Lecture 8: Stem Cells and Female Fertility (PDF)

🥽 Leture 8: Stem cells and female fertility Part 1- Oogonial stem cells → Oogonial stem cells- what are they? → Controversies, evidence for and against their existence What is oogonial stem cells (OSCs)?...

🥽 Leture 8: Stem cells and female fertility Part 1- Oogonial stem cells → Oogonial stem cells- what are they? → Controversies, evidence for and against their existence What is oogonial stem cells (OSCs)? Oogonial stem cells (OSCs) are also known as egg precursor cells or female germline stem cells They are cells present in the ovary after birth that have the capacity to proliferate and generate new oocytes BUTTT → OSCs are the most enigmatic and hotly debated cell type in the mammalian ovary, their existence is still not confirmed So what’s the controversy? Leture 8: Stem cells and female fertility 1  So a central dogma in reproductive biology is that: In mammalian females, all oocytes are generated during fetal life Females can not make new oocytes after birth (fixed pool theory) - once the primordial follicles store is depleted, it’s gone! There are no germline stem cells in the adult female** But then: Johnson et al (2004, 2005) claimed that neo-oogenesis takes place in adult ovary Stem cells present in ovarian surface epithelium (OSE) or the bone marrow (BM) that could make new oocytes ^^ BUT this work was widely criticised- serious methodological flaws, data not definitive, bias interpretation, other possible explanations ignored, conclusions only weakly supported by data And now (15 years later): Still INTENSE debate as to both their existence and whether or not they actually contribute to the ovarian reserve Triggered an enormous amount of research Most researchers remain doubtful EVIDENCE FOR Paper 1: Johnson et al, Nature, 2004 Objective: The study aimed to investigate whether adult mouse ovaries contain stem cells capable of generating new eggs throughout the reproductive life. Key Findings and Methods Leture 8: Stem cells and female fertility 2  1. Follicle Death and New Follicle Formation: Observation: The researchers noted significant follicle death throughout the reproductive life of mice. Conclusion: They inferred that the high rate of follicle death suggested the presence of stem cells that could produce new follicles to maintain fertility. 2. Histological Observations: Findings: The study identified large ovoid cells in the ovarian epithelium that did not fit the typical characteristics of primordial follicles. Proposal: These cells were proposed to be oogonial stem cells, as they were not surrounded by granulosa cells and were distinct from primordial follicles. 3. Cell Markers: Markers Used: The researchers used germ line markers (e.g., MVH/DDX4) and proliferation markers (e.g., BRDU) to identify potential stem cells. Results: They observed cells that were positive for both MVH/DDX4 and BRDU, suggesting these cells were proliferating germ line cells, possibly oogonial stem cells. 4. Myotic Markers: Findings: The study detected markers of meiotic prophase I (e.g., SCP3, Spell 11, DMC1) in adult ovaries using PCR. Conclusion: These markers, typically associated with embryonic development, indicated that there might be newly forming primordial follicles, supporting the idea of ongoing germ cell production. 5. Additional Experiments: Buculfon Treatment: Method: Buculfon, known to deplete pre-meiotic cells in males, was used to treat mice. Leture 8: Stem cells and female fertility 3  Findings: The follicle pool was depleted, leading to the conclusion that Buculfon destroyed stem cells. Criticism: Buculfon also has the potential to kill oocytes directly, raising doubts about whether the depletion was due to loss of stem cells or destruction of existing follicles. Grafting Experiment: Method: Wild-type ovaries were grafted into transgenic female mice expressing GFP. Findings: GFP-positive germ cells formed follicle structures with GFP-negative somatic cells. Interpretation: This was used as evidence that stem cells were present, but the rationale was considered vague and less direct. Main Conclusion: The paper concluded that adult mouse ovaries contain oogonial stem cells capable of producing new oocytes. This finding was based on histological observations, marker studies, and experimental depletion. Paper 2: Johnson et al, Cell, 2005 Objective: The study aimed to determine whether oogonial stem cells (OGSCs) in adult ovaries are derived from bone marrow rather than the ovarian epithelium. Key Findings and Methods 1. Doxorubicin Treatment and Follicle Replenishment: Doxorubicin: A chemotherapy drug known to deplete primordial follicles by killing them. Leture 8: Stem cells and female fertility 4  Observation: After treating mice with Doxorubicin and allowing some time for recovery, the researchers observed a subsequent increase in follicle numbers. Conclusion: This increase was proposed as evidence that new follicles were being generated, suggesting a potential source of these new follicles. 2. Germ Line Markers in Bone Marrow: Markers Identified: The researchers found expression of germ line markers (e.g., Oct4, Phylogelous, No-box, Dazzle, Stella, DDX4) in bone marrow. These markers are typically associated with proliferating germ cells. Implication: The presence of these markers in bone marrow supported the hypothesis that bone marrow could be a source of new oocytes. 3. Bone Marrow Transplant Studies: Method: Mice previously sterilized by chemotherapy underwent bone marrow transplantation. Results: Following transplantation, there was evidence of follicle regeneration and oocyte production in these mice. Genetic Deficiency Testing: They also tested in mice genetically deficient in bone marrow and observed similar results. Findings: The follicle pool was restored, and donor-derived oocytes were present in the female mice. 4. Histological Data: Observations: The study presented histological images showing primordial, primary, pre-antral, and total follicle counts in mice treated with Doxorubicin, with and without bone marrow transplantation. Results: After bone marrow transplantation, follicles were observed at various developmental stages, suggesting that the bone marrow-derived cells contributed to follicle regeneration. 5. Fertility Testing: Leture 8: Stem cells and female fertility 5  Limitations: The study did not test for the fertility of the mice following bone marrow transplantation, so the functional implications of the new follicles were not assessed. Main Conclusion: The paper proposed that oogonial stem cells in adult ovaries might originate from bone marrow rather than the ovarian epithelium. This was based on observed follicle replenishment after Doxorubicin treatment, the presence of germ line markers in bone marrow, and successful restoration of follicle numbers following bone marrow transplantation. Supportive evidence (from other papers) Isolation of OSCs Sources: Presumptive OSCs have been isolated from the ovaries of adult mice, rats, cows, and humans. Markers Used: The isolation is based on the expression of the germ cell marker DDX4 (also known as MVH), which is indicative of germ cells. To confirm their stemness, OSCs are also co-labeled with stem cell markers such as Oct4 (also known as Pou5f1). These markers help distinguish OSCs from other cell types. Culturing OSCs In Vitro Culture: OSCs have been successfully established in culture. In these cultures, OSCs express germ cell and stem cell markers, but do not express markers associated with mature oocytes. Phenotypic Features: OSCs in culture display characteristics typical of stem cells, including high telomerase activity, which is associated with cellular longevity and proliferation. Differentiation and Follicle Formation Differentiation: OSCs can undergo in vitro differentiation to form large spherical cells, typically greater than 35 μm in diameter. These cells express markers associated with oocytes. Follicle-Like Structures: When OSCs are cultured with granulosa cells, they can form follicle-like structures. This process mimics the natural follicle formation, where OSCs develop into oocyte-like cells Leture 8: Stem cells and female fertility 6  surrounded by granulosa cells, illustrating their potential to generate oocyte-like cells. But: Can they really make functional oocytes that contribute to ovulation and fertility? → paper that proved functionality Title: "Production of Offspring from a Germ Line Stem Cell Derived from Neonatal Ovaries." Methodology 1. Isolation and Labeling: The researchers isolated OSCs from neonatal mouse ovaries. These OSCs were labeled with Green Fluorescent Protein (GFP), allowing them to track the cells later. 2. In Vitro Culturing: The GFP-labeled OSCs were propagated in vitro for multiple passages over an extended period. 3. Transplantation: Leture 8: Stem cells and female fertility 7  After in vitro culture, the GFP-labeled OSCs were injected into the ovaries of recipient mice. Findings 1. Formation of Oocytes: GFP-positive oocytes were generated in the recipient mice. These oocytes were surrounded by GFP-negative somatic cells, indicating that the oocytes originated from the transplanted OSCs and not from the recipient’s own ovarian cells. 2. Histological Evidence: Histological images showed GFP-positive oocytes (in green) within the ovarian tissue, surrounded by granulosa cells (somatic cells). 3. Functional Testing: The females with transplanted GFP-positive OSCs were mated, and the researchers found that: The oocytes were capable of ovulation. The oocytes could be fertilized. They supported embryonic development. They produced live offspring, some of which were GFP- positive, confirming their origin from the transplanted OSCs. 4. Offspring Analysis: The resulting offspring included GFP-positive individuals, demonstrating that the transplanted OSCs could generate functional oocytes contributing to live births. Significance: This study provided compelling evidence that OSCs, when cultured in vitro and then transplanted, can produce functional Leture 8: Stem cells and female fertility 8  oocytes capable of supporting fertility and generating live offspring. This finding is significant as it suggests that OSCs have the potential to contribute to reproductive processes and could be explored for therapeutic applications in fertility treatments. EVIDENCE AGAINST Contradictory evidence against oogonial stem cells Follicle Counting and Mathematical Modeling Findings: Studies involving follicle counting and mathematical modeling suggest that females are born with a sufficient number of oocytes or follicles to last their entire reproductive lifespan. Contradiction: This challenges the idea that continuous follicle renewal is necessary, which was proposed by Johnson et al. (2004) based on observed follicle loss. Menopause Findings: Menopause occurs when the number of primordial follicles diminishes to a level insufficient to support new follicle development, ovulation, and hormone production. Contradiction: If OSCs were present and functional, they could theoretically replenish the oocyte pool, potentially preventing menopause or delaying its onset. Selective Ablation of Oocytes Findings: Research involving selective ablation of oocytes in transgenic mice has shown no evidence of postnatal oogenesis or new follicle formation after the destruction of the existing oocyte pool. Contradiction: This suggests that if OSCs exist, they might not be contributing to the formation of new follicles, contradicting the idea that OSCs could replenish oocyte numbers after depletion Criticisms of OSC Isolation Methods Leture 8: Stem cells and female fertility 9  DDX4 Marker: The marker DDX4, used in isolating OSCs, is a cytoplasmic protein, and its use in isolation techniques (like FACS) might not be accurate because FACS requires surface markers. Reproducibility: Other researchers have struggled to replicate Johnson et al.’s findings or to isolate OSCs using the reported methods, suggesting potential issues with the original research techniques. Failure to Detect Meiotic Markers Findings: Several studies have failed to detect meiotic markers such as STRA8, SCP3, Spo11, and DMC1 in adult ovaries. Contradiction: These markers are associated with early meiotic stages, which are crucial for the transition of stem cells into oocytes. Their absence in adult ovaries suggests that there might not be ongoing meiotic processes or new oocyte formation from OSCs. Parabiotic Studies Findings: Studies involving parabiosis (joining of two organisms) have shown that OSCs do not originate from bone marrow. Contradiction: This contradicts earlier findings suggesting that bone marrow-derived OSCs could contribute to oocyte production. Study 1: Ovulated oocytes in adult mice derive from non-circulating germ cells Background and Objective This study, published in 2006, aimed to investigate the hypothesis proposed by Johnson et al. (2005), which suggested that bone marrow-derived germ cells might contribute to the formation of new oocytes in adult female mice. To test this hypothesis, the researchers used a model known as parabiosis, where two mice, one transgenic (expressing GFP) and one non-transgenic (wild type), were surgically joined so that they shared a common circulatory system. The goal was to determine whether circulating germ cells from the bone marrow could travel through the shared Leture 8: Stem cells and female fertility 10  vasculature and contribute to oocyte production in the ovary of the partner mouse. Methodology 1. Parabiosis Setup: Transgenic Mouse (GFP+): Expresses green fluorescent protein (GFP), allowing its cells to be easily tracked. Non-Transgenic Mouse: A wild type, non-fluorescent mouse. The two mice were surgically joined to develop a common circulatory system, ensuring that any circulating cells, including potential germ cells, could move between the two mice. 2. Hypothesis Testing: Hypothesis: If circulating germ cells contribute to oocyte formation, then the ovaries of the parabiosed mice should contain both host-derived and partner-derived oocytes. Specifically, the GFP+ mouse would produce GFP+ oocytes, and if the hypothesis were true, the non-GFP mouse would also ovulate GFP+ oocytes, indicating germ cell transfer. Superovulation: After allowing time for the vasculature to fully join, the researchers induced superovulation in the mice to retrieve oocytes and examine their origin. 3. Follicle Depletion Experiment: Pretreatment with Cyclophosphamide and Busulfan: The non- transgenic mice were pretreated with these chemotherapeutic agents to deplete their ovarian follicle reserves. Purpose: To determine if the depletion would stimulate the repopulation of the ovary with oocytes from the GFP+ partner, testing the idea that OSCs might only become active under conditions of ovarian reserve depletion. Findings 1. Oocyte Origin: Result: Only host-derived oocytes were found in both the GFP+ and non-GFP mice. The GFP+ mouse only ovulated GFP+ Leture 8: Stem cells and female fertility 11  oocytes, and the non-GFP mouse only ovulated non-GFP oocytes. Conclusion: There was no evidence that circulating germ cells contributed to the ovarian reserve, as no GFP+ oocytes were found in the non-transgenic mouse. 2. Follicle Depletion Response: Result: Even after depleting the follicle reserves of the non- transgenic mice, no GFP+ oocytes were recovered from them. Conclusion: This suggests that bone marrow-derived oogonial stem cells do not contribute to the generation of new oocytes, even when the ovarian reserve is depleted. Conclusion → This study provided strong evidence against the hypothesis that bone marrow-derived germ cells contribute to oocyte formation in adult mice. Leture 8: Stem cells and female fertility 12  Study 2: Single-Cell Analysis of Human Ovarian Cortex Objective: The research leverages advanced methodologies, such as single- cell RNA sequencing, to analyze the cellular composition of the human ovarian cortex—the outer layer of the ovary where primordial follicles and any potential OSCs are thought to reside. Methodology 1. Sample Collection: Human Ovarian Cortex Samples: The study used ovarian cortex samples from 21 human patients. Some of these samples were obtained from individuals undergoing sex change procedures, providing complete ovaries for analysis. Leture 8: Stem cells and female fertility 13  Histological Examination: The researchers confirmed the integrity of the tissue by examining its histology, identifying the presence of primordial follicles in the ovarian cortex. 2. Tissue Processing: Dissection and Preservation: The medullary region of the ovary was removed, and the remaining cortical tissue was chopped into smaller pieces and frozen for future use. Single-Cell Suspension: After thawing the tissue, it was processed into single-cell suspensions. The cells were filtered to remove any existing primordial follicles. 3. Cellular Analysis: Transcriptomic Profiling: Using the 10X Genomics platform, the study analyzed the transcriptomes of over 24,000 cells from the ovarian cortex. This approach provided a comprehensive snapshot of the different cell types present. Cell Surface Antigen Profiling: The researchers also analyzed the cells for specific surface markers using fluorescence- activated cell sorting (FACS). 4. Oogonial Stem Cell (OSC) Investigation: DDX4 Antibody Labeling: Cells were labeled with the DDX4 antibody, a marker traditionally used to identify OSCs in previous studies. The study aimed to determine whether the cells isolated by this antibody were truly OSCs or something else. Transcriptome Analysis of Cultured Cells: Cells were cultured under conditions known to support OSCs, and their transcriptomes were analyzed using SmartSeq2 technology. Key Findings 1. Identification of Cell Populations: The analysis identified six distinct cell populations in the ovarian cortex, including oocytes, granulosa cells, immune cells, endothelial cells, perivascular cells, and stromal cells. 2. Absence of Oogonial Stem Cells: Leture 8: Stem cells and female fertility 14  No Evidence of OSCs: The study found no cells with transcriptional profiles indicative of oogonial stem cells in the adult human ovarian cortex. Misidentification of Cells: The cells that had been captured using the DDX4 antibody, previously thought to be OSCs, were actually perivascular cells, not stem cells. Part 2 - The development of oocytes from pluripotent stem cells OVERVIEW Embryonic stem cells (ESCs) cells or induced pluripotent stem cells (iPSCs) could be used to generate granulosa cells and oocytes in the lab These oocytes and granulosa cells could be used to build new follicles that could subsequently be transplanted back into the ovary or combined with IVM and IVF/ICSI protocols to create embryos Huge therapeutic potential for women that do not have oocytes (e.g. genetic conditions, diseases, environmental toxicants, cancer treatments) The field faces huge obstacles before this becomes a viable fertility preservation option. Requires protocols for the differentiation of oocytes and granulosa cells from stem cells and in vitro systems that support the development of follicles to maturity. Each step needs to be fully optimised to recapitulate follicle development, as it occurs in vivo. Embryonic stem cells and induced pluripotent stem cells can be used to make oocytes in mice Hayashi and Saitou (2011) reported the production of live pups from sperm derived from iPSCs The following year, ESCs or iPSCs were used to produce primordial germ cell-like cells (PGCLCs) in vitro, and these were subsequently Leture 8: Stem cells and female fertility 15  transplanted into mice to produce oocytes. Hikabe et al. (2016) extended these protocols to derive mature, fertilizable, developmentally competent oocytes from stem cells entirely in vitro. The oocytes are functional i.e. produce healthy pups, efficiency is extremely low. Study 1: Reconstitution in vitro of the entire cycle of the mouse female germ line. Background and Objective This study, published in Nature in 2016, successfully reconstituted the entire cycle of mouse female germ line development in vitro. The aim was to generate functional oocytes (egg cells) from stem cells outside the body, showcasing the potential of using pluripotent stem cells, such as induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs), to create fertilizable oocytes. Methodology: A Three-Step Process The study followed a well-defined three-step process to achieve the reconstitution: 1. In Vitro Differentiation: Stem Cell Source: The process began with pluripotent stem cells, either ESCs from blastocysts or iPSCs reprogrammed from somatic cells. Primordial Germ Cell-Like Cells (PGCLCs): These pluripotent stem cells were differentiated into PGCLCs using a specialized Leture 8: Stem cells and female fertility 16  culture medium that included growth factors such as basic fibroblast growth factor (bFGF), activin A (ActA), bone morphogenetic protein 4 (BMP4), kit ligand (KL), leukemia inhibitory factor (LIF), and epidermal growth factor (EGF). Reaggregated Ovary Formation: The PGCLCs were mixed with embryonic ovarian cells to form a reaggregated ovary-like structure in vitro. This structure included both germ cells (PGCLCs) and somatic cells (similar to granulosa cells), which are essential for supporting oocyte development. Oocyte Development: Within the reaggregated ovary, PGCLCs differentiated into oocytes. These oocytes progressed through meiotic prophase I to the diplotene stage, forming early follicles without undergoing arrest. This process took approximately three weeks. 2. In Vitro Growth (IVG): Secondary Follicle Development: The early follicles were then grown in vitro, transitioning from the primary to the secondary follicle stage over 11 days. During this phase, the oocytes grew larger and accumulated the necessary RNA and proteins, supported by the surrounding granulosa cells. Growth Factor Stimulation: The growth of secondary follicles was further stimulated by adding growth factors such as growth differentiation factor 9 (GDF-9), bone morphogenetic protein 15 (BMP15), and follicle-stimulating hormone (FSH). This led to the formation of cumulus-oocyte complexes, where the oocytes reached the germinal vesicle (GV) stage. 3. In Vitro Maturation (IVM): Maturation to Metaphase II (MII): The cumulus-oocyte complexes were then transferred to standard in vitro maturation (IVM) conditions, where they were cultured for 16 hours. This facilitated the development of the fully grown GV-stage oocytes to the metaphase II (MII) stage, which is the stage at which oocytes are ready for ovulation. Fertilization: The MII-stage oocytes were then capable of being fertilized in vitro. Leture 8: Stem cells and female fertility 17  Overall, the complete process from stem cell to fertilizable mouse oocyte took almost 33 days. Outcomes Phenotypically Normal Offspring: The pups that were produced following in vitro fertilization (IVF) using these in vitro-generated MII oocytes appeared phenotypically normal. They were fertile and remained healthy for at least 11 months of age, demonstrating that it is possible to generate viable offspring from oocytes created entirely in a laboratory setting. Epigenetic Analysis: The researchers performed an analysis of select imprinted loci, which are specific genomic regions where gene expression is controlled by epigenetic mechanisms. The results suggested that the epigenetic status of the offspring generated from in vitro-grown oocytes was very similar to that of normal wild-type Leture 8: Stem cells and female fertility 18  mice, indicating that the process does not significantly disrupt normal epigenetic patterns. Challenges with Embryonic Development: Embryonic development from in vitro-generated oocytes was often arrested at the cleavage stage, a critical early stage of embryo development. Early and late gestation resorptions (failure of pregnancy where the embryo is absorbed back into the body) were common. ^^ This suggests that while the oocytes were capable of being fertilized and initiating development, they were often of suboptimal quality, leading to high failure rates. Low Success Rate: The success rate of full-term development using two-cell embryos from in vitro-generated oocytes was reported to be around 3.5%. This is substantially lower than the success rate of 61.7% typically achieved using oocytes generated in vivo (within a living organism). This stark contrast highlights the current limitations in the methodology used to produce oocytes entirely in vitro. Future Directions: The study concluded that although the process is feasible, it is far from optimal. The low efficiency and reduced quality of the in vitro-generated oocytes point to the need for ongoing refinement of the culture conditions and methodologies. Future research will likely focus on improving these conditions to increase the success rate and quality of oocytes produced in vitro. Can we make human oocytes? Follicle development and oocyte maturation in humans is a lengthy process. Unlike mice, where the entire cycle can be mimicked in vitro in a relatively shorter timeframe, the extended duration required for Leture 8: Stem cells and female fertility 19  human oocyte development makes it difficult to sustain the necessary conditions in a laboratory setting. Currently, our ability to support the full in vitro development of human follicles is quite limited. This is due to several factors: Physical and Molecular Interactions: Successful oocyte maturation relies heavily on complex interactions between the oocyte and surrounding granulosa cells. These interactions are crucial for providing the necessary nutrients, growth factors, and hormonal support throughout the stages of follicular development. Dynamic Needs: The requirements for nutrients, growth factors, and hormonal support change dynamically throughout the process of follicle development. Replicating these dynamic conditions in vitro is challenging and requires precise control over the culture environment. The successes achieved with in vitro oocyte development in mice do not directly translate to human oocyte development. The differences in physiology between species mean that methods effective in mice may not work as well in humans. Obtaining human ovarian material for research is more difficult due to ethical, legal, and practical constraints, which further limits the progress in this area. Current Progress and Limitations: Partial Success in Human Models: While fully replicating the entire process from the primordial follicle stage to mature oocytes in vitro remains challenging, there has been progress in specific stages. For example, researchers have been able to grow human follicles through phase two of the development process, and in vitro maturation (IVM) of oocyte complexes is already a common practice in fertility clinics. Concerns with In Vitro Manipulation: Despite these advancements, extensive in vitro manipulation raises concerns about the viability, genetic integrity, and epigenetic programming of the gametes produced. Prolonged Leture 8: Stem cells and female fertility 20  culture and manipulation can lead to issues such as genetic abnormalities or improper epigenetic modifications, which may affect the health and viability of the resulting embryos. Study 2: Metaphase II oocytes from human unilaminar follicles grown in a multi-step culture system Key Points of the Study: 1. Unilaminar Follicles: The study focused on human unilaminar follicles, specifically primordial or primary follicles, which are the earliest stages of follicle development. These follicles were cultured using a multi- step system designed to mimic the natural progression of follicle maturation in the human ovary. 2. Three-Step Culture Process: The researchers used a three-step in vitro culture system to grow the oocytes from the primordial follicle stage through to the maturation stage (MII). This process successfully supported the oocytes' development, allowing them to undergo meiotic maturation, a critical step in preparing the oocytes for potential fertilization. 3. Meiotic Maturation: The oocytes were able to reach the MII stage, demonstrating that complete in vitro maturation is possible. However, there were notable concerns about the quality of the oocytes produced, which highlights the need for further optimization of the culture conditions. Observations and Concerns: 1. Large Polar Bodies: Presence of large polar bodies in the MII oocytes. Polar bodies are small cells that result from the asymmetric division of oocytes during meiosis. Ideally, polar bodies should be small, indicating that the majority of the cytoplasmic material Leture 8: Stem cells and female fertility 21  remains in the oocyte, which is crucial for its viability. The presence of large polar bodies suggests that the division was not as asymmetric as it should be, leading to the loss of important cytoplasmic components, which may compromise the oocyte's quality. 2. Cumulus Cell Expansion: Limited expansion of the cumulus cells surrounding the oocytes. Cumulus cells play a crucial role in oocyte development and maturation, and their proper expansion is a marker of healthy oocytes. In this study, the cumulus cells did not expand as expected, indicating that the in vitro conditions may not fully support the natural maturation process. 3. Comparison to Healthy Oocytes: The study provided a comparison between the in vitro-grown oocytes and what is considered a healthy, good-quality oocyte. Ideally, a mature oocyte should be large with a very small polar body, and the cumulus cells should be fully expanded. The in vitro-grown oocytes in this study, however, showed deviations from this ideal, suggesting that while the process is feasible, the oocytes produced may not yet be of optimal quality for successful fertilization and development. Conclusion and Future Directions: The study by Telfer's group is a significant milestone, demonstrating that it is possible to grow human oocytes entirely in vitro from the primordial follicle stage to the MII stage. However, the issues observed with large polar bodies and inadequate cumulus Leture 8: Stem cells and female fertility 22  cell expansion indicate that the current in vitro methods need further refinement to produce oocytes of higher quality. Future research will likely focus on optimizing the culture conditions to better mimic the natural environment of the human ovary, improving the viability and developmental potential of in vitro-grown oocytes for use in fertility treatments. Leture 8: Stem cells and female fertility 23