Flor Sánchez

Molecular control of oogenesis

Oogenesis is a complex process regulated by a vast number of intra- and extra-ovarian factors. Oogonia, which originate from primordial germ cells, proliferate by mitosis and form primary oocytes that arrest at the prophase stage of the first meiotic division until they are fully-grown. Within primary oocytes, synthesis and accumulation of RNAs and proteins throughout oogenesis are essential for oocyte growth and maturation; and moreover, crucial for developing into a viable embryo after fertilization. Oocyte meiotic and developmental competence is gained in a gradual and sequential manner during folliculogenesis and is related to the fact that the oocyte grows in interaction with its companion somatic cells. Communication between oocyte and its surrounding granulosa cells is vital, both for oocyte development and for granulosa cells differentiation. Oocytes depend on differentiated cumulus cells, which provide them with nutrients and regulatory signals needed to promote oocyte nuclear and cytoplasmic maturation and consequently the acquisition of developmental competence.The purpose of this article is to summarize recent knowledge on the molecular aspects of oogenesis and oocyte maturation, and the crucial role of cumulus-cell interactions, highlighting the valuable contribution of experimental evidences obtained in animal models. This article is part of a Special Issue entitled: Molecular Genetics of Human Reproductive Failure.

Quantification of oocyte-specific transcripts in follicle-enclosed oocytes during antral development and maturation in vitro

Oocyte cytoplasmic maturation is influenced by the quantity of synthesized RNA and proteins accumulated and stored during growth. Transcriptional repression and degradation of transcripts occur during oocyte nuclear maturation, and prolonged transcriptional arrest might compromise RNA stores for early development. RNA quantification of key genes in oocytes might be valuable when setting up in vitro cultures that lack the normal hormonal interplay found in vivo. This study quantifies gene expression levels in relation to follicle culture time and time of oocyte maturation in a mouse model. RNA levels of Gdf-9, Bmp-15, Mater, Zar-1, Npm-2 and Fgf-8 were measured in germinal vesicle oocytes along fixed times during in vitro follicle development. For all genes, the highest mRNA levels were detected in oocytes in the pre-antral follicle stage. Antrum formation was associated with a progressive shutdown in transcription leading to mRNA values lower than those in vivo preovulatory oocytes by extending period of in vitro culture. In contrast to in vitro-matured oocytes, the in vivo oocytes from 22- and 29-day-old prepubertal animals obtained after pregnant mares serum gonadotrophin and human chorionic gonadotrophin priming did not down-regulate transcripts upon maturation stimulus except for Mater. These findings show that oocyte gene expression patterns under in vitro conditions can, at certain times, mimic what is reported to occur under in vivo conditions. Moreover, they also show that meiotically competent oocytes kept in a prolonged transcriptionally inactive stage express altered levels of key transcripts compared with in vivo in both immature and mature oocytes.

Different Follicle-Stimulating Hormone Exposure Regimens During Antral Follicle Growth Alter Gene Expression in the Cumulus-Oocyte Complex in Mice

Follicle-stimulating hormone (FSH) and oocyte-secreted factors influence granulosa cell differentiation and follicle development. Whereas FSH stimulates the expression of mural cell transcripts, oocyte-secreted factors regulate specific cumulus cell genes and suppress the appearance of mural cell transcripts. This study addresses the extent to which clinically relevant changes in FSH doses applied during antral follicle development in vitro could alter the expression of oocyte and cumulus cell transcripts. A 12-day culture system in which mouse ovarian preantral follicles can grow to preovulatory follicles was used. The following three FSH regimens were considered: 1) continuous exposure to an FSH level of 10 mIU/ml (control), 2) decreasing concentrations of FSH (low FSH), and 3) an FSH level of 25 mIU/ml (high FSH) as soon as the antrum is formed. Transcripts in oocytes (Gdf9, Bmp15, and Fgf8) and in cumulus cells (Amh, Lhcgr, Ar, and Pfkp) were quantified by real-time PCR. Under high FSH, the three oocyte transcripts were upregulated, while in cumulus cells a shutdown of the Amh signal and substantial increases in Lhcgr and Ar expression were measured. In contrast, low FSH tended to reduce Lhcgr to levels comparable to those in vivo. Levels of Pfkp were not affected by FSH doses. These results demonstrate that a 2.5-fold increase in FSH changes both oocyte and cumulus cell transcript levels. Conversely, a decrease in FSH does not affect transcript levels but seems to limit inappropriate Lhcgr expression. Modulating FSH within physiological ranges during the antral phase of culture alters cumulus cell differentiation.

Oocyte and Cumulus Cell Transcripts from Cultured Mouse Follicles Are Induced to Deviate from Normal In Vivo Conditions by Combinations of Insulin, Follicle-Stimulating Hormone, and Human Chorionic Gonadotropin

Gonadotropins and insulin are major regulators of cell proliferation, differentiation, and survival in cultured mouse ovarian follicles. Applications of variable doses of insulin in combination with follicle-stimulating hormone (FSH) and luteinizing hormone (LH) were studied at the gene expression level in oocytes and cumulus cells. Early preantral follicles grown over 9 days were sequentially exposed to combinations of doses of insulin, FSH, and human chorionic gonadotropin (hCG). From culture Day 1 to 6 (preantral stage), two insulin concentrations (5 ng/ml and 5 μg/ml) were combined with 10 mIU/ml FSH. From Days 6 to 9 (antral stage), the three variable gonadotropin treatments set under each insulin condition were 10 mIU/ml FSH, 25 mIU/ml FSH, and 25 mIU/ml FSH plus 3 mIU/ml hCG. The Gdf9, Bmp15, Fgf8, Dazl, Pou5f1, and Pik3ca mRNA transcripts were quantified in oocytes, and the Amh, Lhcgr, Hsd3b1, Vegfa, and Insig1 mRNA transcripts were quantified in cumulus cells. In vivo controls were unprimed and eCG (equine chorionic gonadotropin)-primed prepubertal female mice. During the preantral stage, none except the Amh transcripts was regulated by insulin. Oocyte transcripts were not affected by the variable gonadotropin treatments on the last culture day but were upregulated in the combination of high insulin plus 25 mIU/ml FSH. Under low insulin conditions, high FSH levels increased levels of Lhcgr and Vegfa expression, and hCG abated this effect. However, under high insulin conditions, hCG upregulated levels of Lhcgr, Vegfa, and Insig1 mRNA. High insulin concentrations upregulated Hsd3b1 transcripts. These results demonstrate that in an in vitro follicle culture, a near physiological insulin background yields oocyte and cumulus cell transcript levels that are more similar to those in vivo. Gene expression in the cumulus–oocyte complex during antral growth is differentially regulated by exposure to high levels of gonadotropins, depending on the background insulin concentration in culture medium.

Human cumulus-enclosed germinal vesicle oocytes from early antral follicles reveal heterogeneous cellular and molecular features associated with in vitro maturation capacity

STUDY QUESTION Are oocyte size, chromatin remodelling, transcriptional activity and mitochondrial distribution in human immature oocytes from early antral follicles retrieved for in vitro maturation (IVM) associated with the acquisition of meiotic competence? SUMMARY ANSWER Oocyte size, chromatin compaction, cessation of RNA synthesis and mitochondria rearrangement around the nucleus are associated with the oocytes potential to resume meiosis in vitro. WHAT IS KNOWN ALREADY Information on oocyte features that confer meiotic competence in human mainly derives from germinal vesicle (GV) oocytes that failed to resume meiosis following an hCG trigger after ovulation induction cycles. Characterization of cumulus-enclosed GV oocytes from small antral follicles prior to IVM provides knowledge on the initial oocyte status and suggests culture requirements in order to promote oocyte competence in vitro. STUDY DESIGN, SIZE, DURATION Prospective collection of 107 oocytes immediately after retrieval (before IVM) and of 293 GV oocytes that had failed to resume meiosis (after IVM). PARTICIPANTS/MATERIALS, SETTING, METHODS Human oocytes were collected from women with polycystic ovary syndrome (PCOS), receiving in total 450 IU of highly purified-hMG for IVM treatment (patients) or who donated oocytes for IVM research (donors). Oocytes at GV-stage were retrieved from follicles <10 mm (range 2-10 mm) diameter, before IVM (oocytes at retrieval) or those that failed to mature after IVM (meiotically incompetent). Oocytes were allocated for either mitochondrial staining, by incubating in mitotracker red and then fixed; or for nascent RNA staining, which was assessed by fluorescent labelling (Click-iT(®) RNA Assay). In every case, oocyte diameter was recorded and chromatin was stained after oocyte fixation. GV-stage oocytes were analysed by confocal laser-scanning microscopy and their characteristics were compared and related to their meiotic competence. MAIN RESULTS AND THE ROLE OF CHANCE Analysis of oocytes at the immature GV-stage revealed that oocytes at retrieval were significantly larger than those that failed to resume meiosis after IVM (112.7 versus 109.6 µm, P < 0.0001). Oocytes assessed at retrieval showed that 50.6% had a condensed chromatin configuration (perinucleolar chromatin rim) and were consistently transcriptionally silent. This rate matched maturation rates in our current in vitro culture system (49%). However, oocytes that had not reinitiated meiosis after 30 h IVM demonstrated, apart of being smaller in diameter, significantly higher rates of dispersed or intermediate chromatin (P = 0.005). Analysis of mitochondrial distribution revealed that many oocytes at retrieval displayed mitochondrial internalization towards the nucleus (12/30) or a perinuclear mitochondrial distribution (6/30). These mitochondrial patterns were observed more rarely in GV incompetent oocytes following 30 h IVM (16/98 and 1/98, respectively). LIMITATIONS, REASONS FOR CAUTION Most of the analyses involved the use of invasive techniques. Hence, despite the fact that these data deliver essential information on the intrinsic oocyte maturational and developmental status, a direct match with embryological outcomes could not be established. WIDER IMPLICATIONS OF THE FINDINGS The evidence described here can aid in tailoring IVM systems in order to promote completion of nuclear and cytoplasmic maturation of unexpanded cumulus-oocyte complexes. STUDY FUNDING/COMPETING INTERESTS This study was supported by research grants by the Institute for the Promotion of Innovation by Science and Technology in Flanders, project numbers IWT 130327 and 110680; the Fund for Research Flanders, project number FWO G.0343.13, the Belgian Foundation Against Cancer (HOPE project) and COOK Medical. None of the authors has any competing interest to declare.

In vitro follicle growth under non-attachment conditions and decreased FSH levels reduces Lhcgr expression in cumulus cells and promotes oocyte developmental competence

PurposeThe in-vitro environment influences oocyte competence and gene expression in cumulus cells and oocytes. Effects of culturing under non-attachment conditions and varying follicle exposure to FSH were investigated at the mRNA level and on oocyte developmental capacity.MethodsQuantitative PCR analysis of Gdf9, Mater, Nmp2 (in oocytes), Lhcgr and Amh (in cumulus cells), and oocyte developmental competence after in vitro follicle culture were evaluated.ResultsFollicle survival (98.7%) and polar body rate (94%) were similar for all conditions. Estradiol and progesterone production were significantly lower in non-attachment follicles (10-fold and 3-fold, respectively). Under non-attachment conditions, a higher two-cell rate (69.9%) and total blastocyst yield (48.5%) were obtained and, by decreasing FSH levels during culture, Lhcgr transcripts were significantly reduced to levels similar to in-vivo. Levels of oocyte-specific transcripts were not significantly influenced by in-vitro conditions.ConclusionNon-attachment conditions influence follicle steroid secretory capacity and, together with dynamic FSH doses, positively influence cumulus cell gene expression and oocyte developmental competence.

An improved IVM method for cumulus-oocyte complexes from small follicles in polycystic ovary syndrome patients enhances oocyte competence and embryo yield

STUDY QUESTION Are meiotic and developmental competence of human oocytes from small (2-8 mm) antral follicles improved by applying an optimized IVM method involving a prematuration step in presence of C-Type Natriuretic Peptide (CNP) followed by a maturation step in presence of FSH and Amphiregulin (AREG)? SUMMARY ANSWER A strategy involving prematuration culture (PMC) in the presence of CNP followed by IVM using FSH + AREG increases oocyte maturation potential leading to a higher availability of Day 3 embryos and good-quality blastocysts for single embryo transfer. WHAT IS KNOWN ALREADY IVM is a minimal-stimulation ART with reduced hormone-related side effects and risks for the patients, but the approach is not widely used because of an efficiency gap compared to conventional ART. In vitro systems that enhance synchronization of nuclear and cytoplasmic maturation before the meiotic trigger are crucial to optimize human IVM systems. However, previous PMC attempts have failed in sustaining cumulus-oocyte connections throughout the culture period, which prohibited a normal cumulus-oocyte communication and precluded an adequate response by the cumulus-oocyte complex (COC) to the meiotic trigger. STUDY DESIGN, SIZE, DURATION A first prospective study involved sibling oocytes from a group of 15 patients with polycystic ovary syndrome (PCOS) to evaluate effects of a new IVM culture method on oocyte nuclear maturation and their downstream developmental competence. A second prospective study in an additional series of 15 women with polycystic ovaries characterized and fine-tuned the culture conditions. PARTICIPANTS/MATERIALS, SETTING, METHODS Fifteen women with PCOS (according to Rotterdam criteria) underwent IVM treatment after 3-5 days of highly purified human menopausal gonadotropin (HP-hMG) stimulation and no human chorionic gonadotropin (hCG) trigger before oocyte retrieval. A first study was designed with sibling oocytes to prospectively evaluate the impact of an IVM culture method: 24 h PMC with CNP + 30 h IVM with FSH and AREG, on embryo yield, in comparison to the standard (30 h) IVM clinical protocol (Group I, n = 15). A second prospective study was performed in 15 women with polycystic ovaries, to characterize and optimize the PMC conditions (Group II, n = 15). The latter study involved the evaluation of oocyte meiotic arrest, the preservation of cumulus-oocyte transzonal projections (TZPs), the patterns of oocyte chromatin configuration and cumulus cells apoptosis following the 24 and 46 h PMC. Furthermore, oocyte developmental potential following PMC (24 and 46 h) + IVM was also evaluated. The first 20 good-quality blastocysts from PMC followed by IVM were analysed by next generation sequencing to evaluate their aneuploidy rate. MAIN RESULTS AND THE ROLE OF CHANCE PMC in presence of CNP followed by IVM using FSH and AREG increased the meiotic maturation rate per COC to 70%, which is significantly higher than routine standard IVM (49%; P ≤ 0.001). Hence, with the new system the proportion of COCs yielding transferable Day 3 embryos and good-quality blastocysts increased compared to routine standard IVM (from 23 to 43%; P ≤ 0.001 and from 8 to 18%; P ≤ 0.01, respectively). CNP was able to prevent meiosis resumption for up to 46 h. After PMC, COCs had preserved cumulus-oocyte TZPs. The blastocysts obtained after PMC + IVM did not show increased aneuploidy rates as compared to blastocysts from conventional ART. LIMITATIONS REASONS FOR CAUTION The novel IVM approach in PCOS patients was tested in oocytes derived from small antral follicles which have an intrinsically low developmental potential. Validation of the system would be required for COCs from different (larger) follicular sizes, which may involve further adjustment of PMC conditions. Furthermore, considering that this is a novel strategy in human IVM treatment, its global efficiency needs to be confirmed in large prospective randomized controlled trials. The further application in infertile patients without PCOS, e.g. cancer patients, remains to be evaluated. WIDER IMPLICATIONS OF THE FINDINGS The findings of this pilot study suggest that the efficiency gap between IVM and conventional IVF can be reduced by fine-tuning of the culture methods. This novel strategy opens new perspectives for safe and patient-friendly ART in patients with PCOS. STUDY FUNDING/COMPETING INTEREST(S) IVM research at the Vrije Universiteit Brussel has been supported by grants from: the Institute for the Promotion of Innovation by Science and Technology in Flanders (Agentschap voor Innovatie door Wetenschap en Technologie-IWT, project 110680); the Fund for Research Flanders (Fonds Wetenschappelijk Onderzoek-Vlaanderen-FWO, project G.0343.13), the Belgian Foundation Against Cancer (HOPE project, Dossier C69). The authors have no conflicts of interest.

Mouse Cumulus-Oocyte Complexes from In Vitro-Cultured Preantral Follicles Suggest an Anti-Luteinizing Role for the EGF Cascade in the Cumulus Cells

The mammalian ovulatory process is a fairly complex succession of events that leads to the release of a competent oocyte. The luteinizing hormone (LH) triggers the cascade of events, which starts with the production of secondary messengers in the follicular wall and ends with the release of a fertilizable oocyte. Most of these events can be reproduced using in vitro models, which offer a wide range of possibilities for study strategies. Although it is accepted that epidermal growth factor receptor (EGFR) activation is required for transmission of the LH-initiated signal, we hypothesized that LH receptor activation might also play a role in oocyte meiotic resumption and cumulus cell response, because the current mouse preantral follicle in vitro model expresses functional LH receptor. To separate the LH-mediated response and the epidermal growth factor (EGF)-mediated response (following LH stimulus), in vitro-grown mouse ovarian follicles were stimulated for ovulation with a combination of human chorionic gonadotropin (hCG) plus galardin (inhibitor for the release of endogenous EGF-like factors) or hCG plus galardin plus EGF. Results suggest that the stimulation provided by LH (hCG) is insufficient to induce a maximum oocyte meiotic resumption and that EGFR activation is also required. Analysis of transcript levels of Egfr, Ereg, Cyp19a1, Hsd3b1, Adamts1, and Has2 in cumulus cells further indicate that the triggers for the EGFR cascade preserve the expression profile of the studied transcripts. Therefore, it is proposed that within this in vitro mouse model, EGF signaling during ovulation might protect the cumulus cells from the potential luteinizing effects of LH.

Immature Oocytes from Unprimed Juvenile Mice Become a Valuable Source for Embryo Production When Using C-Type Natriuretic Peptide as Essential Component of Culture Medium

ABSTRACT C-type natriuretic peptide (CNP) and its receptor natriuretic peptide receptor 2 (NPR2) play a paramount role in the maintenance of oocyte meiotic arrest in antral follicles via the regulation of the intra-oocyte levels of cyclic guanosine monophosphate and cyclic adenosine monophosphate. We investigated the potential of CNP 1) to maintain oocyte meiotic arrest during a prolonged prematuration culture and 2) to sustain acquisition of developmental competence of immature cumulus-oocyte complexes (COCs). Compact COCs were collected from small antral follicles of prepubertal unprimed mice and placed in prematuration culture under different CNP-supplemented media conditions. A preliminary analysis showed a dose-dependent effect of CNP on the maintenance of meiotic arrest. A dose of 25 nM maintained oocytes under meiotic arrest for 24 h, and this period was extended to 48 h in the presence of estradiol. Analysis of transzonal projections of COCs cultured with CNP indicated that oocyte-cumulus connections were well preserved after the prolonged prematuration culture. Furthermore, CNP medium supplemented with FSH and GDF9 promoted oocyte growth and induced a shift in oocyte chromatin configuration from a predominantly dispersed to a condensed configuration. Following in vitro maturation, oocytes cultured under CNP were capable of extruding the first polar body at a high rate (around 80%). Blastocyst formation was significantly improved when oocytes were cultured under CNP-supplemented medium containing FSH and GDF9. This study reports for the first time a prolonged prematuration culture system, with CNP as the pivotal factor, that can efficiently maintain oocytes retrieved from unprimed prepubertal mice under meiotic arrest while promoting their acquisition of developmental competence.

Survey of Fertility Preservation Options Available to Patients With Cancer Around the Globe

Purpose Oncofertility focuses on providing fertility and endocrine-sparing options to patients who undergo life-preserving but gonadotoxic cancer treatment. The resources needed to meet patient demand often are fragmented along disciplinary lines. We quantify assets and gaps in oncofertility care on a global scale. Methods Survey-based questionnaires were provided to 191 members of the Oncofertility Consortium Global Partners Network, a National Institutes of Health–funded organization. Responses were analyzed to measure trends and regional subtleties about patient oncofertility experiences and to analyze barriers to care at sites that provide oncofertility services. Results Sixty-three responses were received (response rate, 25%), and 40 were analyzed from oncofertility centers in 28 countries. Thirty of 40 survey results (75%) showed that formal referral processes and psychological care are provided to patients at the majority of sites. Fourteen of 23 respondents (61%) stated that some fertility preservation services are not offered because of cultural and legal barriers. The growth of oncofertility and its capacity to improve the lives of cancer survivors around the globe relies on concentrated efforts to increase awareness, promote collaboration, share best practices, and advocate for research funding. Conclusion This survey reveals global and regional successes and challenges and provides insight into what is needed to advance the field and make the discussion of fertility preservation and endocrine health a standard component of the cancer treatment plan. As the field of oncofertility continues to develop around the globe, regular assessment of both international and regional barriers to quality care must continue to guide process improvements.Purpose Oncofertility focuses on providing fertility and endocrine-sparing options to patients who undergo life-preserving but gonadotoxic cancer treatment. The resources needed to meet patient demand often are fragmented along disciplinary lines. We quantify assets and gaps in oncofertility care on a global scale. Methods Survey-based questionnaires were provided to 191 members of the Oncofertility Consortium Global Partners Network, a National Institutes of Health–funded organization. Responses were analyzed to measure trends and regional subtleties about patient oncofertility experiences and to analyze barriers to care at sites that provide oncofertility services. Results Sixty-three responses were received (response rate, 25%), and 40 were analyzed from oncofertility centers in 28 countries. Thirty of 40 survey results (75%) showed that formal referral processes and psychological care are provided to patients at the majority of sites. Fourteen of 23 respondents (61%) stated that some fertility preservation services are not offered because of cultural and legal barriers. The growth of oncofertility and its capacity to improve the lives of cancer survivors around the globe relies on concentrated efforts to increase awareness, promote collaboration, share best practices, and advocate for research funding. Conclusion This survey reveals global and regional successes and challenges and provides insight into what is needed to advance the field and make the discussion of fertility preservation and endocrine health a standard component of the cancer treatment plan. As the field of oncofertility continues to develop around the globe, regular assessment of both international and regional barriers to quality care must continue to guide process improvements.