluronidase can be inactivated as needed. Stripper tips include 200 micron and/or 400 micron for fine washing.

Next, the oocytes in the vesicular stage (GV) and metaphase I (MI) are divided into approximately 50,000 to 100,000 granulosa cells (for example, 50,000 to 60,000 cells, 60,000 to 70,000 cells, 70,000 to 80,000 cells, 80,000 to 90,000 cells, or 90,000 to 100,000 cells, for example, 50,000 cells, 55,000 cells). Co-culture with 0, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 cells (for example, specific granulosa cells described herein, hiPSC-derived granulosa cells, or steroid-producing granulosa cells). Metaphase II (MII) oocytes (for example, oocytes with polar bodies in the periuterine coel) can be appropriately frozen for storage. Co-culture of oocytes and granulosa cells for approximately 12 to 120 hours (for example, 12 to 24 hours, 12 to 36 hours, 24 to 48 hours, 36 to 60 hours, 54 to 72 hours, 68 to 96 hours, 96 to 120 hours, for example, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours, 46 hours, 48 hours, 50 hours, Perform the following durations: 52 hours, 54 hours, 56 hours, 58 hours, 60 hours, 62 hours, 64 hours, 66 hours, 68 hours, 70 hours, 72 hours, 74 hours, 76 hours, 78 hours, 80 hours, 82 hours, 84 hours, 86 hours, 88 hours, 90 hours, 92 hours, 94 hours, 96 hours, 98 hours, 100 hours, 102 hours, 104 hours, 106 hours, 108 hours, 110 hours, 112 hours, 114 hours, 116 hours, 118 hours, or 120 hours.

Following co-culture, one or more oocytes are used for assisted reproductive technology (ART) procedures. For example, oocytes can be used for intracytoplasmic sperm injection (ICSI).

Example 4. Administration of follicle-inducing agents This example demonstrates the administration of follicle-inducing agents to the subjects.

A 30-year-old woman undergoes a blood test that detects an estradiol level of 20–50 pg/mL (e.g., 20–30 pg/mL, 25–35 pg/mL, 30–40 pg/mL, 35–45 pg/mL, or 40–50 pg/mL, e.g., 20 pg/mL, 21 pg/mL, 22 pg/mL, 23 pg/mL, 24 pg/mL, 25 pg/mL, 30 pg/mL, 35 pg/mL, 40 pg/mL, 45 pg/mL, or 50 pg/mL). The subject receives multiple injections of an induction agent over 1–4 days (e.g., day 1, day 2, day 3, or day 4) (but no more than 5 days). The subject may receive multiple injections over several days, such as five injections of one or more induction agents. For example, the target dose is 300 IU to 700 IU per injection (for example, 300-500 IU, 400-600 IU, 500-700 IU, 300-350 IU, 350-400 IU, 400-450 IU, 450-500 IU, 500-550 IU, 550-600 IU, 600-650 IU, 650-700 IU, for example). , receive a 3-day stimulation (at least one injection per day) using rFSH in doses of 300 IU, 325 IU, 350 IU, 375 IU, 400 IU, 425 IU, 450 IU, 475 IU, 500 IU, 525 IU, 550 IU, 575 IU, 600 IU, 625 IU, 650 IU, 675 IU, or 700 IU. In other cases, subjects receive injections using 200–700 μg or 2,500–10,000 IU (e.g., 200–500 μg, 300–600 μg, 400–700 μg, 200–300 μg, 300–400 μg, 400–500 μg, 500–600 μg, or 600–700 μg) of hCG as an inducer. In yet another example, the subject receives one or more doses (e.g., 1, 2, 3, 4, or 5 times) of clomiphene citrate in doses of 50–150 mg (e.g., 50–75 mg, 60–80 mg, 75–100 mg, 90–115 mg, 110–130 mg, 125–150 mg, e.g., 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 110 mg, 120 mg, 130 mg, 140 mg, 150 mg).

Example 5. Materials and Methods of Examples 6-8 The inventors have developed human ovarian supporting cells (OSCs) generated from human induced pluripotent stem cells (hiPSCs) that have the ability to reproduce dynamic ovarian function in vitro. Here, the inventors are studying the potential of such OSCs, collected from a simple gonadotropin-stimulated cycle, to improve human oocyte maturation as a co-culture system applicable to IVM. The inventors demonstrate that OSC-IVM significantly improves maturation rates compared to available IVM systems. Most importantly, the inventors demonstrate that OSC-supported IVM oocytes are capable of forming robust euploid blastocysts, which is an important marker of their clinical utility. Taken together, these findings represent a novel approach to IVM that is widely applicable to the implementation of modern IVF.

Specifically, to determine whether in vitro maturation (IVM) of human oocytes can be improved by co-culturing ovarian supporting cells (OSCs) derived from human induced pluripotent stem cells (hiPSCs), oocyte donors were recruited and subjected to simplified gonadotropin stimulation with and without hCG induction agents. The cumulus-oocyte complex (COC) was then assigned to either OSC-IVM conditions or an IVM control using only the culture medium.

Under informed consent, oocyte donors aged 19–37 years were recruited and selected for donation, using anti-Müllerian hormone (AMH) levels exceeding 1 ng/mL as the selection criterion. The OSC-IVM culture conditions consisted of 100,000 OSCs in a suspension culture containing human chorionic gonadotropin (hCG), recombinant follicle-stimulating hormone (rFSH), androstenedione, and doxycycline. The IVM control lacked OSCs and contained either the same supplements or only FSH and hCG.

The primary endpoints were mid-stage II (MII) formation rate and morphological quality. A limited cohort of oocytes was further used for fertilization and blastocyst formation using PGT-A analysis. OSC-IVM demonstrated a statistically significant improvement in MII formation rate compared to the medium-alone control. OSC-IVM also demonstrated a statistically significant improvement in MII formation rate compared to the commercially available IVM control. There was no significant difference in oocyte morphological quality between OSC-IVM and the control. OSC-IVM improved maturation, fertilization, cleavage, blastocyst formation, high-quality blastocyst formation, and euploid blastocyst formation compared to the commercially available IVM control.

In conclusion, the novel OSC-IVM platform is an effective tool for the maturation of human oocytes obtained from simplified gonadotropin-stimulated cycles, leading to improved blastocyst formation. OSC-IVM demonstrates broad applicability to various stimulation regimens, including simplified IVF induced with hCG and conventional IVM cycles without induction, making it a highly useful tool for modern infertility treatment.

i. Collection of Cumulus Oculum Oocyte Complex (COC) Participant Age, IRB, and Informed Consent Participants were enrolled in the study under informed consent (CNRHA 47/428973.9/22, IRB number 20225832, Western IRB, and protocol number GC-MSP-01, respectively) through the Ruber Clinic (Madrid, Spain), Spring Fertility Clinic (New York, USA), and Planor Clinic (Lima, Peru). The age range of the participants was 19 to 37 years. Oocytes collected from the Ruber Clinic and Planor Clinic were used only for maturation analysis, while oocytes collected from Spring Fertility were used for embryogenesis analysis.

Stimulation Characteristics: In preparation for the aspiration of immature oocytes in Experiment 1, 25 subjects received stimulation for 3-4 days using 300-600 IU of rFSH and an hCG inducer. AMH levels were >1 ng/mL (see below). In preparation for the aspiration of immature oocytes in Experiment 2, 21 subjects received administration of 200 IU of rFSH and an hCG inducer for 3 consecutive days. To increase the number of donors that produce more oocytes, an AMH level >1.5 ng/mL was used as a selection criterion (see below). In Experiment 2, with the goal of subsequent embryo formation, 6 subjects received administration of clomiphene citrate (100 mg) 3-5 times, with or without an hCG inducer, along with 1-2 further administrations of 150 IU of rFSH. An AMH level >2.0 ng was used as a selection criterion (see below). Gonadotropin injections were initiated on day 2 of the natural cycle or day 5 after discontinuation of oral contraceptives. A complete table of donor stimulation regimens for each donor in the study is shown in Table 1 below.

ii. Small follicle aspiration for collection of immature cumulus oocyte complexes. Oocytes for co-culture experiments were collected 36 hours after induction injection (10,000 IU hCG) using transvaginal ultrasound with a needle guide on the probe. Aspiration was performed using ASP medium (VITROLIFE®) and a double-lumen 19 gauge needle without follicular flushing (the double-lumen needle was chosen for the additional rigidity provided by the second channel in the needle). Follicular contents were collected through the aspiration needle and tube using vacuum pump aspiration (100 mm Hg) and injected into a 15 mL round-bottom polystyrene centrifuge tube. Under conditions where the final outcome was embryogenesis, aspiration was performed 36 hours after induction injection (10,000 IU hCG) or 48 hours after the last rFSH injection for non-induction cycles. Without performing follicular flushing, follicular contents were collected by aspiration using a single-lumen 19-gauge needle or a single-lumen 20-gauge needle, with vacuum pump aspiration (approximately 200 mmHg), via a tube connection to a 15 mL round-bottom polystyrene centrifuge tube. In all cases, when the follicle was collapsing, rapidly rotating the aspiration needle around its long axis induced a curettage effect, assisting in the release of COC into the aspirated fluid. Although the follicles were not flushed, during the oocyte collection procedure, the aspiration needle was removed from the subject and flushed frequently to suppress coagulation and needle occlusion.

Follicular aspirates were examined in the laboratory using a dissecting microscope. Because the aspirates tended to contain more blood than typical IVF follicular aspirates, they were washed with HEPES medium (G-MOPS Plus, VITROLIFE®) to minimize coagulation. In many cases, the aspirates were further filtered using a 70-micron cell strainer (FALCON®, Corning) to improve the oocyte search process. The COCs were transferred to dishes containing LAG medium (Medicult, COOPERSURGICAL®) using a sterile Pasteur pipette until ready for use in the IVM procedure. The number of aspirated COCs represented approximately 40% of the antral follicles identified in the target ovaries on the start date.

iii. Preparation of Ovarian Supporting Cells (OSCs) OSCs were prepared from human induced pluripotent stem cells (hiPSCs) according to a transcription factor (TF)-oriented protocol. OSCs were produced in multiple batches, cryopreserved in vials containing 120,000–150,000 live cells, and stored in liquid nitrogen in CryoStor CS10 cell freezing medium (STEMCELL TECHNOLOGIES®).

Culture dishes (4+8 dishes, BIRR) for oocyte maturation experiments were prepared the day before oocyte collection by placing 100 μL of culture medium and additional components in droplets under mineral oil. On the morning of oocyte collection, the frozen OSCs were thawed at 37°C for 2-3 minutes (in a heated beads or water bath), resuspended in OSC-IVM medium, and removed by centrifugation and pelletizing twice to remove residual cryoprotectant. Equilibrated OSC-IVM medium was used for the final resuspendion. Next, to allow for culture equilibrium and medium preparation, 2-4 hours before adding oocytes, 50 μL of the droplets were replaced with 50 μL of OSC suspension, and OSCs were seeded at a concentration of 100,000 OSCs per 100 μL droplet.

iv. In vitro maturation COCs were maintained in pre-incubation LAG medium (Medicult, COOPERSURGICAL®) at 37°C for 2-3 hours after collection and before being introduced into in vitro maturation conditions. Two different experimental comparisons were performed to achieve the following objectives.

Experiment 1 (OSC Activity): The purpose of this comparison was to determine whether stimulated OSCs were the active components of the co-culture system. For this purpose, the culture media for the experimental and control conditions were prepared according to the manufacturer Medicult's recommendations. Androstenedione and doxycycline (both necessary for OSC activation/stimulation) were further supplemented with the same media formulation to compare maturation results in the presence or absence of OSCs (see Table 2 below).

Experiment 2 (Clinical Applicability of OSC): The purpose of this experiment was to compare the effectiveness of the OSC-IVM system with a commercially available in vitro maturation system (Medicult IVM). For this purpose, a control group condition was prepared and supplemented according to the manufacturer Medicult's recommendations, while the culture medium for OSC-IVM was prepared using all supplements (see Table 2 below).

Subject Description (Experiment 1): 132 oocytes were collected from 25 subjects (average age 25 years) who received simple gonadotropin stimulation. 49 were used for OSC-IVM co-culture and 83 for control culture. Co-cultures were performed in parallel under experimental and control conditions, where possible. When performed in parallel, COCs were distributed evenly. Even distribution means that COCs with clearly large cumulus oophores, small cumulus oophores, or spread cumulus oophores were distributed as evenly as possible between the two conditions. If selective distribution of different COC sizes was not possible, COCs were distributed as randomly as possible between one or two conditions. In this comparison, due to the small number of oocytes collected per subject, it was often impossible to effectively distribute oocytes between conditions simultaneously. COC was subjected to these in vitro maturation conditions for a total of 24–28 hours at 37°C in a Trigas incubator where the _CO2 was adjusted so that the pH of the bicarbonate buffer medium was 7.2–7.3 and the _O2 level was maintained at 5%.

Subject Description (Experiment 2): For the IVM outcome endpoint, 21 subjects were recruited for comparison. 143 COCs were collected for comparison, with 70 allocated for the IVM control and 73 for the OSC-IVM condition. Co-culture was performed in parallel for all subjects under both experimental and control conditions. As mentioned above, the COCs were evenly distributed between the two conditions. The COCs were subjected to these in vitro maturation conditions for a total of 28 hours at 37°C in a Trigas incubator with the _CO2 adjusted to a bicarbonate buffer pH of 7.2–7.3 and the _O2 level maintained at 5%. In vitro maturation and subsequent embryogenesis were performed to evaluate the developmental capacity of oocytes treated in the OSC co-culture system compared to oocytes treated in commercially available IVM medium. For embryogenesis, a small cohort of oocyte donors was recruited and donor sperm was used for fertilization. For comparison with the evaluation criteria for embryo results, six additional subjects were recruited and accepted. Forty-six COCs were collected for comparison, with 21 allocated for the medium-IVM control and 25 for the OSC-IVM conditions. Co-culture was carried out in parallel under experimental and control conditions. As previously mentioned, the COCs were evenly distributed between the two conditions. The COCs were subjected to these in vitro maturation conditions for a total of 28 hours at 37°C in a Trigas incubator where the _CO2 was adjusted to a bicarbonate buffer medium pH of 7.2–7.3 and the _O2 level was maintained at 5%. Embryogenesis proceeded in parallel, with the groups kept separate, and culture continued until day 7 after IVM.

v. Evaluation of in vitro maturation After the 24-28 hour in vitro maturation period, the COCs were subjected to hyaluronidase treatment to remove surrounding cumulus cells and corona cells. After hyaluronidase treatment, the cumulus cells were stored for future analysis, and the oocytes were evaluated for maturation according to the following criteria.

GV – Typically, an oocyte contains a single nucleolus within the oocyte.

MI – The oocyte lacks a nuclear vesicle, and the polar body is absent in the perivitelline space between the oocyte and the zona pellucida.

MII – The oocyte lacks a nuclear vesicle, and the polar body is present in the perivitelline space between the oocyte and the zona pellucida.

vi. Morphological Score of Oocytes After IVM, oocytes were collected from the culture dish, cumulus cells and OSCs were removed, evaluated for maturation assessment, and then individually imaged by digital microscopy. After imaging, oocytes were rapidly frozen in 0.2 mL PCR tubes pre-filled with 5 μL of DPBS. The images were then scored according to the Total Oocyte Score (TOS) evaluation system. Oocytes were scored by one trained embryologist and assigned scores of -1, 0, and 1 for morphology, cytoplasmic particle size, perivitelline space (PVS), zona pellucida (ZP) size, polar body (PB) size, and oocyte diameter. Zona pellucida and oocyte diameter were measured using ECHO® Revolve Microscope software and ImageJ image analysis software (2.9.0/1.53t). A total quality score is assigned to oocytes from the sum of all categories, ranging from -6 to +6, with higher scores indicating better morphological quality.

vii. Processing of oocytes after morphological scoring For oocytes used solely for evaluating oocyte maturation, flash freezing was performed after evaluation of in vitro maturation and any further morphological scoring. Flash freezing was performed by placing each oocyte into a 0.25 mL PCR tube containing 5 μL of DPBS. After capping the tubes, they were immersed in liquid nitrogen until bubbling stopped. The PCR tubes were then stored at -80°C for future molecular analysis.

For the oocytes used to create embryos, mature oocytes were immediately used for intracytoplasmic sperm injection (ICSI), and embryonic development was carried out until the blastocyst stage. Oocytes from this study were not used for embryo transfer, transplantation, or reproductive purposes.

viiii. In vitro fertilization and embryo culture A cohort of six subjects was used for in vitro maturation and subsequent embryo development. COCs from these subjects were subjected to the conditions used in Experiment 2 (treatment by OSC co-culture using all adjuvants and commercially available IVM treatment as a control). All COCs were cultured for 28 hours, then decontaminated, MII formation was evaluated, and micrographs were taken. Individual oocytes under each condition were injected with sperm on day 1 after collection (intracytoplasmic sperm injection (ICSI)). Following ICSI, the oocytes were cultured at 37°C in a Trigas incubator with a culture medium designed for embryo culture (Global Total, COOPERSURGICAL®, Bedminster, NJ), with the _CO2 adjusted to a pH of 7.2–7.3 in bicarbonate buffer medium and the _O2 level maintained at 5%. The following day, 12–16 hours after ICSI, fertilization was evaluated, and oocytes with one or two pronuclei were cultured until day 3. Digested embryos underwent laser zona pellucida perforation and were developed to the blastocyst stage. Blastocysts were scored according to the Gardner scale, and then trophectoderm biopsy was performed for preimplantation genetic testing for aneuploidy (PGT-A). If judged to be of high quality (3CC rating or higher), they were cryopreserved.

The trophectoderm biopsy samples were transferred to 0.25 mL PCR tubes and sent to a reference laboratory (JUNO GENETICS®, Basking Ridge, NJ) for comprehensive chromosomal analysis (preimplantation genetic testing for aneuploidy, PGT-A) using next-generation sequencing (NGS) based on single nucleotide polymorphisms (SNPs) for all 46 chromosomes.

ix. Data Analysis and Statistics Egg maturation results were analyzed using the Python statistical packages pandas (1.5.0), scipy (1.7.3), and statsmodels (0.13.2). Maturation percentages by donor group were analyzed using linear regression according to the IVM environment (OSC-IVM or culture control). T-test statistics were calculated by comparing cell line incubation results with the culture control, and then used to calculate p-values. Bar graphs show the mean values for each population, and error bars represent the standard error (SEM) of the mean.

Example 6. hiPSC-derived OSCs effectively promote the maturation of human oocytes following co-culture. To obtain immature COCs for IVM, a protocol similar to the above IVM studies, a simplified IVF or hCG-stimulated IVM, i.e., minimal gonadotropin stimulation for 3–4 days, and in most cases, an hCG inducer was used. This simplified stimulation program resulted in a mixed cohort of mostly immature oocytes (GV and MI), especially when hCG was included, but proliferated cumulus COCs were also obtained, which may have contained MII oocytes. The demographics and treatment plans of the oocyte donors are shown in Table 3 for each experimental group. Overall, the results show that oocytes could be recovered from non-polycystic ovary syndrome (non-PCOS/PCOS) donors, although the yield was lower than that of conventional controlled ovarian hyperstimulation cycles. In Experiment 1, oocytes from each donor were assigned to either the control IVM or the OSC-IVM arm. Age, body mass index (BMI), and total COC collected did not differ significantly between groups in Experiment 1. In Experiment 2, the control group and the OSC-IVM group for both evaluation items included the same donor group, because oocytes were evenly distributed among multiple conditions for each donor. In Experiment 2, age and BMI differed significantly compared to Experiment 1, and the total number of COCs collected per donor decreased, but the difference was not statistically significant. A schematic diagram of the OSC-IVM conditions is shown in Figure 8A, and a representative image of OSC co-culture is shown in Figure 8B.

Previously, we demonstrated that OSCs derived from hiPSCs are mainly composed of granulosa cells and ovarian stromal cells. In response to in vitro hormonal stimulation, these OSCs produce growth factors and steroids necessary for interaction with oocytes and cumulus cells. To investigate whether hiPSC-derived OSCs can functionally promote human oocyte maturation in vitro, we established a co-culture system with these cells and newly collected cumulus-encircling oocytes and evaluated the maturation rate after 24–28 hours (see Materials and Methods, Experiment 1). In this comparison, due to the small number of oocytes collected per donor, it was not possible to consistently divide oocytes between both conditions simultaneously; therefore, each group mainly consisted of oocytes from non-overlapping donor groups, and pairwise comparisons were not used. Surprisingly, oocytes treated with IVM using OSCs showed a significant improvement in maturation results (approximately 1.5 times) compared to the control group (Figure 9A). More specifically, the maturation rate of the OSC-IVM group was 68% ± 6.83% SEM, compared to 46% ± 8.51% SEM in the culture medium control (Figure 9A, p = 0.02592, asymmetric t-test). The maturation rate of OSC-IVM compared to the control was statistically significant. These results support the idea that hiPSC-derived OSCs possess functional activity in the in vitro co-culture system, as indicated by the significantly higher egg maturation rate.

Next, we investigated whether hiPSC-derived OSCs also affected the total oocyte score (TOS) results. Interestingly, the evaluation scores (Figure 9B) did not show a statistically significant difference between the two groups (asymmetric t-test, p = 0.2909), indicating that the mature MII oocyte results were of comparable morphological quality between the two IVM conditions. Overall, these data demonstrate that OSC co-culture improves maturation without negatively impacting the morphological quality of human oocytes, highlighting the potential of hiPSC-derived OSCs to be used as a high-performance system for cumulus-inlaid oocyte IVM.

Example 7. Oocyte maturation rate in OSC-IVM is superior to that of commercially available IVM systems. To further investigate whether OSC-IVM can be used as a viable system for maturing human oocytes in a clinical setting, our OSC co-culture system was compared with a commercially available IVM standard. The commercially available IVM standard was used without modification, as described in its clinical use instructions (Medicult IVM). A sibling oocyte study was conducted to compare the MII formation rate and oocyte morphological quality after 28 hours of in vitro maturation in both systems (Materials and Methods, Experiment 2). In particular, in OSC-IVM, the average MII formation rate was approximately 1.6 times higher in the overall donor, with 68% ± 6.74% mature oocytes compared to the control condition of 43% ± 7.90% (Figure 10A, p = 0.0349, asymmetric t-test). The maturation rate of OSC-IVM compared to the commercially available IVM control was statistically significant. Contrary to previous observations, co-culturing with hiPSC-derived OSCs did not affect the oocyte morphological quality between groups as measured by TOS, and the oocytes exhibited comparable visual morphological characteristics (Figure 10B, p = 0.9420, asymmetric t-test). These results indicate that OSC-IVM significantly outperforms commercially available IVM culture media in terms of MII formation rate without any apparent adverse effects on oocyte morphological quality, suggesting its beneficial application to human IVM.

Example 8. Immature cumulus encirclement oocytes matured by OSC-IVM and stimulated by simplified gonadotropin are developmentally qualified for embryogenesis. The inventors attempted to investigate the developmental qualification of oocytes treated with the OSC-IVM system by evaluating euploid blastocyst formation compared to commercially available IVM controls. Using a limited cohort of six subjects who received simplified stimulation (see Materials and Methods, Experiment 2, Tables 2 and 3), OSC-IVM-treated oocytes were examined to determine whether they were capable of fertilization, cleavage, and euploid blastocyst formation. The results of these embryos were compared with those obtained from oocytes treated with commercially available IVM medium. With OSC-IVM, the average MII formation rate of mature oocytes across the donor was approximately 1.2 times higher at 60% ± 15.4% compared to 52% ± 8% under control conditions (Figure 11A, Table 4). Mature oocytes from both treatment groups were subjected to ICSI, and fertilized oocytes were cultured until day 7 of development. OSC-assisted MIIs tended to show improved fertilization rates, cleavage rates, blastocyst rates, and rates of usable quality blastocyst formation (52%, 52%, 40%, and 28%) compared to commercially available IVM controls (38%, 38%, 24%, and 19%) as a percentage of input COC numbers (Figure 11A, Table 4). When examined on an incremental basis, OSC-IVM oocytes showed improved rates of fertilization and blastocyst formation, while division to fertilized oocytes was similar to that of commercially available IVM controls. Overall, under both conditions, the inventors found that all fertilized oocytes subsequently divided. Surprisingly, the PGT-A results show that 100% of the transplantable-quality blastocysts produced by OSC-IVM were euploid, compared to only 25% with commercially available IVM systems. While these results are not statistically significant, likely due to the small sample sizes in each group, these findings demonstrate that OSC-IVM produces high-quality, developmentally qualified, healthy, and mature oocytes. These results further reveal that OSC-IVM can produce healthy euploid embryos from simplified stimulation cycles at a higher rate than commercially available IVM conditions, strongly supporting the clinical applicability of this novel system for IVM ART.

Example 9: Materials and Methods of Examples 10-12 The inventors have demonstrated that human ovarian supporting cells (OSCs) generated from human induced pluripotent stem cells (hiPSCs) exhibit the ability to reproduce dynamic ovarian function in vitro. Here, the inventors investigate in detail the use of these OSCs as a co-culture system to better mimic the in vitro ovarian environment and to promote IVM to rescue naked immature oocytes obtained from a normal gonadotropin-stimulated cycle. The inventors demonstrate that OSC-IVM significantly improves the oocyte maturation rate compared to spontaneous maturation in a matched control medium. Furthermore, oocytes matured in combination with OSC-IVM are transcriptionally more similar to normal IVF metaphase II (MII) oocytes compared to oocytes spontaneously matured in a control medium. In summary, these findings reveal a novel approach to improve mature MII oocyte outcomes in modern IVF procedures by utilizing an optimized IVM system that better mimics the in vitro ovarian environment.

Specifically, to determine whether rescue in vitro maturation (IVM) of human oocytes can be enhanced by co-culturing with ovarian supporting cells (OSCs) derived from human induced pluripotent stem cells (hiPSCs), we collected vena cava (GV) oocytes and metaphase I (MI) oocytes from infertility patients undergoing standard ovarian stimulation and assigned them to either the control or treatment group.

Oocyte donors aged 25–45 years provided immature oocytes with informed consent, without additional selection criteria. The 24–28 hour OSC-IVM culture conditions consisted of 100,000 OSCs in a suspension culture containing human chorionic gonadotropin (hCG), recombinant follicle-stimulating hormone (rFSH), androstenedione, and doxycycline. The IVM control lacked OSCs but contained the same supplements.

The primary endpoints were MII formation rate and morphological quality. Furthermore, the location of the metaphase spindle assembly and the transcriptome profile of oocytes were evaluated compared to in vivo matured oocyte controls. OSC-IVM resulted in a statistically significant improvement in MII formation rate compared to the medium-IVM control. There was no significant difference in oocyte morphological quality between OSC-IVM and the medium-IVM control. OSC-IVM resulted in MII oocytes with no cases of spindle absence and no significant difference in placement compared to in vivo matured MII controls. OSC-IVM-treated MII oocytes showed a transcriptome maturation signature significantly more similar to IVF-MII controls compared to medium-IVM control MII oocytes.

i. Collection of Immature Oocytes 47 oocyte donors were enrolled in the study using informed consent (IRB number 20222213, Western IRB). The age range of the subjects was 25 to 45 years, with an average age of 35 years. Oocytes were collected at several in vitro fertilization and egg freezing clinics in New York City (IRB number 20222213, Western IRB). Infertility patients who provided immature oocytes that would otherwise be discarded signed informed consent provided by the clinics authorizing their use for research purposes. Patients received age-appropriate regulated ovarian stimulation using gonadotropin-releasing hormone (GnRH) analogs (agonists or antagonists), or received injections of recombinant or highly purified urinary gonadotropins (recombinant FSH, human menopausal gonadotropins), followed by administration of ovulation-inducing agents (human chorionic gonadotropin (hCG) or GnRH agonists). 34–36 hours after the injection(s) of the ovulation-inducing agent(s), oocytes were collected from the patients under conscious sedation using standard clinical procedures.

After briefly exposing the collected oocytes to hyaluronidase, adherent cumulus cells were mechanically removed by repeatedly aspirating and discharging each cumulus-oocyte complex using a small-pore pipette. The maturation of the undressed oocytes was evaluated by observing the polar body or uronic vesicle. Immature oocytes (GV or MI), which would normally be discarded, were allocated to the research study. All immature oocytes collected daily from the clinic were pooled, placed in LAG medium (Medicult, COOPERSURGICAL®) in 5 mL round-bottom tubes, and transferred from the clinic to our laboratory in a 37°C transport incubator.

In several experiments, immature (GV and MI) oocytes obtained from similar in vitro fertilization and oocyte freezing cycles were vitrified and stored at the clinic. The cryopreserved oocytes were transported from the clinic to the laboratory in liquid nitrogen and stored until use. Subsequently, the oocytes were thawed using the standard Kitasato protocol for vitrified or slow-frozen oocytes (VITROLIFE®, USA), and their maturity status was assessed as GV or MI, used for comparison of in vitro maturation conditions.

A limited number of MII oocytes, previously stored for research purposes and obtained from conventional controlled ovarian hyperstimulation, were provided as controls for this study (IVF-MII). These oocytes were transferred from the tissue vault to our laboratory and thawed using either the standard Kitasato protocol for vitrified oocytes (KITAZATO®, USA) or the slow freeze-thaw protocol for previously slow-frozen oocytes (VITROLIFE®, USA), and used for biofluorescence imaging and transcriptome analysis.

ii. Preparation of Ovarian Supporting Cells (OSCs) OSCs derived from human induced pluripotent stem cells (hiPSCs) were prepared according to the transcription factor (TF)-oriented protocol described previously. OSCs were produced in multiple batches and cryopreserved in vials containing 120,000–150,000 cells, and stored in the gas phase of liquid nitrogen in CryoStor® CS10 cell freezing medium (STEMCELL TECHNOLOGIES®). Culture dishes for oocyte maturation experiments (4+8 dishes, BIRR) were prepared the day before oocyte harvesting by placing culture medium and additional components in 100 μL droplets under mineral oil (LifeGuard, LIFEGLOBAL GROUP®). On the morning of oocyte collection, the cryopreserved OSCs were thawed at 37°C for 2-3 minutes (in a heated beads or water bath), resuspended in OSC-IVM medium, and removed residual cryoprotectant by centrifugation and pelletization twice. Equilibrated OSC-IVM medium was used for the final resuspendion. Next, to allow for culture equilibrium and culture medium preparation, 50 μL of the droplet was replaced with 50 μL of the OSC suspension 2-4 hours before adding the oocytes, and OSCs were seeded at a concentration of 100,000 OSCs per 100 μL droplet (Figure 12A). The OSCs were cultured in suspension surrounding the naked oocytes in microdroplets under oil. IVM culture was continued for 24-28 hours, after which the oocytes were removed from the culture, imaged, and collected for molecular analysis.

iii. In vitro maturation Immature oocytes were maintained in pre-incubation LAG medium (Medicult, COOPERSURGICAL®) at 37°C for 2-3 hours before being introduced into in vitro maturation conditions (either medium-IVM or OSC-IVM) after collection.
One experimental condition was considered.

Experiment (OSC Activity): The purpose of this comparison was to determine whether stimulated OSCs were active components or contributing factors to the co-culture system. For this purpose, the culture media for the experimental and control conditions were prepared according to the manufacturer Medicult's recommendations, and further supplemented with androstenedione and doxycycline (both necessary for OSC activation/stimulation) to compare maturation results in the presence or absence of OSCs in the same medium formulation (see Table 5 below).

Oocytes were collected from 56 patients and pooled into 29 independent cultures. Of the total 141 oocytes, 82 were used in OSC-IVM and 59 were used in medium-IVM. Culturing was carried out in parallel under experimental and control conditions, where possible. Immature oocytes from each donor pool were distributed evenly between the two conditions at a time, with no more than 15 cells cultured per cycle. Specifically, immature oocytes (GV and MI) were distributed as evenly and randomly as possible between the two conditions. Due to the small and highly variable number of available immature oocytes provided as discarded donations, it was often not possible to run both conditions in parallel from the same oocyte source. Immature oocytes were matured in vitro at 37°C for a total of 24–28 hours in a Trigas incubator with _CO2 adjusted to a pH of 7.2–7.3 in bicarbonate buffered medium and an _O2 level maintained at 5%.

iv. Evaluation of in vitro maturation At the end of in vitro culture, oocytes were collected from the culture dish, mechanically decontaminated, and residual OSCs were washed off. Next, the maturation status of the oocytes was individually evaluated according to the following criteria.

GV – Typically, an oocyte contains a single nucleolus within the oocyte.

MI – The oocyte lacks a nuclear vesicle, and the polar body is absent in the perivitelline space between the oocyte and the zona pellucida.

MII – The oocyte lacks a nuclear vesicle, and the polar body is present in the perivitelline space between the oocyte and the zona pellucida.

After in vitro maturation evaluation and morphological scoring, oocytes were individually imaged using digital microscopy, and the metaphase II spindle was examined by fluorescence imaging as needed. Oocytes from this study were not used for embryogenesis, embryo transfer, transplantation, or reproductive purposes.

v. Morphological Score of Oocytes Oocytes collected after IVM were individually imaged using digital photographic microscopy with an ECHO® Revolve inverted fluorescence microscope using phase-contrast imaging. The images were then evaluated according to the Total Oocyte Score (TOS) evaluation system. A trained embryologist was blinded, and oocytes were assigned scores of -1, 0, and 1 for each of the following criteria: morphology, cytoplasmic particle size, perivitelline space (PVS), zona pellucida (ZP) size, polar body (PB) size, and oocyte diameter. The zona pellucida and oocyte diameter were measured using ECHO® Revolve Microscope software and image analysis software FIJI (2.9.0/1.53t). A total quality score was assigned to the oocyte from the sum of all categories, ranging from -6 to +6, with higher scores indicating better morphological quality.

vi. Investigation of the arrangement of the metaphase II spindle and its polar body. Previously vitrified, deprived immature oocytes were thawed and equally distributed in OSC-IVM and medium-IVM conditions, then cultured for 28 hours. Additional provided MII oocytes were collected, stained, and microtubules of the meiotic spindle were visualized by fluorescence microscopy as an IVF control (IVF-MII) (Figure 14A-B). MII oocytes were incubated for 1 hour in 2 μM α-tubulin dye (ABBERIOR® Live AF610) in the presence of 10 μM verapamil (ABBERIOR® Live AF610). Next, the spindle position was visualized using a fluorescence microscope (ECHO® Revolve microscope, TxRED filter block EEX:560/40 EM:630/75 DM:585). The angle between the first polar body and the spindle of IVM oocytes was determined using FIJI software (with the vertex centered on the oocyte). This measurement was also performed in a cohort of IVF MII oocytes (n=34) as a control reference population.

vii. Cryopreservation of oocytes for subsequent molecular analysis After morphological examination was completed, oocytes were individually placed in 0.25 mL tubes containing 5 μL of Dulbecco's phosphate-buffered saline (DPBS) and rapidly frozen in liquid nitrogen. After the formation of nitrogen bubbles stopped, the tubes were stored at -80°C until subsequent molecular analysis.

viiii. Preparation of Single Oocyte Transcriptomics Library and RN Sequence Determination The RNA sequencing library was prepared using a combination of NEBNEXT® Single Cell/Low Input RNA Library Prep Kit for ILLUMINA® and NEBNEXT® Multiplex Oligos for ILLUMINA® (96 Unique Dual Index Primer Pairs) (NEB #E6440S) according to the manufacturer's instructions. Briefly, oocytes frozen in 5 μL of DPBS and stored at -80°C were thawed, lysed in lysis buffer, and then the RNA was processed for reverse transcriptase and template switching. cDNA was amplified by PCR for 12–18 cycles and then sized and purified using KAPA® Pure Beads (Roche). cDNA inputs were normalized across samples. Following fragmentation and end preparation, a NEBNEXT® Unique Dual Index Primer Pair adapter was ligated, and samples were enriched using 8 cycles of PCR. Libraries were cleaned up with KAPA® Pure Beads, quantified using Quant-iT PicoGreen dsDNA Reagent and Kit (Invitrogen), and then equal amounts of cDNA were pooled from each oocyte library. If necessary, final KAPA® Pure bead size selection was performed on the pool and quantified using the Qubit dsDNA HS kit (Invitrogen). After confirming the library size distribution (peak at approximately 325 bp) using the Bioanalyzer HS DNA kit (Agilent), the library pool was subjected to RNA sequencing analysis using the ILLUMINA® MiSeq with either the MiSeq Micro V2 (2 x 150 bp) or MiSeq V2 (2 x 150 bp) kit, according to the manufacturer's instructions.

ix. Analysis of Oocyte Transcriptomics Data ILLUMINA® sequencing files (bcl files) were converted to fastq read files using Illumina® bcl2fastq (v2.20) software, deployed via BaseSpace, using standard parameters for low-input RNA-seq of individual oocytes. Gene transcription counts of the low-input RNA-seq data were aligned to the Homo sapiens GRCH38 (v2.7.4a) genome using STAR (v2.7.10a) to generate gene count files, which were annotated using ENSEMBL. Gene counts were compiled into a sample gene matrix file (h5). Computer analysis was performed using data structures and methods from the Scanpy (v1.9.1) package as a basis. The number of gene transcripts was normalized to 10,000 per sample and converted to log-log (ln) + 1. Principal component analysis was performed using the Scanpy package method, focusing on 30 PCA components. Integration and project (batch) correction were performed using BBKNN. Projection to two dimensions was performed using the Uniform Manifold Projection (UMAP) method. Cluster discovery was performed using the Ledien method with a resolution of 0.5.

To define the predictive transcriptome profile of normal MII oocytes, a cohort of in vivo matured IVF-MII samples (n=34) was used as a baseline, and differential gene expression was used to compare this baseline set with a subset of GV cells after IVM. The top 50 differentially expressed genes were collected for each comparison using both the Wilcoxon rank-sum test and cosine similarity-based marker gene identification (COSG) methods. No other MI or MII oocyte sets were used as baselines, as these marker genes were developed to ensure minimal bias in other MII transcriptome profiling. This method generated MII signature marker gene expression profiles for GV and IVF cells that failed to mature. Cells were scored for each marker gene set using the Scanpy gene marker scoring method.

To visualize the cells identified by the inventors in the signature marker space, marker scores were plotted as a two-dimensional space. Next, the space was manually divided into quadrants based on morphological maturation results and Leiden clusters. Clusters were annotated considering their distribution in the score space and their presence in each quadrant, relating IVM maturation results to the overall transcriptome profile.

x. Data Analysis and Statistics The egg maturation results were analyzed using the Python statistical packages pandas (1.5.0), scipy (1.7.3), and statsmodels (0.13.2). Maturation rates by donor group were analyzed by linear regression according to the IVM environment, either OSC-IVM or medium-IVM. T-test statistics were calculated by comparing OSC-IVM and medium-IVM, and then used to calculate p-values using Welch's correction for unequal variances. One-way ANOVA was used to compare two or more groups in the spindle apparatus position analysis. Chi-squared analysis was used to compare the population composition of the Leiden group in transcriptome analysis of three sample conditions. Bar graphs show the mean for each population, and error bars represent the standard error (SEM) of the mean.

Example 10. Human PSC-derived OSCs effectively promote human oocyte maturation in a co-culture system with deprived oocytes. It has been demonstrated that hiPSC-derived OSCs are mainly composed of granulosa cells and ovarian stromal cells. These OSCs respond to in vitro hormonal stimulation, i.e., FSH, to produce growth factors and steroids and express adhesion molecules necessary for interaction with oocytes and cumulus cells. To investigate whether hiPSC-derived OSCs have the function of promoting human oocyte maturation in vitro, a co-culture system was established with these cells and newly collected deprived immature oocytes as a method for rescuing immature deprived oocytes, and the maturation rate was evaluated after 24-28 hours (Figure 12).

First, we investigated whether OSC-IVM affected the maturation rate of dehydrated oocytes compared to oocytes stored in medium-IVM conditions containing the same culture medium and all supplements but without OSC. The maturation rate was determined for each oocyte culture group under each condition. Surprisingly, oocytes treated with OSC-IVM showed a significantly improved maturation rate (approximately 1.7 times). Specifically, the maturation rate of the OSC-IVM group was 62% ± 5.57% SEM, compared to 37% ± 8.96% SEM in the medium-IVM group (Figure 13A, p = 0.0138, asymmetric t-test). Furthermore, the morphological quality of MII oocytes obtained under both IVM conditions was evaluated by assessing the total oocyte score (TOS). No significant difference was observed between the two groups (Figure 13B, p = 0.5725, asymmetric t-test), suggesting that in vitro maturation of dehydrated oocytes does not affect the morphological characteristics of MII cells. Overall, these data demonstrate that OSC co-culture improves the oocyte maturation rate of human oocytes without adverse morphological quality compared to spontaneous maturation observed in control IVM medium, highlighting the potential of using hiPSC-derived OSCs to rescue immature, naked oocytes produced by in vitro fertilization procedures.

Example 11. OSC-IVM promotes high-quality assembly of the meiotic spindle in IVM oocytes. Previous studies have shown that the assembly of the meiotic spindle, more specifically both the presence and angle of the spindle relative to PB1, are important indicators of oocyte quality related to fertilization and developmental ability, with the presence of a spindle with a small angle to PB1 indicating improved quality. As a comparative measure of oocyte quality, we attempted to determine the relative positions of the meiotic spindle apparatus and the first polar body in OSC-treated oocytes compared to MII oocytes collected from in vitro fertilization cycles (IVF-MII) (Figure 14). Oocytes that spontaneously matured under medium-IVM conditions were also included as a control (Figure 14A). The spindle angle did not differ significantly between conditions (MII OSC-IVM: 22 ⁰ ±5.2 SEM, MII medium-IVM: 15 ⁰ ±5.7 SEM, IVF-MII: 41 ⁰ ±8.3 SEM, p = 0.1155, ANOVA), suggesting that the spindle position is not impaired by in vitro maturation of denatured oocytes. Interestingly, the only condition in which no cases of spindle absence were observed was the condition containing OSC-IVM-derived MII oocytes (Figure 14B). Further research is needed to validate this observation, but it may suggest the formation of high-quality oocytes. Overall, these results indicate that MII oocytes matured in vitro with OSCs retain spindle angle values comparable to those of MII oocytes directly harvested from IVF-treated cells. This suggests that, based on these parameters, IVM applied to rescue vegetatively neutered immature oocytes does not adversely affect oocyte quality.

Example 12. OSC-IVM promotes the maturation of MII oocytes with high transcriptome similarity to in vivo matured MII oocytes. Single oocyte transcriptome analysis was performed to further compare the quality and maturity of OSC-IVM oocytes with cohorts of IVF-MII control oocytes and medium-IVM oocytes. Transcriptome analysis provides a holistic view of oocyte gene expression and is a powerful means of representing the state, function, and general characteristics of cells. It is initiated by combining datasets containing 1) naked immature oocytes after 24-28 hours of co-culture with OSC (OSC-IVM), 2) naked immature oocytes maintained in in vitro maturation medium control (Media-IVM), and 3) MII oocytes collected from a normal IVF cycle (IVF-MII). Next, UMAP plots were generated, and individual oocytes were annotated with conditions (OSC-IVM, medium-IVM, and IVF-MII) and maturation outcomes (GV, MI, MII) (Figure 15A). This analysis revealed that maturation status is the primary factor in oocyte segregation across the entire transcriptome space, suggesting that transcriptional profiles are excellent predictors of oocyte maturation status. MII oocytes are mainly represented in the large cluster in the upper right half of the plot (Figure 15A Maturation). GV oocytes are mainly projected into the small cluster in the lower left half of the plot. Thus, classification in UMAP is a combination of two projection dimensions. As expected, MII oocytes collected from IVF (IVF-MII) show close grouping with MII from both OSC-IVM and medium-IVM. Similarly, GV from OSC-IVM and medium-IVM show close proximity to each other and are farther away from MII oocytes. In contrast, MI oocytes are scattered between both groups, which is likely an intermediate maturation stage and may be a result of their very small number compared to the other two maturation stages (GV and MII).

Next, to assess the quality of rescued/matured MII cells in vitro, reference transcriptome signatures of matured MII oocytes were generated using standard methods. To establish a standard, genetic scores for the IVF MII maturation signature were created using MII oocytes taken from conventional ovarian hyperstimulation IVF samples (IVF-MII). In parallel, genetic scores for the unmatured signature of IVM GV were generated using stalled GV obtained from IVM conditions (OSC-IVM and medium-IVM) (Figure 15B). These two genetic signatures were used to capture the relative positive control of IVM, i.e., successful maturation outcomes like IVF, and the negative control of IVM, i.e., oocytes that stall as GV.

To better understand the subtle differences in transcriptomes among mature MII oocytes, we used the Leiden algorithm to further subcluster our samples into groups sharing closer transcriptome profiles. Three clusters (0, 2, and 3) were identified within the MII oocyte population. One cluster (1) consisted almost entirely of GVs. As expected, the GV maturation signature was strongly expressed in cluster 1. Similarly, the MII maturation signature included MIIs from both IVF and IVM, which accounted for a higher proportion in clusters 0 and 2. Therefore, we designated cluster 1 as representing the non-mature transcriptome profile of (GV). Clusters 0 and 2, on the other hand, represent profiles similar to the IVF MII maturation transcriptome profile. Interestingly, cluster 3 showed lower expression for the non-mature signatures of both IVF MIIs and IVM GVs. This may indicate a transitional state between immature and mature development, where neither signature is significantly upregulated, or it may result from stagnation, shutdown, or oocyte dysfunction of cellular activity.

In Figure 15C, the inventors evaluate the quality of individual oocytes against their IVF-MII maturation signature (y-axis) and IVM-GV non-mature signature (x-axis). For visual clarity, the signature dimension plot is divided into labeled quadrants to help distinguish between classification groups. As expected, the inventors found that most oocytes morphologically classified as GV clustered in the lower right quadrant (IV), with high scores for the non-mature GV signature and low scores for the IVF-MII maturation signature. In contrast, individual oocytes from the IVF-MII condition clustered in the upper left quadrant (I) (approximately 91%), with high scores for the MII maturation signature and low scores for the non-mature GV signature. Notably, OSC-IVM MII (blue + symbol) was found almost entirely (approximately 79%) in the upper left quadrant (I) along with IVF-MII oocytes, suggesting strong transcriptome similarity between these two groups. In contrast, MII from medium-IVM was more frequently located in the lower left quadrant (III) (approximately 46%), showing lower scores for both the MII maturation signature and the GV maturation signature. Interestingly, this lower left quadrant (III) largely contained cells derived from cluster 3, which were morphologically classified as MII despite their weak MII maturation signature. This morphological and transcriptome divergence suggests that these oocytes may be in a low-activity state, in a pre-maturation transitional or pending state. To evaluate confounding variables in our transcriptome analysis, we assessed the expression of cell cycle, apoptosis, and oxidative stress genes. No significant patterns were detected, indicating that the oocytes were neither biased nor stressed (Figure 14). This observation suggests that MII oocytes derived from OSC-IVM are transcriptionally more similar to oocytes under IVF-MII conditions compared to medium-IVM controls.

Finally, to determine the proportion of MII oocytes with a strong IVF-MII maturation signature under each experimental condition, we calculated the proportion of cells in clusters 0 and 2 identified as containing oocytes with an "IVF-like MII" signature (Figure 15D). As expected, the majority (91%) of MII oocytes from the IVF-MII condition were classified into clusters 0 and 2. Co-culturing with OSCs generated 79% of MII oocytes with an "IVF-like MII" profile (clusters 0 and 2), as the positive effect on maturation continues to be demonstrated. In contrast, only 56% of MII oocytes obtained from the medium-IVM condition were present in the "IVF-like MII" profile clusters. This population distribution is significantly different from random ( ^χ² test, α = 0.00632). Overall, the inventors conclude that OSC-IVM supports the formation of MII oocytes with high transcriptome similarity to IVF-mature MII oocytes, and strongly support the use of this novel approach to rescue veiled immature oocytes from IVF treatment.

Example 13. Granulosa cells support germ cell development within ovaloids. Current methods for inducing and culturing human primordial germ cell-like cells (hPGCLCs) generate immature, pre-migration primordial germ cells (PGCs) lacking expression of gonadal PGC markers such as DAZL. During fetal development, PGCs mature through interaction with gonadal somatic cells, and DAZL plays a crucial role in the downregulation of pluripotency factors and involvement in gamete formation. This process has recently been replicated in vitro using mouse fetal ovarian somatic cells, allowing hPGCLCs to develop to an oogonia-like stage. We hypothesized that in vitro-derived human granulosa cells might play a similar role, potentially eliminating interspecies developmental discrepancies. Therefore, we combined granulosa cells and hPGCLCs to form ovarian organoids, which we named ovaloids.

To generate ovaloids, these two cell types were aggregated in low-binding U-bottom wells and subsequently transferred to gas-liquid interface transwell culture. For comparison, mouse embryonic ovarian somatic cells were isolated according to the previously described protocol and aggregated with hPGCLCs. Immunofluorescence revealed that expression of the maturation marker DAZL began in a subset of OCT4+ hPGCLCs on day 4 of co-culture with hiPSC-derived granulosa cells (Figure 16A). In contrast, potent DAZL expression in co-culture with mouse cells was not observed until day 32 (Figure 16B), and weaker expression was observed on day 26. Similarly, in a previous study using the same hPGCLC strain and anti-DAZL antibody, DAZL expression was observed only after 77 days of co-culture with mouse embryonic testicular somatic cells.

The proportion of DAZL+ cells reached its peak on day 14 in human oocytes and on day 38 in mouse oocytes (Figure 16C). In human oocytes, the proportion of OCT4+ cells decreased after day 8. In mouse oocytes, the proportion of OCT4+ cells also decreased over time. In human oocytes, DAZL+OCT4- cells appeared in addition to DAZL+OCT4+ cells on day 16 (Figure 16E), and after day 38, the total number of DAZL+ cells exceeded the number of OCT4+ cells (Figure 16C). Downregulation of OCT4 in DAZL+ oogonia occurs in vivo during the second trimester of human fetal ovarian development, but the transition of DAZL to cytoplasmic localization, which has been reported to occur at this stage, was not observed. Expression of TFAP2C, an early PGC marker, decreased during ovarian culture (Figure 16D) and almost completely disappeared by day 8. In contrast, SOX17 expression was still detectable on day 8, and OCT4 and DAZL expression continued until day 54 (Figure 16A, C).

This system allowed hPGCLCs to rapidly develop to the gonadal stage, but long-term culture reduced the number of germ cells in hiPSCs and mouse-derived obaroids (Figure 16C), indicating that either of these cells were dying or differentiating into other strains. Unlike mouse-derived obaroids, hiPSC-derived obaroids cultured in Transwell gradually flattened and widened, and by day 38, most of them had disintegrated.

Nevertheless, these long-term experiments observed the formation of empty follicle-like structures composed of cubic AMHR2+FOXL2+granulosa cells (Figure 17A), suggesting that TF can promote follicle formation even in the absence of oocytes. The formation of follicle-like structures was first observed on day 16 (Figure 16E), and by day 26, the largest of these structures had grown to a diameter of 1–2 mm (Figure 17B). By day 70, the follicles had developed into follicles of various sizes, mainly small, simple follicles (Figure 17C), but also including antral follicles (Figure 17D). The cells on the outer surface of the follicles showed NR2F2-positive staining, a marker for ovarian stromal and follicular membrane cells (Figures 17C, D).

To further investigate gene expression in hPGCLCs and somatic cells in this system, scRNA-seq was performed on ovaloids isolated on days 2, 4, 8, and 14 of culture, and the cells were clustered according to gene expression. As expected, the largest cluster (cluster 0) contained cells expressing granulosa markers such as FOXL2, WNT4, and CD82 (Figure 17A, B). Cells expressing secondary/antral granulosa cell markers such as FSHR and CYP19A1 were also found within this cluster, but in much smaller numbers. A smaller cluster (cluster 1) expressing the ovarian stromal marker NR2F2 was also present. NR2F2 is expressed by both stromal and follicular cells, but the cells in cluster 1 did not express 17α-hydroxylase (CYP17A1). This indicates that they were unable to produce androgens and were not follicular cells.

Clusters of hPGCLCs expressing marker genes such as CD38, KIT, PRDM1, TFAP2C, PRDM14, NANOG, and POU5F1 were also observed. In particular, X-chromosome lncRNAs XIST, TSIX, and XACT were all expressed more highly in hPGCLCs compared to other clusters (approximately 80-fold, 20-fold, and 2900-fold on average, respectively) (Figure 18B). This indicates that in hPGCs, the X reactivation process associated with the high expression of both XIST and XACT is initiated in hPGCLCs. The X-chromosome HPRT1 gene, known to be more highly expressed in cells with two active X chromosomes, was also upregulated by approximately three times.

Next, the in vitro-generated obaroids were compared to a reference atlas of human fetal ovarian development. Using Scanpy Ingest, the samples were integrated into the atlas, and each cell was annotated with the cell type closest to the in vivo data (Figure 18C). The follicles consisted primarily of granulosa, gonadal mesenchyme, and pregranulosa lineages (Figure 18D), with a small amount of coelomic epithelium. The proportion of granulosa cells increased from day 2 to day 8, which may indicate maturation of the somatic cell population. As expected, nerve cells, immune cells, smooth muscle cells, and erythrocytes present in the fetal ovary were completely absent from our obaroids. Epithelial cells, endothelial cells, and perivascular cells were detected, but at very low frequencies (less than 1%), which may indicate a low rate of off-target differentiation.

Furthermore, during the experiment, we examined fractions of the entire germ cell population and fractions of cells expressing the gonadal germ cell markers DAZL and DDX4 (Figure 18D). Germ cell populations were defined based on the integration of fetal ovarian atlases. This population increased from day 2 to day 4, but then decreased. In comparison, the proportions of DAZL+ and DDX4+ cells also increased from day 2 to day 4, but remained nearly constant from day 4 to day 14 (Figure 18D). Differential gene expression analysis and gene ontology enrichment were performed by comparing DAZL+ and DAZL- cells. Upregulated genes (log2fc>2, n=221) were most prevalent for terms related to general developmental processes, but also included terms related to adhesion and migration (e.g., "amoeba-type cell migration"), as well as terms related to the development of the reproductive system. The downregulated genes (log2fc < -2, n = 6451) were strongly associated with metabolic processes and mitotic cells. These data suggest that DAZL+ cells in our OVA-roid downregulate their metabolism and growth. This is consistent with the known role of DAZL in suppressing PGC growth.

Example 14. Preclinical trials Preclinical trials of the OSC-IVM system were conducted using a control with matched cell culture media in a sibling oocyte test. This test included both human naked immature oocytes collected after standard gonadotropin stimulation and intact immature COCs collected after minimal gonadotropin stimulation. The control condition contained the same culture medium formulation as the OSCs-IVM condition, with the only difference between the conditions being the presence of OSCs in the OSC-IVM. The results showed that the OSC-IVM system statistically significantly improved the oocyte maturation rate, as determined by the presence of polar bodies, by approximately 15% in naked oocytes with standard treatment (Figure 19A) and approximately 17% in intact COCs with minimal stimulation (Figure 19B). OSC-IVM was compared to the clinically approved Medicult-IVM system, which is marketed for use in intact COCs after minimal stimulation.

In approved sibling oocyte studies, OSC-IVM statistically significantly improved oocyte maturation rates by approximately 28% on average per study donor compared to Medicult-IVM (Figure 19C).

It was also determined whether OSC co-culture improves oocyte quality. While there is no widely accepted method for determining "oocyte quality," research has shown that certain morphological and molecular characteristics can be used to predict oocyte quality because they correlate with improved embryogenesis and birth rates in IVF. One such measure is the Total Oocyte Score (TOS), which is derived from a manual qualitative assessment of six morphological characteristics of mature oocytes: oocyte size, zona pellucida size, color/shape, cytoplasmic grain size, polar body quality, and PVS quality. Another indicator of quality is spindle assembly arrangement. This has been shown to be a reliable indicator of oocyte quality by measuring the angle between polar body 1 (PB1) and the spindle, with a decrease in angle correlating with improved oocyte quality. Finally, certain genetic markers identified by transcriptome analysis correlate with oocyte quality. These assess indicators such as oxidative stress, embryogenetic capacity, and DNA damage. Here, all three indicators were employed to determine whether OSC-IVM could improve oocyte quality compared to a culture-adjusted control. Using a limited number of deprived immature oocytes and IVF in vivo MII controls, it was determined that the OSCs described herein tended to improve oocyte quality compared to the culture-adjusted control and showed similarity to in vivo MII oocytes in terms of morphological quality (Figure 20A). Similarly, OSC-IVM showed an average reduction in the angle between PB1 and the spindle compared to the culture-adjusted control and IVF in vivo MII, and no cases of spindle deficiency were observed in OSC-IVM MII (Figure 20B). Furthermore, differential gene expression analysis (DGEA) revealed that
OSCs-IVM oocytes showed high similarity to MII oocytes in the body, demonstrating that they are expected to express major embryonic potential genes (Figure 20C-D).

Furthermore, to investigate the toxicity of OSC co-culture, both human and porcine animal models were studied. Using a human preclinical model, OSCs-IVM conditions were performed, and oocyte results were evaluated, specifically oocytes considered "degraded," meaning they were in a state of rapid apoptosis or cell death. The results showed that OSC-IVM did not result in a significant increase in oocyte degradation rates in human oocytes compared to Medicult-IVM medium alone (Figure 21A). Similarly, the ability of mature porcine oocytes to form blastocysts in the presence of the OSC-IVM product was also evaluated. As shown in the figure, the inventors were able to successfully fertilize and generate blastocysts from porcine oocytes under OSC-IVM conditions (Figure 21B). While these porcine studies were not designed to test efficacy, they demonstrate that OSCs-IVM is non-toxic to oocytes and does not interfere with embryogenesis.

The above was a detailed description of exemplary embodiments of the present invention. Various modifications and additions can be made without departing from the spirit and scope of the invention. To provide numerous feature combinations in related new embodiments, features from each of the various embodiments described above may be combined as needed with features from other described embodiments. Furthermore, although several distinct embodiments are described above, what is described herein is merely illustrative of the application of the principles of the present invention. Furthermore, while certain methods shown herein may be illustrated and/or described to be performed in a specific order, that order can be significantly altered within the ordinary art to achieve the methods, systems, apparatus, and software described herein. Therefore, this description is intended to be interpreted as illustrative only and not intended to limit the scope of the invention.

Exemplary embodiments have been disclosed above and illustrated in the accompanying drawings. As will be apparent to those skilled in the art, various modifications, omissions, and additions can be made to those specifically disclosed herein without departing from the spirit and scope of the present invention.

Specific Embodiments Other Embodiments All publications, patents, and patent applications referenced herein are incorporated by reference, with each individual publication or patent application being specifically and individually incorporated by reference.

While the present invention has been described in relation to its specific embodiments, it goes without saying that the invention is subject to further modification, and this application covers all changes, uses, or modifications, including developments from the invention that generally adhere to the principles of the invention, derive from known or customary practices in the art to which the invention belongs, and are applicable to the essential features described herein and subject to the claims.

Other embodiments are described in the claims.

Claims (319)

An in vitro composition comprising one or more ovarian supporting cells (OSCs) and one or more diluents or excipients, wherein the composition optionally promotes the maturation of one or more oocytes. The composition according to claim 1, wherein the one or more OSCs comprise one or more granulosa cells. The composition according to claim 2, wherein one or more OSCs express FOXL2, AMHR2, CD82, or any combination thereof. The composition according to any one of claims 1 to 3, wherein one or more OSCs express one or more genes selected from GJA1, MDK, BBX, HES4, PBX3, YBX3, BMPR2, CD46, COL4A1, COL4A2, LAMC1, ITGAV, and ITGB. The composition according to claim 3 or 4, wherein one or more OSCs further express one or more genes selected from FOXO1, CDH1, CYP19A1, RARRES2, NOTCH2, NRG1, BMPR1B, EGFR (ERBB1), and ERBB4. The composition according to claim 3 or 4, wherein one or more OSCs further express one or more genes selected from RARRES2, NOTCH2, NOTCH3, ID3, and BMPR2. The composition according to claim 3 or 4, wherein one or more OSCs further express the gene CDH2 and/or NOTCH2. The composition according to any one of claims 1 to 7, wherein one or more OSCs do not exhibit significant expression of RARRES2. The composition according to any one of claims 1 to 8, wherein one or more OSCs express NR2F2. The composition according to any one of claims 1 to 9, wherein one or more OSCs include ovarian stromal cells. The composition according to any one of claims 1 to 10, wherein one or more OSCs include granulosa cells and ovarian stromal cells. The composition according to any one of claims 1 to 11, wherein one or more OSCs contain more than 60% granulosa cells, more than 70% granulosa cells, more than 80% granulosa cells, more than 90% granulosa cells, or more than 95% granulosa cells. The composition according to any one of claims 1 to 12, wherein the one or more OSCs are obtained by differentiation of a population of iPSCs, and optionally, the iPSCs are hiPSCs. The aforementioned hiPSC is the following transcription factor (i) RUNX2,
(ii) NR5A1,
(iii) GATA4,
(iv) FOXL2,
(v) Any combination of two of the transcription factors,
(vi) any combination of three of the transcription factors, or (vii) one or more combinations of all four of the transcription factors, according to claim 13. The composition according to claim 13 or 14, wherein the expression or overexpression of one or more transcription factors is induced by a doxycycline-responsive transcriptional regulator. The composition according to any one of claims 13 to 15, wherein the hiPSC comes into contact with a Wnt/β-catenin pathway activator. The composition according to claim 16, wherein the Wnt/β-catenin pathway activator is a Rho-related protein kinase (ROCK) inhibitor, a glycogen synthase kinase-3 (GSK3) inhibitor, or a combination thereof. The composition according to any one of claims 1 to 17, wherein at least one of the one or more OSCs is enclosed. The composition according to claim 18, wherein one or more OSCs are encapsulated in alginate, laminin, collagen, vitronectin, chitosan, hyaluronic acid, poly-D-lactone, or any mixture thereof. The composition according to claim 19, wherein one or more OSCs are encapsulated in laminin, and optionally, the laminin is laminin-521. The system or composition according to claim 19 or 20, wherein one or more OSCs are encapsulated in vitronectin. The composition according to any one of claims 1 to 21, wherein the one or more OSCs have the expression of one or more genes associated with pluripotency that is low or undetectable compared to iPSCs. The composition according to claim 22, wherein one or more genes related to pluripotency include NANOG. The composition according to claim 23, wherein one or more genes related to pluripotency include POU5F1. The composition according to any one of claims 1 to 24, wherein at least a portion of the OSC produces one or more growth factors. The composition according to claim 25, wherein the one or more growth factors include insulin-like growth factor (IGF), stem cell factor (SCF), epidermal growth factor (EGF), leukemia suppressor factor (LIF), vascular endothelial growth factor (VEGF), bone morphogenetic protein (BMP), C-type natriuretic peptide (CNP), or any combination thereof. The composition according to claim 25 or 26, wherein at least a portion of the one or more growth factors is secreted. The composition according to any one of claims 1 to 27, wherein one or more OSCs generate one or more steroids. The composition according to claim 28, wherein the one or more steroids include estradiol, progesterone, or a combination thereof. The composition according to claim 28 or 29, wherein one or more steroids are produced in response to hormonal stimulation. The composition according to claim 30, wherein the hormonal stimulation includes FSH, androstenedione therapy, or a combination thereof. The composition according to any one of claims 28 to 31, wherein at least a portion of the one or more steroids is secreted. The composition according to any one of claims 1 to 32, wherein one or more OSCs are cryopreserved. The composition according to any one of claims 1 to 33, further comprising an in vitro maturation (IVM) medium. The composition according to claim 34, wherein the IVM medium comprises a cell culture medium. The composition according to claim 34 or 35, wherein the IVM medium comprises Medicult-IVM medium. The IVM medium comprises one or more supplements, as described in any one of claims 34 to 36. The one or more supplements mentioned above are
(i) Human serum albumin (HSA) at a concentration of approximately 5 to 15 mg/mL (optional), and at a concentration of 10 mg/mL (optional).
(ii) Recombinant follicle-stimulating hormone (rFSH) at a concentration of approximately 70 mIU/mL to approximately 80 mIU/mL, and at an optional concentration of 75 mIU/mL.
(iii)Optionally, human chorionic gonadotropin (hCG) at concentrations of approximately 95 mIU/mL to approximately 105 mIU/mL, and optionally at a concentration of 100 mIU/mL.
(iv)Optionally, androstenedione at concentrations of approximately 495 ng/mL to approximately 505 ng/mL, and further optionally, at a concentration of 500 ng/mL.
(v) Optionally, doxycycline at concentrations of approximately 0.5 μg/mL to approximately 1.5 μg/mL, and optionally at a concentration of 1 μg/mL.
The composition according to claim 37, or comprising any combination of the one or more supplements. The composition according to any one of claims 1 to 38, wherein the one or more oocytes are collected from a donor. The composition according to claim 39, wherein the donor is approximately 19 to 45 years of age. The subject is subjected to ovarian stimulation, as described in claim 39. The composition according to claim 41, wherein the ovarian stimulation comprises treatment with gonadotropin-releasing hormone (GnRH). The composition according to claim 42, wherein the ovarian stimulation comprises treatment with one or more GnRH analogs. The composition according to claim 43, wherein one or more GnRH analogs are GnRH agonists or antagonists. The ovarian stimulation comprises one or more ovulation-inducing agents, according to any one of claims 41 to 44. The composition according to claim 45, wherein one or more ovulation-inducing agents include human chorionic gonadotropin (hCG). The composition according to claim 45 or 46, wherein the one or more ovulation-inducing agents comprise a GnRH agonist, and optionally, the GnRH agonist is leuprolide. The ovarian stimulation is the composition according to any one of claims 41 to 47, comprising FSH treatment. The ovarian stimulation is a composition according to any one of claims 41 to 47, wherein the ovarian stimulation does not include FSH treatment. The FSH treatment is the composition according to claim 48, comprising 300 international units (IU) to 700 IU of FSH. The FSH treatment is the composition according to claim 50, comprising 400 IU to 600 IU of FSH. The composition according to any one of claims 48, 50, and 51, wherein the FSH treatment comprises one, two, three, or more FSH injections, and optionally, the FSH treatment comprises multiple injections, each injection comprising a dose of FSH of about 100 IU to about 200 IU. The composition according to any one of claims 41 to 52, wherein the ovarian stimulation further comprises the administration of clomiphene citrate, optionally, the clomiphene citrate is administered in one or more injections for a maximum of eight days, and optionally, each injection comprises a dose of 50 mg to 150 mg. The ovarian stimulation further comprises one or more hCG-inducing agents, according to any one of claims 41 to 53. The composition according to claim 54, wherein the one or more hCG-inducing agents comprise 2,500 IU to 10,000 IU of hCG or about 200 μg to about 700 μg of hCG, and optionally, the hCG is administered to the subject in a dose of about 400 μg to about 600 μg, and optionally, the hCG is administered to the subject in a dose of about 500 μg per dose. The composition according to any one of claims 1 to 55, wherein one or more oocytes are located within a cumulus-oocyte complex (COC). The composition according to any one of claims 1 to 56, wherein the one or more oocytes comprises one or more deprived immature oocytes. The composition according to claim 57, wherein all of the one or more oocytes are developed immature oocytes. The composition according to any one of claims 1 to 56, wherein one or more oocytes are not veiled. The composition according to any one of claims 1 to 59, wherein the one or more oocytes include one or more oocytes containing nucleus vesicles (GVs). The composition according to any one of claims 1 to 60, wherein one or more of the oocytes include one or more oocytes in metaphase I (MI). The composition according to any one of claims 1 to 61, wherein one or more of the oocytes include one or more oocytes in metaphase II (MII). The composition according to any one of claims 1 to 62, wherein at least a portion of the one or more oocytes comprises one or more previously vitrified oocytes. The composition according to any one of claims 1 to 63, wherein at least a portion of the one or more oocytes comprises one or more previously cryopreserved oocytes. The composition according to any one of claims 1 to 64, wherein the one or more oocytes are co-cultured with the one or more OSCs. The composition according to claim 65, wherein, before and/or after the co-culture, one or more oocytes are evaluated for parameters selected from the group consisting of total oocyte score, maturation rate of oocytes from the GV to MII stage, maturation rate of oocytes from the GV to MI stage, maturation rate of oocytes from the MI to MII stage, mean oocyte shape, mean oocyte size, mean oocyte quality, mean perivitelline space (PVS) quality, mean zona pellucida (ZP) quality, and mean polar body quality. The composition according to claim 66, wherein the one or more co-cultured oocytes have substantially the same morphological qualities as oocytes matured in vivo, and the morphological qualities include oocyte size, oocyte zona pellucida size, oocyte color, oocyte shape, oocyte cytoplasm particle size, oocyte polar body quality, and oocyte PVS quality. The composition according to claim 66 or 67, wherein the one or more co-cultured oocytes have an improved maturation rate compared to oocytes in a culture that does not contain the one or more OSCs. The composition according to any one of claims 66 to 68, wherein the one or more co-cultured oocytes have a metaphase II spindle that is substantially in the same position as that of a mature oocyte in the body. The composition according to any one of claims 65 to 69, wherein the one or more co-cultured oocytes have substantially the same transcriptome profile as oocytes matured in the body. The composition according to any one of claims 65 to 70, wherein the one or more oocytes are co-cultured with the one or more OSCs for about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, or about 36 hours. The composition according to claim 71, wherein one or more oocytes are co-cultured with one or more OSCs for approximately 24 to 28 hours. The composition according to claim 71 or 72, wherein the one or more oocytes co-cultured with the one or more OSCs form one or more undifferentiated germ cells after contact with one or more mature spermatids. The composition according to any one of claims 65 to 73, wherein the one or more oocytes are co-cultured in direct contact with the one or more OSCs. The composition according to any one of claims 65 to 74, wherein one or more oocytes do not come into direct contact with the OSC. The culture system is suspension culture, according to any one of claims 65 to 75. The culture system is an adherent culture, according to any one of claims 65 to 75. A method for culturing oocytes, wherein one or more oocytes are co-cultured with one or more ovarian supporting cells (OSCs). A method for preparing one or more oocytes previously collected from a human subject for use in assisted reproductive technology (ART) procedures, the method comprising co-culturing the one or more oocytes with one or more OSCs. A method for generating mature oocytes for use in ART treatment, the method comprising co-culturing one or more oocytes previously collected from a human subject with a population of ovarian supporting cells differentiated from one or more iPSCs. A method for inducing oocyte maturation in vitro, comprising co-culturing one or more oocytes with a population of ovarian supporting cells differentiated from one or more iPSCs, wherein the co-culturing is optionally performed for approximately 6 to 120 hours. A method for generating mature oocytes for use in ART treatment, wherein the method is:
(a) Differentiating one or more iPSCs to generate one or more OSCs,
(b) Collect one or more immature oocytes from the subject,
(c) The method comprising co-culturing one or more oocytes with one or more OSCs to generate one or more mature oocytes. A method for promoting the maturation of oocytes of a subject who has undergone ART treatment and has previously been administered one or more follicle-inducing agents during the follicle induction period, wherein the method is:
(a) Collecting one or more immature oocytes from the subject,
(b) Co-culturing one or more oocytes with one or more OSCs differentiated from iPSCs, thereby generating one or more mature oocytes,
(c) The method comprising isolating one or more mature oocytes. The method according to any one of claims 78 to 83, wherein, before and/or after the co-culture, one or more oocytes are evaluated for parameters selected from the group consisting of total oocyte score, oocyte maturation rate from GV to MII stage, oocyte maturation rate from GV to MI stage, oocyte maturation rate from MI to MII stage, mean oocyte shape, mean oocyte size, mean oocyte quality, mean perivitelline space (PVS) quality, mean zona pellucida (ZP) quality, and mean polar body quality. The method according to claim 84, wherein the one or more co-cultured oocytes have substantially the same morphological qualities as oocytes matured in vivo, and the morphological qualities include oocyte size, oocyte zona pellucida size, oocyte color, oocyte shape, oocyte cytoplasmic grain size, oocyte polar body quality, and oocyte PVS quality. The method according to any one of claims 84 or 85, wherein the one or more co-cultured oocytes have an improved maturation rate compared to oocytes in a culture that does not contain the one or more OSCs. The method according to claim 86, wherein the one or more co-cultured oocytes have an improved maturation rate compared to oocytes matured in the body. The method according to any one of claims 84 to 87, wherein the one or more co-cultured oocytes have a metaphase II spindle that is substantially in the same position as that of a mature oocyte in the body. The method according to any one of claims 78 to 87, wherein the one or more co-cultured oocytes have substantially the same transcriptome profile as oocytes matured in the body. The method according to any one of claims 78 to 89, wherein one or more OSCs express FOXL2, AMHR2, CD82, or any combination thereof. The method according to any one of claims 78 to 90, wherein one or more OSCs express one or more genes selected from GJA1, MDK, BBX, HES4, PBX3, YBX3, BMPR2, CD46, COL4A1, COL4A2, LAMC1, ITGAV, and ITGB. The method according to claim 90 or 91, wherein one or more OSCs further express one or more genes selected from FOXO1, CDH1, CYP19A1, RARRES2, NOTCH2, NRG1, BMPR1B, EGFR (ERBB1), and ERBB4. The method according to claim 90 or 91, wherein the one or more OSCs further express one or more genes selected from RARRES2, NOTCH2, NOTCH3, ID3, and BMPR2. The method according to claim 90 or 91, wherein one or more OSCs further express the CDH2 gene and/or the NOTCH2 gene, but do not exhibit significant expression of the RARRES2 gene. The method according to any one of claims 78 to 94, wherein one or more OSCs include granulosa cells. The method according to any one of claims 78 to 90, wherein one or more OSCs express NR2F2. The method according to any one of claims 78 to 90 and 96, wherein the one or more OSCs include ovarian stromal cells. The method according to any one of claims 78 to 97, wherein the one or more OSCs include granulosa cells and ovarian stromal cells. The method according to any one of claims 78 to 98, wherein one or more OSCs comprises more than 60% granulosa cells, more than 70% granulosa cells, more than 80% granulosa cells, more than 90% granulosa cells, or more than 95% granulosa cells. The method according to any one of claims 78 to 99, wherein the one or more OSCs are obtained by differentiation of a population of iPSCs, and optionally, the population of iPSCs is hiPSCs. The aforementioned hiPSC is the following transcription factor (i) RUNX2,
(ii) NR5A1,
(iii) GATA4,
(iv) FOXL2,
(v) Any combination of two of the transcription factors,
(vi) expressing or overexpressing any combination of three of the transcription factors, or (vii) expressing or overexpressing one or more combinations of all four of the transcription factors. The method according to claim 101, wherein the expression or overexpression of one or more transcription factors is induced by a doxycycline-responsive transcriptional regulator. The method according to any one of claims 100 to 102, wherein the hiPSC comes into contact with a Wnt/β-catenin pathway activator. The method according to claim 103, wherein the Wnt/β-catenin pathway activator is a Rho-related protein kinase (ROCK) inhibitor, a glycogen synthase kinase-3 (GSK3) inhibitor, or a combination thereof. The method according to any one of claims 78 to 104, wherein one or more OSCs are enclosed. The method according to claim 105, wherein one or more OSCs are encapsulated in alginate, laminin, collagen, vitronectin, chitosan, hyaluronic acid, poly-D-lactone, or a mixture thereof. The method according to claim 106, wherein one or more OSCs are encapsulated in laminin, and optionally the laminin is laminin-521. The method according to claim 106 or 107, wherein one or more OSCs are encapsulated in vitronectin. The method according to any one of claims 78 to 108, wherein the one or more OSCs have the expression of one or more genes associated with pluripotency that is low or undetectable compared to iPSCs. The method according to claim 109, wherein one or more genes related to pluripotency include NANOG. The method according to claim 110, wherein one or more genes related to pluripotency include POU5F1. The method according to any one of claims 78 to 111, wherein one or more of the OSCs produce one or more growth factors. The method according to claim 112, wherein the growth factor includes IGF, SCF, EGF, LIF, VEGF, BMP, CNP, or any combination thereof. The method according to claim 112 or 113, wherein at least a portion of the one or more growth factors is secreted. The method according to any one of claims 78 to 114, wherein the one or more OSCs produce one or more steroids. The method according to claim 115, wherein the one or more steroids include estradiol, progesterone, or a combination thereof. The method according to claim 115 or 116, wherein the one or more steroids are produced in response to hormonal stimulation of the OSC. The method according to claim 117, wherein the hormonal stimulation includes exposure to FSH, androstenedione, or a combination thereof. The method according to any one of claims 115 to 118, wherein at least a portion of the one or more steroids is secreted. The method according to any one of claims 78 to 119, wherein one or more OSCs were previously cryopreserved. The method according to any one of claims 78 to 120, further comprising culturing one or more OSCs together with one or more oocytes in in vitro maturation (IVM) medium. The method according to claim 121, wherein the IVM medium includes a cell culture medium. The method according to claim 121 or 122, wherein the IVM medium comprises Medicult-IVM medium. The method according to any one of claims 121 to 123, wherein the IVM medium comprises one or more supplements. The one or more supplements mentioned above are
(i) Human serum albumin (HSA) at a concentration of approximately 5 to 15 mg/mL (optional), and at a concentration of 10 mg/mL (optional).
(ii) Recombinant follicle-stimulating hormone (rFSH) at a concentration of approximately 70 mIU/mL to approximately 80 mIU/mL, and at an optional concentration of 75 mIU/mL.
(iii)Optionally, human chorionic gonadotropin (hCG) at concentrations of approximately 95 mIU/mL to approximately 105 mIU/mL, and optionally at a concentration of 100 mIU/mL.
(iv)Optionally, androstenedione at concentrations of approximately 495 ng/mL to approximately 505 ng/mL, and further optionally, at a concentration of 500 ng/mL.
(v) Optionally, doxycycline at concentrations of approximately 0.5 μg/mL to approximately 1.5 μg/mL, and optionally at a concentration of 1 μg/mL.
The method according to claim 124, or the method comprising any combination of the one or more supplements. The method according to any one of claims 78 to 125, wherein the one or more oocytes are collected from a donor subject. The method according to claim 126, wherein the subject is approximately 19 to 45 years of age. The method according to claim 126, wherein the subject is receiving ovarian stimulation. The method according to claim 128, wherein the ovarian stimulation includes treatment with gonadotropin-releasing hormone (GnRH). The method according to claim 129, wherein the ovarian stimulation comprises treatment with one or more GnRH analogs. The method according to claim 130, wherein the GnRH analog is a GnRH agonist or antagonist. The ovarian stimulation is the method according to any one of claims 128 to 131, comprising one or more ovulation-inducing agents. The method according to claim 132, wherein the one or more ovulation-inducing agents include hCG. The method according to claim 132 or 133, wherein the one or more ovulation-inducing agents include a GnRH agonist, and optionally the GnRH agonist is leuprolide. The ovarian stimulation is the method according to any one of claims 128 to 134, comprising FSH treatment. The method according to claims 128-134, wherein the ovarian stimulation does not include FSH treatment. The FSH treatment according to claim 135, comprising 300 IU to 700 IU of FSH. The FSH treatment according to claim 137, comprising 400 IU to 600 IU of FSH. The method according to any one of claims 135, 137, and 138, wherein the FSH treatment comprises one, two, three, or more FSH injections, and optionally, the FSH treatment comprises multiple injections, each injection comprising a dose of FSH of approximately 100 IU to approximately 200 IU. The method according to any one of claims 128 to 139, wherein the ovarian stimulation comprises the administration of clomiphene citrate, and optionally, the clomiphene citrate is administered in one or more doses for a maximum of eight days, and optionally, each dose is 50 mg to 150 mg. The ovarian stimulation method according to any one of claims 128 to 140, comprising one or more hCG-inducing agents. The method according to claim 141, wherein the one or more hCG-inducing agents comprise 2,500 IU to 10,000 IU of hCG or about 200 μg to about 700 μg of hCG, and optionally, the hCG is administered to the subject in a dose of about 400 μg to about 600 μg, and optionally, the hCG is administered to the subject in a dose of about 500 μg per dose. The method according to any one of claims 78 to 142, wherein the one or more oocytes are located within a cumulus-oocyte complex (COC). The method according to any one of claims 78 to 143, wherein the one or more oocytes include one or more deprived immature oocytes. The method according to claim 144, wherein all of the one or more oocytes are developed immature oocytes. The method according to any one of claims 78 to 143, wherein one or more oocytes are not veiled. The method according to any one of claims 78 to 146, wherein the one or more oocytes include one or more oocytes containing nucleus vesicles (GVs). The method according to any one of claims 78 to 147, wherein the one or more oocytes include one or more oocytes in metaphase I (MI). The method according to any one of claims 78 to 148, wherein the one or more oocytes include one or more oocytes in metaphase II (MII). The method according to any one of claims 78 to 149, wherein the one or more oocytes comprises one or more previously vitrified oocytes. The method according to any one of claims 78 to 150, wherein the one or more oocytes include one or more previously cryopreserved oocytes. The method according to any one of claims 78 to 151, wherein the one or more oocytes are co-cultured with the one or more OSCs for about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, or about 36 hours. The method according to claim 152, wherein the one or more oocytes are cultured with the one or more OSCs for about 24 to about 28 hours. The method according to claim 152 or 153, wherein the one or more oocytes co-cultured with the one or more OSCs form one or more undifferentiated germ cells after contact with one or more mature spermatids. The method according to any one of claims 78 to 154, wherein the one or more oocytes are co-cultured in direct contact with the one or more OSCs. The method according to any one of claims 78 to 155, wherein one or more oocytes do not come into direct contact with one or more OSCs. The method according to any one of claims 78 to 156, wherein the co-culture is a suspension co-culture. The method according to claims 78 to 156, wherein the co-culture is an adherent co-culture. A method for promoting the differentiation of one or more induced pluripotent stem cells (iPSCs) into one or more ovarian supporting cells (OSCs), wherein the method is:
(a) Culturing one or more iPSCs in vitro,
(b) In one or more iPSCs, the expression or overexpression of one or more transcription factors including FOXL2, NR5A1, RUNX2, GATA4, or any combination thereof is induced, thereby generating differentiated cells.
(c) Determine that the differentiated cells obtained from (b) exhibit a gene expression profile similar to the gene expression profile of one or more OSCs,
(d) The method comprising co-culturing one or more of the identified OSCs with one or more oocytes previously collected from a subject, thereby maturing the one or more oocytes. A method for generating one or more ovarian supporting cells (OSCs) from one or more induced pluripotent stem cells (iPSCs), wherein the method is:
(a) Culturing one or more iPSCs in vitro,
(b) In one or more iPSCs, the expression or overexpression of one or more transcription factors including FOXL2, NR5A1, RUNX2, GATA4, or any combination thereof is induced, thereby generating differentiated cells.
(c) Determine that the differentiated cells obtained from (b) exhibit a gene expression profile similar to the gene expression profile of one or more OSCs,
(d) The method comprising co-culturing one or more of the identified OSCs with one or more oocytes previously collected from a subject, thereby maturing the one or more oocytes. A method for preparing a composition comprising one or more ovarian supporting cells (OSCs), wherein the method is:
(a) Culturing one or more iPSCs in vitro,
(b) In one or more iPSCs, the expression or overexpression of one or more transcription factors including FOXL2, NR5A1, RUNX2, GATA4, or any combination thereof is induced, thereby generating differentiated cells.
(c) Determine that the differentiated cells obtained from (b) exhibit a gene expression profile similar to the gene expression profile of one or more OSCs,
(d) The method comprising co-culturing one or more of the identified OSCs with one or more oocytes previously collected from the subject, thereby maturing one or more oocytes. The method according to any one of claims 159 to 161, wherein the iPSC is a human iPSC (hiPSC). The iPSC is previously cryopreserved, according to the method of any one of claims 159 to 162. The method according to any one of claims 159 to 163, wherein the co-culture is performed in an in vitro maturation (IVM) medium. The method according to claim 164, wherein the IVM medium includes a cell culture medium. The method according to claim 164 or 165, wherein the IVM medium comprises Medicult-IVM medium. The method according to any one of claims 164 to 166, wherein the IVM medium comprises one or more supplements. The one or more supplements mentioned above are
(i) Human serum albumin (HSA) at a concentration of approximately 5 to 15 mg/mL (optional), and at a concentration of 10 mg/mL (optional).
(ii) Recombinant follicle-stimulating hormone (rFSH) at a concentration of approximately 70 mIU/mL to approximately 80 mIU/mL, and at an optional concentration of 75 mIU/mL.
(iii)Optionally, human chorionic gonadotropin (hCG) at concentrations of approximately 95 mIU/mL to approximately 105 mIU/mL, and optionally at a concentration of 100 mIU/mL.
(iv)Optionally, androstenedione at concentrations of approximately 495 ng/mL to approximately 505 ng/mL, and further optionally, at a concentration of 500 ng/mL.
(v) Optionally, doxycycline at concentrations of approximately 0.5 μg/mL to approximately 1.5 μg/mL, and optionally at a concentration of 1 μg/mL.
The method according to claim 167, or comprising any combination of the one or more supplements. The method according to any one of claims 159 to 168, wherein the induction from iPSC to OSC is performed for approximately 1 to 10 days, and optionally, the induction is performed for approximately 5 days. The method according to any one of claims 159 to 169, wherein the iPSC is cultured in a medium containing a matrix. The method according to claim 170, wherein the matrix comprises alginate, laminin, collagen, vitronectin, chitosan, hyaluronic acid, poly-D-lactone, or a mixture thereof. The method according to claim 171, wherein the matrix comprises laminin, and optionally the laminin is laminin-521. The method according to claim 171 or 172, wherein the matrix comprises vitronectin. The method according to any one of claims 159 to 173, wherein the iPSC is reprogrammed using a transposase method to carry one or more inducible transcription factors. The method according to any one of claims 159 to 173, wherein the iPSC is transformed by electroporation, liposome-mediated transformation, or virus-mediated gene transfer. The method according to any one of claims 159 to 175, wherein the expression or overexpression of one or more transcription factors is induced by a doxycycline-responsive transcriptional regulator. The method according to any one of claims 159 to 176, wherein the iPSC comes into contact with a Wnt/β-catenin pathway activator. The method according to claim 177, wherein the Wnt/β-catenin pathway activator is a Rho-related protein kinase (ROCK) inhibitor, a glycogen synthase kinase-3 (GSK3) inhibitor, or a combination thereof. The method according to any one of claims 159 to 178, wherein one or more OSCs express FOXL2, AMHR2, CD82, or any combination thereof. The method according to any one of claims 159 to 179, wherein one or more OSCs express one or more genes selected from GJA1, MDK, BBX, HES4, PBX3, YBX3, BMPR2, CD46, COL4A1, COL4A2, LAMC1, ITGAV, and ITGB. The method according to claim 179 or 180, wherein one or more OSCs further express one or more genes selected from FOXO1, CDH1, CYP19A1, RARRES2, NOTCH2, NRG1, BMPR1B, EGFR (ERBB1), and ERBB4. The method according to claim 179 or 180, wherein the one or more OSCs further express one or more genes selected from RARRES2, NOTCH2, NOTCH3, ID3, and BMPR2. The method according to claim 179 or 180, wherein one or more OSCs further express the CDH2 gene and/or the NOTCH2 gene, and optionally lack significant expression of the RARRES2 gene. The method according to any one of claims 159 to 179, wherein the one or more OSCs comprises one or more granulosa cells. The method according to any one of claims 159 to 179, wherein one or more OSCs express NR2F2. The method according to any one of claims 159 to 179 and 185, wherein the one or more OSCs comprises one or more ovarian stromal cells. The method according to any one of claims 159 to 186, wherein the one or more OSCs include granulosa cells and ovarian stromal cells. The method according to any one of claims 159 to 187, wherein one or more OSCs include more than 60% granulosa cells, more than 70% granulosa cells, more than 80% granulosa cells, more than 90% granulosa cells, or more than 95% granulosa cells. The method according to any one of claims 159 to 188, wherein the one or more OSCs have the expression of one or more genes associated with pluripotency that is low or undetectable compared to iPSCs. The method according to claim 189, wherein one or more genes related to pluripotency include NANOG. The method according to claim 190, wherein one or more genes related to pluripotency include POU5F1. The method according to any one of claims 159 to 191, wherein one or more of the OSCs produce one or more growth factors. The method according to claim 192, wherein the growth factor includes IGF, SCF, EGF, LIF, VEGF, BMP, CNP, or any combination thereof. The method according to claim 192 or 193, wherein at least a portion of the one or more growth factors is secreted. The method according to any one of claims 159 to 194, wherein one or more of the OSCs generate one or more steroids. The method according to claim 195, wherein the one or more steroids include estradiol, progesterone, or a combination thereof. The method according to claim 195 or 196, wherein the one or more steroids are produced in the presence of one or more hormones. The method according to claim 197, wherein the one or more hormones include FSH, androstenedione, or a combination thereof. The method according to any one of claims 194 to 198, wherein one or more steroids are secreted. The method according to any one of claims 159 to 199, wherein the one or more oocytes collected from the subject are immature oocytes. The method according to any one of claims 159 to 200, wherein the co-culture of one or more OSCs and one or more oocytes promotes the maturation of the one or more oocytes. The method according to any one of claims 159 to 201, further comprising collecting one or more oocytes for assisted reproductive technology procedures. The method according to any one of claims 159 to 202, wherein the subject has undergone ovarian stimulation before the collection of one or more oocytes. The method according to claim 203, wherein the ovarian stimulation includes treatment with gonadotropin-releasing hormone (GnRH). The method according to claim 203 or 204, wherein the ovarian stimulation comprises treatment with one or more GnRH analogs. The method according to claim 205, wherein the one or more GnRH analogs are GnRH agonists or antagonists. The ovarian stimulation is the method according to any one of claims 203 to 206, comprising one or more ovulation-inducing agents. The method according to claim 207, wherein the one or more ovulation-inducing agents include hCG. The method according to claim 207 or 208, wherein the one or more ovulation-inducing agents comprise a GnRH agonist, and optionally the GnRH agonist is leuprolide. The ovarian stimulation is the method according to any one of claims 203 to 209, comprising FSH treatment. The method according to any one of claims 203 to 209, wherein the ovarian stimulation does not include FSH treatment. The method according to claim 210, wherein the FSH treatment comprises 300 IU to 700 IU of FSH. The method according to claim 212, wherein the FSH treatment comprises 400 IU to 600 IU of FSH. The method according to any one of claims 210, 212, and 213, wherein the FSH treatment comprises one, two, three, or more FSH injections, and optionally, the FSH treatment comprises multiple injections, each injection comprising a dose of FSH of approximately 100 IU to approximately 200 IU. The method according to any one of claims 203 to 214, wherein the ovarian stimulation further comprises the administration of clomiphene citrate, optionally administered in one or more doses for a maximum of eight days, and optionally each dose being 50 mg to 150 mg. The method according to any one of claims 203 to 215, wherein the ovarian stimulation further comprises one or more hCG-inducing agents. The composition according to claim 216, wherein the one or more hCG-inducing agents comprise 2,500 IU to 10,000 IU of hCG or about 200 μg to about 700 μg of hCG, and optionally, the hCG is administered to the subject in a dose of about 400 μg to about 600 μg, and optionally, the hCG is administered to the subject in a dose of about 500 μg per dose. The method according to any one of claims 159 to 217, wherein the one or more oocytes are located within a cumulus-oocyte complex (COC). The method according to any one of claims 159 to 218, wherein the one or more oocytes include one or more deprived immature oocytes. The method according to claim 219, wherein all of the one or more oocytes are veiled immature oocytes. The method according to any one of claims 159 to 217, wherein one or more oocytes are not denucleated before or after the co-culture. The method according to any one of claims 159 to 221, wherein the one or more oocytes include one or more oocytes containing nucleus vesicles (GVs). The method according to any one of claims 159 to 222, wherein the one or more oocytes include one or more oocytes in metaphase I (MI). The method according to any one of claims 159 to 223, wherein the one or more oocytes include one or more oocytes in metaphase II (MII). The method according to any one of claims 159 to 224, wherein at least a portion of the one or more oocytes comprises one or more previously vitrified oocytes. The method according to any one of claims 159 to 225, wherein at least a portion of the one or more oocytes comprises one or more previously cryopreserved oocytes. The method according to any one of claims 159 to 226, wherein, before and/or after the co-culture, one or more oocytes are evaluated for parameters selected from the group consisting of total oocyte score, oocyte maturation rate from GV to MII stage, oocyte maturation rate from GV to MI stage, oocyte maturation rate from MI to MII stage, mean oocyte shape, mean oocyte size, mean oocyte quality, mean perivitelline space (PVS) quality, mean zona pellucida (ZP) quality, and mean polar body quality. The method according to claim 227, wherein the one or more co-cultured oocytes have substantially the same morphological qualities as oocytes matured in vivo, and the morphological qualities include oocyte size, oocyte zona pellucida size, oocyte color, oocyte shape, oocyte cytoplasmic grain size, oocyte polar body quality, and oocyte PVS quality. The method according to claim 227 or 228, wherein the one or more co-cultured oocytes have an improved maturation rate compared to oocytes in a culture that does not contain the one or more OSCs. The method according to any one of claims 227 to 229, wherein the one or more co-cultured oocytes have metaphase II spindles located substantially in the same position as those of mature oocytes in the body. The method according to any one of claims 159 to 230, wherein the one or more co-cultured oocytes have substantially the same transcriptome profile as oocytes matured in vivo. The method according to any one of claims 159 to 231, wherein the one or more oocytes are co-cultured with the one or more OSCs for approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, approximately 24 hours, approximately 25 hours, approximately 26 hours, approximately 27 hours, approximately 28 hours, approximately 29 hours, approximately 30 hours, approximately 31 hours, approximately 32 hours, approximately 33 hours, approximately 34 hours, approximately 35 hours, or approximately 36 hours. The method according to claim 232, wherein the one or more oocytes are cultured with the one or more OSCs for about 24 to about 28 hours. The method according to any one of claims 232 or 233, further comprising isolating one or more MII-stage oocytes from a co-culture comprising one or more oocytes and one or more OSCs collected from the subject. The method according to any one of claims 232 to 234, wherein the one or more oocytes co-cultured with the one or more OSCs form one or more undifferentiated germ cells after contact with one or more mature spermatids. The method according to any one of claims 159 to 235, wherein the one or more oocytes are co-cultured in direct contact with the one or more OSCs. The method according to claims 159 to 235, wherein the one or more oocytes do not come into direct contact with the one or more OSCs. The method according to any one of claims 159 to 237, wherein the co-culture is a suspension co-culture. The method according to any one of claims 159 to 237, wherein the co-culture is an adherent co-culture. A cell culture system comprising one or more ovarian supporting cells (OSCs), wherein the system promotes the maturation of one or more oocytes. The system according to claim 240, wherein the one or more OSCs include one or more granulosa cells. The system according to claim 241, wherein one or more OSCs express FOXL2, AMHR2, CD82, or any combination thereof. The system according to any one of claims 240 to 242, wherein one or more OSCs express one or more genes selected from GJA1, MDK, BBX, HES4, PBX3, YBX3, BMPR2, CD46, COL4A1, COL4A2, LAMC1, ITGAV, and ITGB. The system according to claim 242 or 243, wherein one or more OSCs further express one or more genes selected from FOXO1, CDH1, CYP19A1, RARRES2, NOTCH2, NRG1, BMPR1B, EGFR (ERBB1), and ERBB4. The system according to claim 242 or 243, wherein one or more OSCs further express one or more genes selected from RARRES2, NOTCH2, NOTCH3, ID3, and BMPR2. The system according to claim 242 or 243, wherein one or more OSCs further express the genes CDH2 and/or NOTCH2. The system according to any one of claims 240 to 246, wherein one or more OSCs do not exhibit significant expression of RARRES2. The system according to any one of claims 240 to 247, wherein one or more OSCs express NR2F2. The system according to any one of claims 240 to 248, wherein one or more OSCs include ovarian stromal cells. The system according to any one of claims 240 to 249, wherein one or more OSCs include granulosa cells and ovarian stromal cells. The system according to any one of claims 240 to 250, wherein one or more OSCs include more than 60% granulosa cells, more than 70% granulosa cells, more than 80% granulosa cells, more than 90% granulosa cells, or more than 95% granulosa cells. The system according to any one of claims 240 to 251, wherein the one or more OSCs are obtained by differentiation of a population of iPSCs, and optionally, the iPSCs are hiPSCs. The aforementioned hiPSC is the following transcription factor (i) RUNX2,
(ii) NR5A1,
(iii) GATA4,
(iv) FOXL2,
(v) Any combination of two of the transcription factors,
(vi) expressing or overexpressing any combination of three of the transcription factors, or (vii) expressing or overexpressing one or more combinations of all four of the transcription factors. The system according to claim 252 or 253, wherein the expression or overexpression of one or more transcription factors is induced by a doxycycline-responsive transcriptional regulator. The system according to any one of claims 252 to 254, wherein the hiPSC comes into contact with a Wnt/β-catenin pathway activator. The system according to claim 255, wherein the Wnt/β-catenin pathway activator is a Rho-related protein kinase (ROCK) inhibitor, a glycogen synthase kinase-3 (GSK3) inhibitor, or a combination thereof. The combination system according to any one of claims 240 to 256, wherein at least one of the one or more OSCs is enclosed. The system according to claim 257, wherein the OSC(s) are encapsulated in alginate, laminin, collagen, vitronectin, chitosan, hyaluronic acid, poly-D-lactone, or any mixture thereof. The system according to claim 258, wherein the OSC(s) are encapsulated in laminin, and optionally the laminin is laminin-521. The OSC is encapsulated in vitronectin, according to the system or system according to claim 258 or 259. The system according to any one of claims 240 to 260, wherein one or more OSCs have the expression of one or more genes associated with pluripotency that is low or undetectable compared to iPSCs. The system according to claim 261, wherein one or more genes related to pluripotency include NANOG. The system according to claim 262, wherein one or more genes related to pluripotency include POU5F1. The system according to any one of claims 240 to 263, wherein at least a portion of the OSCs produces one or more growth factors. The system according to claim 264, wherein the one or more growth factors include insulin-like growth factor (IGF), stem cell factor (SCF), epidermal growth factor (EGF), leukemia suppressor factor (LIF), vascular endothelial growth factor (VEGF), bone morphogenetic protein (BMP), C-type natriuretic peptide (CNP), or any combination thereof. The system according to claim 264 or 265, wherein at least a portion of the one or more growth factors is secreted. The system according to any one of claims 240 to 266, wherein one or more of the OSCs generate one or more steroids. The system according to claim 267, wherein the one or more steroids include estradiol, progesterone, or a combination thereof. The system according to claim 267 or 268, wherein one or more steroids are produced in response to hormonal stimulation. The system according to claim 269, wherein the hormonal stimulation includes FSH, androstenedione therapy, or a combination thereof. The system according to any one of claims 267 to 270, wherein at least a portion of the one or more steroids is secreted. The system according to any one of claims 240 to 271, wherein one or more OSCs are cryopreserved. The system according to any one of claims 240 to 272, further comprising an in vitro maturation (IVM) medium. The system according to claim 273, wherein the IVM medium includes a cell culture medium. The system according to claim 273 or 274, wherein the IVM medium comprises Medicult-IVM medium. The IVM medium comprises one or more supplements, according to any one of claims 273 to 275. The one or more supplements mentioned above are
(i) Human serum albumin (HSA) at a concentration of approximately 5 to 15 mg/mL (optional), and at a concentration of 10 mg/mL (optional).
(ii) Recombinant follicle-stimulating hormone (rFSH) at a concentration of approximately 70 mIU/mL to approximately 80 mIU/mL, and at an optional concentration of 75 mIU/mL.
(iii)Optionally, human chorionic gonadotropin (hCG) at concentrations of approximately 95 mIU/mL to approximately 105 mIU/mL, and optionally at a concentration of 100 mIU/mL.
(iv)Optionally, androstenedione at concentrations of approximately 495 ng/mL to approximately 505 ng/mL, and further optionally, at a concentration of 500 ng/mL.
(v) Optionally, doxycycline at concentrations of approximately 0.5 μg/mL to approximately 1.5 μg/mL, and optionally at a concentration of 1 μg/mL.
The system according to claim 276, or comprising any combination of the one or more supplements. The system according to any one of claims 240 to 277, wherein the one or more oocytes are collected from a donor. The system according to claim 278, wherein the donor target is approximately 19 to 45 years of age. The system according to claim 278, wherein the subject is receiving ovarian stimulation. The system according to claim 280, wherein the ovarian stimulation includes treatment with gonadotropin-releasing hormone (GnRH). The system according to claim 281, wherein the ovarian stimulation includes treatment with one or more GnRH analogs. The system according to claim 282, wherein one or more GnRH analogs are GnRH agonists or antagonists. The ovarian stimulation system according to any one of claims 280 to 283, comprising one or more ovulation-inducing agents. The system according to claim 284, wherein the one or more ovulation-inducing agents include hCG. The system according to claim 284 or 285, wherein the one or more ovulation-inducing agents comprise a GnRH agonist, and optionally the GnRH agonist is leuprolide. The ovarian stimulation includes FSH treatment, according to any one of claims 280 to 286. The ovarian stimulation system according to any one of claims 280 to 286, wherein the ovarian stimulation does not include FSH treatment. The FSH treatment described above is the system according to claim 287, comprising 300 to 700 IU of FSH. The FSH treatment described above is the system according to claim 289, comprising 400 IU to 600 IU of FSH. The system according to any one of claims 287, 289, and 290, wherein the FSH treatment comprises one, two, three, or more FSH injections, and optionally, the FSH treatment comprises multiple injections, each injection comprising a dose of FSH of approximately 100 IU to approximately 200 IU. The system according to any one of claims 280 to 291, wherein the ovarian stimulation further comprises the administration of clomiphene citrate, optionally administered in one or more doses for a maximum of eight days, and optionally each dose being between 50 mg and 150 mg. The ovarian stimulation further comprises one or more hCG-inducing agents, according to any one of claims 280 to 292. The system according to claim 293, wherein the one or more hCG inducers comprise 2,500 IU to 10,000 IU of hCG or about 200 μg to about 700 μg of hCG, and optionally, the hCG is administered to the subject in a dose of about 400 μg to about 600 μg, and optionally, the hCG is administered to the subject in a dose of about 500 μg per dose. The system according to any one of claims 240 to 294, wherein one or more oocytes are located within a cumulus-oocyte complex (COC). The system according to any one of claims 240 to 295, wherein the one or more oocytes comprises one or more deprived immature oocytes. The system according to claim 296, wherein all of the one or more oocytes are veiled immature oocytes. The system according to any one of claims 240 to 295, wherein one or more oocytes are not veiled. The system according to any one of claims 240 to 298, wherein the one or more oocytes include one or more oocytes containing nucleus vesicles (GVs). The system according to any one of claims 240 to 299, wherein the one or more oocytes include one or more oocytes in metaphase I (MI). The system according to any one of claims 240 to 300, wherein the one or more oocytes include one or more oocytes in metaphase II (MII). The system according to any one of claims 240 to 301, wherein at least a portion of the one or more oocytes comprises one or more previously vitrified oocytes. The system according to any one of claims 240 to 302, wherein at least a portion of the one or more oocytes comprises one or more previously cryopreserved oocytes. The system according to any one of claims 240 to 303, wherein the one or more oocytes are co-cultured with the one or more OSCs. The system according to claim 304, wherein, before and/or after the co-culture, one or more oocytes are evaluated with respect to parameters selected from the group consisting of total oocyte score, oocyte maturation rate from GV to MII stage, oocyte maturation rate from GV to MI stage, oocyte maturation rate from MI to MII stage, mean oocyte shape, mean oocyte size, mean oocyte quality, mean perivitelline space (PVS) quality, mean zona pellucida (ZP) quality, and mean polar body quality. The system according to claim 305, wherein the one or more co-cultured oocytes have substantially the same morphological qualities as oocytes matured in vivo, and the morphological qualities include oocyte size, oocyte zona pellucida size, oocyte color, oocyte shape, oocyte cytoplasmic grain size, oocyte polar body quality, and oocyte PVS quality. The system according to claim 305 or 306, wherein the one or more co-cultured oocytes have an improved maturation rate compared to oocytes in a culture that does not contain the one or more OSCs. The system according to any one of claims 305 to 307, wherein one or more co-cultured oocytes have a metaphase II spindle that is substantially in the same position as that of a mature oocyte in the body. The system according to any one of claims 304 to 308, wherein the one or more co-cultured oocytes have substantially the same transcriptome profile as oocytes matured in vivo. The system according to any one of claims 304 to 309, wherein one or more oocytes are co-cultured with one or more OSCs for approximately 20 hours, approximately 21 hours, approximately 22 hours, approximately 23 hours, approximately 24 hours, approximately 25 hours, approximately 26 hours, approximately 27 hours, approximately 28 hours, approximately 29 hours, approximately 30 hours, approximately 31 hours, approximately 32 hours, approximately 33 hours, approximately 34 hours, approximately 35 hours, or approximately 36 hours. The system according to claim 310, wherein one or more oocytes are co-cultured with one or more OSCs for approximately 24 to 28 hours. The system according to claim 310 or 311, wherein the one or more oocytes co-cultured with the one or more OSCs form one or more undifferentiated germ cells after contact with one or more mature spermatids. The system according to any one of claims 304 to 312, wherein the one or more oocytes are co-cultured in direct contact with the one or more OSCs. The system according to any one of claims 304 to 313, wherein one or more oocytes do not come into direct contact with the OSC. The culture system is a suspension culture system, as described in any one of claims 304 to 314. The culture system is an adherent culture system, as described in any one of claims 304 to 314. A kit comprising a composition and accompanying documentation according to any one of claims 1 to 77, wherein the accompanying documentation instructs the user of the kit to co-culture the population of ovarian supporting cells with one or more oocytes according to the method described in any one of claims 78 to 158. A kit comprising a vial containing a population of iPSCs and an accompanying document, wherein the accompanying document instructs the user of the kit to differentiate the population of iPSCs into one or more ovarian supporting cells according to the method described in any one of claims 159 to 239. A kit comprising a vial containing one or more OSCs and an accompanying document, wherein the accompanying document instructs the user of the kit to culture the cells in the cell culture system described in any one of claims 240 to 316.