Cumulus Complexes Research Articles

The Effect of Simulated Physiological Oocyte Maturation (SPOM) and L-Carnitine on Bovine Oocyte Developmental Competence

Background: Simulated Physiological Oocyte Maturation (SPOM) mimics in vitro the physiological events of oocyte maturation in the presence of cAMP modulators. These modulators increase the intracellular concentrations of cAMP, which inhibits the immediate resumption of meiosis and gives the oocyte more time to gain optimal developmental competence. In addition, L-carnitine helps to increase the energy supply of cells through the β-oxidation of fatty acids. This study aimed to investigate the effect of SPOM and L-carnitine supplementation during In Vitro Maturation (IVM) and In Vitro Culture (IVC) on the developmental competence of bovine oocytes. Methods: Ovarian Cumulus Complexes (COCs) were cultured in the presence or absence of forskolin+IBMX during the first 2 hr of IVM (pre-IVM) with or without L-carnitine (LC) during IVM or IVC in six experimental groups as follows: I) pre-IVM (pre-IVM group), II) pre-IVM with L-carnitine supplementation during IVM (pre-IVM/LC group), III) L-carnitine supplementation during IVM (IVM/LC group), IV) L-carnitine supplementation during in vitro culture (IVC/LC group), V) pre-IVM+ IVC/LC group, and VI) no treatment during IVM and IVC (Control group). The cleavage and blastocyst rates, the blastocysts’ total cells number, and the expression of Nanog, Bax, Oct4, Cdx2, and Ifnt genes in resulting blastocysts were assessed. To assess differences among experimental groups, a one-way analysis of variance was initially employed, followed by post hoc Fisher LSD. The difference between groups was considered statistically significant when p<0.05. Results: The cleavage and blastocyst rates in the Pre-IVM and Pre-IVM/LC groups was higher than control group and other groups (p≤0.05) except for IVC/LC and IVM/LC groups, respectively. The number of blastocyst’s Inner Cell Mass (ICM) in pre-IVM and Pre-IVM/LC groups as well as the ratio of ICM/TE were higher than control group (p<0.05). The expression of OCT4, CDX2, and IFNT increased in both the pre-IVM and pre-IVM/LC groups compared to the control group (p<0.05). Conclusion: In conclusion, the application of SPOM-adapted IVM and L-carnitine during IVM of bovine oocyte improves the quantity and quality of the resulting embryos.

GDF9His209GlnfsTer6/S428T and GDF9Q321X/S428T bi-allelic variants caused female subfertility with defective follicle enlargement

BackgroundAntral follicles consist of an oocyte cumulus complex surrounding by somatic cells, including mural granulosa cells as the inner layer and theca cells as the outsider layer. The communications between oocytes and granulosa cells have been extensively explored in in vitro studies, however, the role of oocyte-derived factor GDF9 on in vivo antral follicle development remains elusive due to lack of an appropriate animal model. Clinically, the phenotype of GDF9 variants needs to be determined.MethodsWhole-exome sequencing (WES) was performed on two unrelated infertile women characterized by an early rise of estradiol level and defect in follicle enlargement. Besides, WES data on 1,039 women undergoing ART treatment were collected. A Gdf9Q308X/S415T mouse model was generated based on the variant found in one of the patients.ResultsTwo probands with bi-allelic GDF9 variants (GDF9His209GlnfsTer6/S428T, GDF9Q321X/S428T) and eight GDF9S428T heterozygotes with normal ovarian response were identified. In vitro experiments confirmed that these variants caused reduction of GDF9 secretion, and/or alleviation in BMP15 binding. Gdf9Q308X/S415T mouse model was constructed, which recapitulated the phenotypes in probands with abnormal estrogen secretion and defected follicle enlargement. Further experiments in mouse model showed an earlier expression of STAR in small antral follicles and decreased proliferative capacity in large antral follicles. In addition, RNA sequencing of granulosa cells revealed the transcriptomic profiles related to defective follicle enlargement in the Gdf9Q308X/S415T group. One of the downregulated genes, P4HA2 (a collagen related gene), was found to be stimulated by GDF9 protein, which partly explained the phenotype of defective follicle enlargement.ConclusionsGDF9 bi-allelic variants contributed to the defect in antral follicle development. Oocyte itself participated in the regulation of follicle development through GDF9 paracrine effect, highlighting the essential role of oocyte-derived factors on ovarian response.

Chronological age enhances aging phenomena and protein nitration in oocyte.

The average age of childbearing has increased over the years contributing to infertility, miscarriages, and chromosomal abnormalities largely invoked by an age-related decline in oocyte quality. In this study, we investigate the role of nitric oxide (NO) insufficiency and protein nitration in oocyte chronological aging. Mouse oocytes were retrieved from young breeders (YB, 8-14 weeks [w]), retired breeders (RB, 48-52w) and old animals (OA, 80-84w) at 13.5 and 17 hours after ovulation trigger. They were assessed for zona pellucida dissolution time (ZPDT); ooplasmic microtubule dynamics (OMD); cortical granule (CG) status and spindle morphology (SM), as markers of oocyte quality. Sibling oocytes from RB were exposed to NO supplementation and assessed for aging phenomena (AP). All oocyte cumulus complexes were subjected to fluorescence nitrotyrosine (NT) immunocytochemistry and confocal microscopy to assess morphology and protein nitration. At 13.5 h from hCG trigger, oocytes from RB compared to YB had significantly increased ZPDT (37.8 ± 11.9 vs 22.1 ± 4.1 seconds [s]), OMD (46.9 vs 0%), CG loss (39.4 vs 0%), and decreased normal SM (30.3 vs 81.3%), indicating premature AP that worsened among oocytes from RB at 17 hours post-hCG trigger. When exposed to SNAP, RB AP significantly decreased (ZPDT: 35.1 ± 5.5 vs 46.3 ± 8.9s, OMD: 13.3 vs 75.0% and CG loss: 50.0 vs 93.3%) and SM improved (80.0 vs 14.3%). The incidence of NT positivity was significantly higher in cumulus cells (13.5 h, 46.7 ± 4.5 vs 3.4 ± 0.7%; 17 h, 82.2 ± 2.9 vs 23.3 ± 3.6%) and oocytes (13.5 h, 57.1 vs 0%; 17 h, 100.0 vs 55.5%) from RB compared to YB. Oocytes retrieved decreased with advancing age (29.8 ± 4.1 per animal in the YB group compared to 10.2 ± 2.1 in RB and 4.0 ± 1.6 in OA). Oocytes from OA displayed increased ZPDT, major CG loss, increased OMD and spindle abnormalities, as well as pronuclear formation, confirming spontaneous meiosis to interphase transition. Oocytes undergo zona pellucida hardening, altered spindle and ooplasmic microtubules, and premature cortical granule release, indicative of spontaneous meiosis-interphase transition, as a function of chronological aging. These changes are also associated with NO insufficiency and protein nitration and may be alleviated through supplementation with an NO-donor.

Follicle Fitness: Key Factors for Ovarian Health and Better Contraceptive Strategies

The ovarian follicle is an important endocrine organ that controls the female reproductive cycle through hypothalamic-pituitary-ovarian hormonal communication, an intricate network that governs reproductive fitness. Using genomics and molecular techniques our research is uncovering the processes by which ovarian folliculogenesis and oocyte development is regulated by the interplay of endocrine and paracrine signals that mediate coordinated growth and remodelling of the follicle, oocyte maturation and ultimately ovulation of a mature oocyte. We are investigating the consequences of dysregulation of hormones or their signalling networks that can lead to reproductive disorders such as polycystic ovary syndrome (PCOS) or impaired fertility through failure of ovulation or oocyte developmental competence. Ovulation is the pivotal event where the mature oocyte, surrounded by the expanded cumulus complex, is released from the follicle into the fallopian tube. This event is a critical target for understanding causes of infertility, developing therapies to reverse these causes, and also developing new contraceptives that do not disrupt endocrine function. In particular we are interrogating the molecular genomics of ovarian responses to the LH-surge and the steroids progesterone, androgen, estrogen, and others. Deciphering this endocrine network enriches our understanding of female reproductive biology and opens new avenues for interventions and fertility treatments.

Three-dimensional architecture of granulosa cell derived from oocyte cumulus complex, revealed by FIB-SEM

The oocyte cumulus complex is mainly composed of an oocyte, the perivitelline space, zona pellucida and numerous granulosa cells. The cumulus granulosa cells (cGCs) provide a particularly important microenvironment for oocyte development, regulating its growth, maturation and meiosis. In this study, we studied the internal structures and cell-to-cell connections of mouse cGCs using focused ion beam scanning electron microscopy (FIB-SEM). We reconstructed three-dimensional models to display characteristic connections between the oocyte and cGCs, and to illustrate various main organelles in cGCs together with their interaction relationship. A special form of cilium identified in granulosa cell was never reported in previous literature.

Deep machine learning applied to support clinical decision-making in the treatment of infertility using assisted reproductive technologies

Introduction. Machine learning (ML) applied to data analysis allows to more accurately and targetedly determine the most significant correctable and non-correctable predictors of onset of pregnancy in assisted reproductive technology (ART) programs in patients of different age groups. Analysis of data using various techniques and comparison of results obtained via two models will determine the most significant factors for onset of pregnancy in the ART program.Aim. To determine the most significant clinical and embryological predictors of onset of pregnancy using standard regression analysis and a decision tree algorithm to predict pregnancy in the ART program.Materials and methods. A total of 1,021 married couples were included in the retrospective study. The study analysed clinical and laboratory test findings and stimulated cycle parameters depending on the effectiveness of the ART program. A regression analysis was carried out and a decision tree algorithm was built using the Gini criterion to determine the most significant factors.Results. We identified “general” signs that require further validation on other models, including ML: the presence/absence of a history of pregnancies, stimulated cycle parameters (oocyte cumulus complex, number of metaphase II (MII) oocytes, number of zygotes), spermogram indicators on the day of puncture, number of high and good quality embryos, as well as the embryo grading.Conclusion. rFSH (follitropin-alpha, Gonal-f) gives a significant result in two of the five available age groups, follitropin-beta, corifollitropin alfa – in one of the five groups only. Building a model that includes not only the couple’s medical history data, but also molecular markers using machine learning methods will not only allow us to most accurately determine the most promising groups of patients for in vitro fertilization (IVF) programs, but also increase the efficiency of ART programs by selecting the highest quality embryo to be transferred.

Oocyte cumulus complex quality and oviduct transportation velocity in systemic autoimmune disease model mice.

Oocyte transportation by the oviduct involves the interaction between ciliated epithelial cells and cumulus cells. To determine whether the quality of cumulus-oocyte complexes (COCs) changes the transportation property of COCs, we compared the transportation velocity of COCs (TVC) by the infundibulum ex vivo with various combinations of infundibula and COCs collected from different mice. We used young and aged C57BL/6N and MRL/MpJ, and MRL/MpJ-Faslpr/lpr mice as the strains with intact female reproductive function and the systemic autoimmune disease model exhibiting oocyte pick-up dysfunction owing to the morphofunctional abnormality of ciliated epithelium, respectively. The TVC of aged MRL strains was less than that of aged C57BL/6N mice, suggesting that aging affects the transportation of COCs in MRL strains. The TVC of aged MRL/MpJ-Faslpr/lpr mice was the least among all examined combinations, whereas the TVC accelerated when the infundibulum or COCs were collected from other strains. These results indicate that the transportation property of COCs is determined not only by the ciliary function in the infundibulum but also by the properties of COCs.

Development of buffalo embryo derived from ICSI: effects of various somatic-cell co-culture

Context Somatic-cell co-culture of intracytoplasmic sperm injection (ICSI) buffalo embryos has not been reported earlier. Aim This study aimed to determine the effects of buffalo oviductal epithelial-cell, granulosa-cell, and cumulus-cell co-culture on in vitro culture of early embryo development as ICSI and post-activation. Methods Selected oocyte–cumulus complexes were cultured for 19–20 h in 50-μL drop of tissue culture medium (TCM199 + 10% buffalo follicular fluid, hCG 50 IU/mL, 0.02% arbitrary units (AU)/mL follicle-stimulating hormone and 1 μg/mL estradiol-17βE2). Oocytes reaching Metaphase II were subjected to ICSI with immobilised spermatozoa. All ICSI oocytes were activated with calcium ionophore for 5 min, followed by cycloheximide for 5 h. The embryos at 6–8-cell stages were co-cultured. Key results The morula, blastocyst, and hatched blastocyst rates when co-cultured with oviductal epithelial cells were 68.18%, 48.18%, and 30.00% respectively. The morula, blastocyst, and hatched blastocyst rates when co-cultured with cumulus cells were 51.49%, 34.33%, and 16.42% respectively. The morula, blastocyst, and hatched blastocyst rates when co-cultured with granulosa cells were 52.14%, 32.48%, and 13.68% respectively. Conclusions In vitro maturation buffalo oocytes can be fertilised in vitro with ICSI and co-cultured with different types of cells. Oviductal epithelial cell co-culture was shown to be superior in supporting in vitro embryo development in this study. Implications The oviductal epithelial cells are easy to prepare and may be used for co-culture to increase the efficiency of in vitro production of buffalo embryos.

Time line in IVF laboratory

The time line in IVF is critical, as the procedures are associated with the time of the ovulation trigger for follicular maturation, followed by sequential procedures in relation to it. Starting from oocyte recovery scheduled 36 hours post-trigger, the laboratory procedures are followed up sequentially. Oocyte cumulus complex (OCC) may be incubated until insemination or ICSI until 40 hours post-trigger. Conventional insemination may be done either short (4–6 hours) or long insemination (overnight). However, short insemination is associated with an increase in the 3-PN rate. OCC for ICSI may be denuded at least after 38 hours post-trigger, and ICSI may be performed within 2 to 4 hours post-denuding. ICSI done within 2 hours post-denuding appears to be yielding better-quality embryos. Time-lapse studies have indicated that it is possible to obtain higher clinical and live birth rates with embryos reaching the blastocyst stage faster than other embryos. Embryo transfers done with Day 3 embryos result in a higher pregnancy rate than Day 2 while blastocyst transfers perform better than day 3 transfers. Mobilization of patients after ET may not affect the outcome.

The oocyte cumulus complex regulates mouse sperm migration in the oviduct

As the time of ovulation draws near, mouse spermatozoa move out of the isthmic reservoir, which is a prerequisite for fertilization. However, the molecular mechanism remains unclear. The present study revealed that mouse cumulus cells of oocytes–cumulus complexes (OCCs) expressed transforming growth factor-β ligand 1 (TGFB1), whereas ampullary epithelial cells expressed the TGF-β receptors, TGFBR1 and TGFBR2, and all were upregulated by luteinizing hormone (LH)/human chorionic gonadotropin (hCG). OCCs and TGFB1 increased natriuretic peptide type C (NPPC) expression in cultured ampullae via TGF-β signaling, and NPPC treatment promoted spermatozoa moving out of the isthmic reservoir of the preovulatory oviducts. Deletion of Tgfb1 in cumulus cells and Tgfbr2 in ampullary epithelial cells blocked OCC-induced NPPC expression and spermatozoa moving out of the isthmic reservoir, resulting in compromised fertilization and fertility. Oocyte-derived paracrine factors were required for promoting cumulus cell expression of TGFB1. Therefore, oocyte-dependent and cumulus cell-derived TGFB1 promotes the expression of NPPC in oviductal ampulla, which is critical for sperm migration in the oviduct and subsequent fertilization.

Pregnancy outcomes in infertile patients undergoing intracytoplasmic sperm injection and two interval programs of hCG-to-oocyte aspiration

This study aimed to determine the outcomes of intracytoplasmic sperm injection (ICSI) if oocyte retrieval was done 32-34 hours or 34:05-36 hours after human chorionic gonadotropin (hCG) injection. A randomized sample involving 186 patients with tubal failure was divided into groups A (96 patients) and B (90 patients). Intracytoplasmic sperm injection was performed on all patients according to described protocols. The number of oocytes retrieved, oocyte cumulus complex quality, number of fertilized eggs, and pregnancy rates were compared between groups. The total of oocytes retrieved in group B was significantly higher than in group A but not significant (P=0.068). The oocyte maturation rate was also significantly higher in the long interval group B than in the short interval group A (P=0.039). There was a significant difference between the two groups in terms of fertilization rate (0.040), and the pregnancy rate in group B was higher than that in group A, but it was not significant (P=0.055). The prolonged interval could also increase the pregnancy rate, but it was not significant. It seems that if the interval between hCG priming and oocyte retrieval is prolonged, the percentage of the number of oocytes retrieved, mature oocytes (MII), and fertilized oocytes increases.

Multifunctional 4D-Printed Sperm-Hybrid Microcarriers for Assisted Reproduction.

Remotely controllable microrobots are appealing for various biomedical in vivo applications. In particular, in recent years, ourgroup has focused on developing sperm-microcarriers to assist sperm cells with motion deficiencies or low sperm count (two of the most prominent male infertility problems) to reach the oocyte toward in-vivo-assisted fertilization. Different sperm carriers, considering their motion in realistic media and confined environments, have been optimized. However, the already-reported sperm carriers have been mainly designed to transport single sperm cell, with limited functionality. Thus, to take a step forward, here, the development of a 4D-printed multifunctional microcarrier containing soft and smart materials is reported. These microcarriers can not only transport and deliver multiple motile sperm cells, but also release heparin and mediate local enzymatic reactions by hyaluronidase-loaded polymersomes (HYAL-Psomes). These multifunctional facets enable in situ sperm capacitation/hyperactivation, and the local degradation of the cumulus complex that surrounds the oocyte, both to facilitate the sperm-oocyte interaction for the ultimate goal of in vivo assisted fertilization.

No evidence for age-related differences in mitochondrial RNA quality in the female germline.

Graphical abstract Mitochondrial quality is implicated as a contributor to declining fertility with aging. We investigated mitochondrial transcripts in oocytes and their associated cumulus cells from mice of different ages using RNA-seq. Mice aged 3 weeks, 9 weeks, and 1 year were superovulated, and 48 h later, oocyte cumulus complexes were collected by follicle puncture. We did not detect any major differences that could be attributed to aging. However, mitochondrial RNA transcripts which deviated from the consensus sequence were found at a higher frequency in cumulus cells than in their corresponding oocyte. Previous investigations have shown that variation in the sequence of mtRNA transcripts is substantial, and at least some of this can be accounted for by post-transcriptional modifications which impact base calling during sequencing. Our data would be consistent with either less post-transcriptional modification in mitochondrial RNA from oocytes than cumulus cells or with lower mtDNA mutational load.Lay summaryWomen become less fertile as they age. Shortage of energy contributes to this, caused by a decline in the quality of mitochondria (the powerhouses of the cell) in the egg. Genes are the blueprint for the cell. They are made of DNA which is copied into an RNA message, or instructions, for making proteins. We counted differences in the RNA message of developing eggs and the cells that support them during development (cumulus cells). We compared the number of these differences in mice of different ages. These age groups represent mice had not reached puberty, those of prime reproductive age, and old mothers. We did not find any differences linked to the age of the mice. However, we did find differences between the egg and the cumulus cells. In most cases, there were lower levels of mutations in eggs than there were in cumulus cells.

O-294 In-vitro maturation of oocytes from ovary tissue and ovarian longevity

Abstract Study question Is it possible to accomplish simple and robust in-vitro maturation (IVM) of oocytes from human ovarian tissue using principles of in vitro gametogenesis? Summary answer Many mature oocytes can be obtained from ovary tissue with simple media and no need for ovarian stimulation. What is known already IVM from ovarian tissue has been difficult in the past. To mature primordial follicles in vitro has become possible in mice utilizing eight ‘core genes’. It has not yet been performed in humans, and will be complicated. However, culturing germinal vesicle oocytes that have already become meiotically competent by in-vivo IVD and IVG would not be expected to be difficult. In addition, it is far easier to obtain many germinal vesicle oocytes with cortical dissection rather than with a needle. Study design, size, duration After the ovarian cortex has been dissected from the medulla and divided into slices for cryopreservation, the ‘spent’ medium in which the dissection took place was examined for free, loose cumulus complexes, and cultured for 24 to 48 hours. A variety of different culture media and gonadotrophin concentrations were employed, based on previously published data from in vitro gametogenesis in mice. Participants/materials, setting, methods A total of 119 female patients between age 2 and 35 years old underwent ovary cryopreservation (as well as in-vitro maturation of oocytes and IVM in the last 13 individuals) over a 24 year period. Up to 22 years later, 17 returned to have their ovary tissue thawed and transplanted back. Main results and the role of chance Every woman had return of ovarian function 5 months after transplant, similar to previous observations. As observed before, AMH concentration rose as FSH fell 4 months later. The grafts continued to work up to 8 years. Of the 17, 13 (76%) became pregnant with intercourse at least once, resulting in 19 healthy live births, including six live births from three women who had leukemia. Of the harvested germinal vesicle oocytes, 35% developed with simple culture media into mature metaphase II oocytes. Maturation of germinal vesicle to MII oocytes was detected between 24 and 48 h of exposure to the HCG-containing media. For most participants, the number of mature oocytes was what would be obtained from ovarian stimulation. Surprisingly, the success of IVM was not related to the specific media or to the concentration of gonadotrophin in the media. A variety of media and concentrations were intentionally used in light of now-established mechanisms of in-vitro oogenesis, to see if this understanding could be used for a simplification of IVM. Limitations, reasons for caution This is an early pilot study only, but the results are strikingly consistent with our extensive work with IVG in mice. Wider implications of the findings This study and the results of in-vitro gametogenesis reveal the limited role of the ovulatory cycle and ovarian stimulation in oocyte development other than for the oocyte to exit the ovary. The normal ovulation cycle is not needed for meiotic competence. Ovarian stimulation is only required for easy oocyte retrieval. Trial registration number Not applicable

P-724 Gene expression profiling of cumulus cells from women with adenomyosis disclose differential pattern to endometriosis: striking the chord for evaluation of a biomarker

Abstract Study question Can metabolic picture of cumulus cell (CC) assess assisted reproductive technology outcome/s in women with adenomyosis/endometriosis and have implications on treatment decisions? Summary answer The differentially expressed genes between uterine pathologies explain the functional gap to identify diagnostic/prognostic markers of CC and further dissect molecular contribution to oocyte maturation. What is known already A healthy intra-follicular environment supports achievement of oocyte developmental competence (ODC) largely via coordinated CC-follicular fluid (FF) crosstalk. Lately, promising studies document CC of endometriosis are involved in important pathways for ODC like oxidative phosphorylation, metabolism, mitochondrial function and acetylation to name a few. Similar presentation/s of adenomyosis to other gynecological diseases including endometriosis makes the diagnosis difficult. Given that the functional integrities of CCs are susceptible to concurrent pathological conditions, understanding the influence of CCs on oocyte quality compared to routine morphological analysis in presence of deleterious systemic or pelvic conditions may impact clinical decisions. Study design, size, duration Twenty-eight consented patients undergoing IVF-ICSI between January to December 2021at Institute of Reproductive Medicine, Kolkata were enrolled. CCs obtained and corresponding oocyte maturation was assessed. They were sub-stratified (n = 9) according to uterine pathologies: adenomyosis (Group A), and endometriosis (Group B) as diagnosed by transvaginal ultrasound (TVUS). Counterpart/s of male sub-fertility, defined by World Health Organization criteria (5th edition) was treated as control (Group C; n = 10). CC from mature MII and immature GV oocytes were compared. Participants/materials, setting, methods CCs collected following oocyte-denudation were evaluated by Trypan Blue viability test. Patient age and embryo development were recorded with respect to each cumulus complex. Real Time-quantitative polymerase-chain-reaction (qRTPCR) was performed for glycolysis (PFKP, PKM2, ALDOA, LDHA, TPI1), cholesterol-biosynthesis (CYP51, MVK, PMVK, EBP) and extracellular-matrix (PTGS2, PTX3, TNFAIP6, VCAN) related gene/s known to have important functions in cumulus expansion and metabolism. Patient characteristics and gene expression were correlated by Spearman’s correlation. P < 0.05 was considered statistically significant. Main results and the role of chance A significant higher (p < 0.001) age (A vs B: 35.77±2.33 vs 25.88± 1.76; A vs C: 35.77±2.33 vs 31.9±1.98) and increased number of oocytes retrieved (A vs B: 11.22±1.56 vs 8.55±0.88) was documented in adenomyosis cohort. Immature oocytes from Group A expressed enhanced cholesterol/steroid biosynthesis, with significant (A vs B; p < 0.01) up-regulation of CYP51, MVK, PMVK, EBP in spite of hyper-estrogenic environment in peripheral blood. On contrary, significant attenuation (p < 0.01) of cholesterol biosynthesis was documented in endometriosis cohort compared to control. Expression of PTGS2, PTX3 and VCAN was down-regulated (p < 0.001) (which together mediate cumulus expansion and oocyte maturation, as well as corpus luteum formation and maintenance and reduce spindle abnormalities) in both Group A and B. Expression/s of CC-PFKP and PKM2 was lower in immature compared to mature oocytes (B vs C; p < 0.01) in endometriosis making the duo responsible for follicular/oocyte growth, and embryonic development. This supports the role in promoting cumulus expansion and oocyte competence. Expression levels of other genes including ALDOA, LDHA, TPI1, and TNFAIP6 were not associated with oocyte maturity, and fertilization in either cohort/s. Interestingly, levels of VCAN, TNFAIP6, PTX3 changed significantly as a function of the subject’s age and embryo development. Limitations, reasons for caution Our findings are limited by the relatively small sample size and inherent biological variability of human samples. Several yet known or unknown factors are presumably involved in the implantation process of adenomyosis and/or endometriosis. Multi-centric systematic studies are needed to corroborate these findings and account for the probable confounding factors. Wider implications of the findings A differential metabolic dependence of CC play essential role in oocyte competence in adenomyosis influencing cumulus expansion and oocyte maturation. This would be useful to do further studies to know how molecular interface of implantation capacity with live-birth rate would be affected to complement embryonic morphological assessment/s. Trial registration number Not Applicable

Embryological Characteristics of Human Oocytes With Agar-Like Zona Pellucida and Its Clinical Treatment Strategy.

Zona pellucida (ZP) abnormalities are the cause of low fertility or infertility, agar-like ZP is more common in abnormal ZP. The purpose of this exploration is to systematically analyze the fertilization competence of agar-like ZP oocytes, the development characteristics of subsequent embryos as well as the results of embryo transfer, aiming to explore effective clinical treatment strategies. A total of 58 patients with agar-like ZP were set as the case group and the control group involved 3866 patients, in which the patients’ oocytes presented normal ZP. BMI, basal hormone levels, and hormone levels were similar in both groups. The case patients suffered significantly longer infertility years than control (p<0.05), and most patients were diagnosed with pelvic inflammatory diseases. A distinct difference was observed in the structure of oocyte corona cumulus complexes between the two groups. The embryo development parameters, which include the rates of cleavage, high-quality embryo, blastocyst, and high-quality blastocyst in the case group were greatly lower than that in the control group (p<0.05). The rates of cumulative clinical pregnancy and live birth were comparable between the two groups. In the subsequent follow-up, thirty-four of the 58 patients receiving intracytoplasmic single sperm injection (ICSI) or early rescue ICSI (R-ICSI) treatment successfully gave birth to babies, and all of the newborns were with no neonatal defects. In addition, the fertilization rate of the R-ICSI group was significantly lower than that of the ICSI group (p<0.05). The occurrence of agar-like ZP impairs the development competence of human oocytes, however, the human oocytes with agar-like ZP can develop into healthy offspring, and an ICSI regimen is the optimal treatment strategy for them.

Cumulus Extracellular Matrix Is an Important Part of Oocyte Microenvironment in Ovarian Follicles: Its Remodeling and Proteolytic Degradation.

The extracellular matrix (ECM) is an essential structure with biological activities. It has been shown that the ECM influences gene expression via cytoskeletal components and the gene expression is dependent upon cell interactions with molecules and hormones. The development of ovarian follicles is a hormone dependent process. The surge in the luteinizing hormone triggers ovulatory changes in oocyte microenvironment. In this review, we discuss how proteolytic cleavage affects formation of cumulus ECM following hormonal stimulation; in particular, how the specific proteasome inhibitor MG132 affects gonadotropin-induced cytoskeletal structure, the organization of cumulus ECM, steroidogenesis, and nuclear maturation. We found that after the inhibition of proteolytic cleavage, gonadotropin-stimulated oocyte–cumulus complexes (OCCs) were without any signs of cumulus expansion; they remained compact with preserved cytoskeletal F-actin-rich transzonal projections through the oocyte investments. Concomitantly, a significant decrease was detected in progesterone secretion and in the expression of gonadotropin-stimulated cumulus expansion–related transcripts, such as HAS2 and TNFAIP6. In agreement, the covalent binding between hyaluronan and the heavy chains of serum-derived the inter-alpha-trypsin inhibitor, essential for the organization of cumulus ECM, was missing.

IVF protocol efficacy in women with expected suboptimal response depending on ovary stimulation mode

Investigation objective IVF protocol efficacy estimation in women with expected suboptimal response depending on ovary stimulation mode. Materials and technique A randomized controlled study embracing results of 51 IVF cycle in women with ovary suboptimal response. The suboptimal response prognostic analysis was performed basing on ≤9 oocyte cumulus complexes obtained in previous IVF programs, the presence of no less than 5–9 antral follicles in both oocytes and amount of anti-Mullerian Hormone ≥0,8 ng/mL. In Group I (n = 25), the stimulation was performed by recombinant corifollitropin alfa combined with highly purified urinary gonadotropin, while in Group II (n = 26) it was made by means of recombinant follitropin/lutropin alfa within the protocol of applying gonadotropin-releasing hormone antagonists. Results The total gonadotropin dose in Group II patients was authentically lower compared to Group I (p˂,01). No statistical difference between the two studied groups was detected concerning the number of obtained oocytes, 2pn zygote, good-quality transferred embryos and clinical pregnancy rate (p>.05). Embryo cryopreservation was performed only for group-II patients. Conclusion Corifollitropin alfa administration combined with highly purified menotropin in IVF cycles for suboptimal responders is quite effective, however, this strategy has no preference over other stimulation modes. The strategy of using recombinant follitropin/lutropin alfa can be promotive to IVF outcomes for suboptimal responders by means of embryo banking. ClinicalTrials.gov Identifier: NCT03177538

The special techniques for Semen analysis and preparation

Infertility can be detected when the couples have not completed pregnancy after a year or more of normal coitus. So, in order to treat infertility, there are many supported reproductive techniques are in practice. The success rate of these techniques depends upon the way by which preparation of the paternal semen sample. Over the past 30 years, the manual has been standard as providing global standards and has been used extensively by research and clinical laboratories throughout the world. The spermatozoa of all placental (eutherian) mammals, including humans, are in a protective, no labile formal at ejaculation and are incapable of fertilization even if they are placed in direct contact with an oocyte. Accordingly, they must undergo a subsequent period of final maturation. during which they acquire the capacity to interact with the oocyte cumulus complex and achieve fertilization. In this article we tried to explain the most important analysis and techniques necessary for semen preparation to produce healthy and normal offspring.

O-112 Outcomes of random-start ovarian stimulation protocols as a possible evidence of the theory of antral follicles continuous recruitment

Abstract Study question We tried to validate the possibility and efficiency of ovarian stimulation (OS) started on any day of the menstrual cycle, based on a theory of continuous recruitment of antral follicles. Summary answer Formation of a pool of follicles with higher sensitivity to gonadotropic stimulation occurs several times during the menstrual cycle (MC). What is known already According to classical concepts and fundamental positions formulated in the middle of the last century, follicular recruitment occurs only once during the menstrual cycle - in the early follicular phase. Nowadays there is increasing evidence to suggest that there are multiple (two or three) antral follicular waves of recruitment during the MC. Also some researchers state that the process of follicle recruitment is continuous. Study design, size, duration This prospective clinical study was conducted at the V.I. Kulakov NMRC for OG&amp;Pof Russia. The study included female cancer patients seeking retrieval and cryopreservation of oocytes and/or embryos before cancer treatment. 240 patients were selected for the study. The patients were divided into 5 groups depending on the cycle day on the moment when ovarian stimulation was initiated. All patients signed an informed consent form approved by the Ethics Committee. Participants/materials, setting, methods The 1st group consisted of patients who started standart OS from 1 to 5 days of the cycle (n = 65); the 2nd - from 6 to 10 (n = 36), the 3rd - from 11 to 15 (n = 45), the 4th - from 16 to 22 (n = 44), the 5th - from 23 to 28 (n = 50). In the late follicular and luteal phase we performed OS without a pituitary modulator. The comparative analysis included features of oo-, embryogenesis and steroidogenesis. Main results and the role of chance The mean age, BMI and AMH were not different among groups. There were no LH rise or OHSS signs noticed in any groups, despite that OS in late follicular and luteal phase of the MC was performed with no GnRH antagonist addition.There was no statistically significant difference in the duration of stimulation, starting doses, total dose of FSH and HMG. The largest number of oocyte cumulus complexes was obtained in the 5th group (11 (9–21) vs 7 (3,5–15,5) in the 1st group, p = 0,030). The greatest number of mature oocytes was obtained in the 4th and 1st groups. In the 2nd group the largest number of immature oocytes was obtained (37 (9.1%)). A smaller number of mature oocytes (165 (61.8%) vs 492 (72.9%) and 314 (77.5%), p = 0.001) was obtained in group 2 (compared with the 1st and the 4th groups), when stimulation was started in the presence of a dominant follicle . These periods coincided with higher estradiol and lower FSH serum levels. Based on our data the optimal moment for effective OS initiation starts with the decrease in serum estradiol which is approximately 48 hours before the menstrual bleeding. Limitations, reasons for caution The presented results could be applied mainly to young patients with high and normal ovarian reserve, who were in the main study group. In patients with low ovarian reserve, short menstrual cycle and early ovulation an issue of favorable time points for the initiation of OS should be resolved individually. Wider implications of the findings The data collected during our research could possibly contribute to future personification of OS protocols. Tailoring the ovarian stimulation protocols to the needs of the patients could decrease time needed for completing the protocol without affecting oocyte yield or their maturity. Trial registration number none