Meiosis II Research Articles

P-237 Fertilization prediction of mature oocyte based on microscopic image of first polar body morphology using YOLOv8

Abstract Study question Can a machine learning-based model predict successful fertilization of oocyte using first polar body morphology? Summary answer With small imbalance oocyte dataset, the most successful model to predict fertilization of mature oocyte is YOLOv8lcls with 97.47% accuracy, 100.00% specificity, and 94.87% sensitivity. What is known already The development of oocytes is crucial in the development and fertility of embryos in In Vitro Fertilization (IVF). There are several internal and external factors that could affect the fertilization competency of oocytes, one of which is the first polar body (PBI). The importance of the quality of PBI in ICSI has long been studied and has been evaluated by scoring systems regarding Istanbul Consensus. However, there is still a debate regarding the influence of PBI on the developmental competency of oocytes. Study design, size, duration This study involved the analysis of retrospective data including the microscopic light image of meiosis II (MII) oocytes at the time of Intracytoplasmic sperm injection (ICSI), along with polar body scoring by embryologists and the fertilization outcome of 1006 MII oocytes from a single public IVF clinic, between 2020-2022. After ICSI, fertilized oocytes were classified into 2 groups: 2PN and non-2PN (e.g., 0PN, 1PN, > =3PN). Participants/materials, setting, methods Light microscopic images of oocytes in the metaphase of meiosis II (MII) stage are captured, followed by the prediction of the likelihood of 2PN development after fertilization through intra-cytoplasmic sperm injection (ICSI). The image has its PBI segmented by the CNN and then cropped out of the original image. The PBI image is then fed into the prediction CNN to obtain the final result. The model performance was determined by evaluating accuracy, sensitivity, and specificity. Main results and the role of chance There were 1006 images of MII oocytes included in this study. After ICSI, 860 oocytes were fertilized resulting in the 2PN stage embryos and were classified as 2PN, whereas 146 oocytes were not fertilized or abnormally fertilized and were classified as non-2PN. Among these images, 782 oocytes received a PBI score of A, 677 of which are 2PN and 105 are non-2PN. The B grade consists of 224 images, of which are 183 2PN, and 41 non-2PN. The dataset was split into 3 subsets for training (70%), validation (10%), and testing (20%). The images are augmented by horizontal flipping to increase the amount of data and allow the model to observe several positions of the PBI. The YOLOv8 segmentation model is applied to segment the PBI and locate its position. The image is then rotated to move the PBI to the top and the PBI is cropped based on its position. Lastly, the cropped PBI image is used to predict the development by using the YOLOv8 Classify model trained on an augmented dataset. The YOLOv8l-cls model achieves the highest accuracy at 97.47%, specificity at 100.00%, and sensitivity at 94.87%. Limitations, reasons for caution The model does not include information on sperm quality, which also essentially contributes to the fertilization process. 2PN stage prediction is just the initial and surrogate outcome in IVF treatment. The model’s interpretation of the PBI score remains inconclusive. Therefore, additional experiments using dataset with balanced PBI grading are recommended. Wider implications of the findings The developed model is the first step in paving the way to establish and implement tools for assisting embryologists in selecting and prioritizing the high-potential oocyte for fertilization. Additional factors such as patient characteristics, other features of oocytes, and male factors should be further integrated to improve model performance. Trial registration number NA

The Relationship Between Serum Growth Differentiation Factor-15 (GDF-15) Levels and Clinical Outcomes in Infertile Women Receiving In-vitro Fertilization Treatment

Objective: It has been reported in many studies that Growth Differentiation Factor-15 (GDF-15) has an important role in physiological or pathological processes. As there is no study in the current scientific literature examining the status of GDF-15 in infertility and its treatment outcomes, we aimed to investigate this.
 Material and methods: According to their ovarian reserve characteristics, 88 infertile women were divided into three groups: normal ovarian reserve (NOR), diminished ovarian reserve (DOR), and polycystic ovary syndrome (PCOS). Estradiol (E2), follicle-stimulating hormone (FSH), luteinizing hormone (LH), anti-mullerian hormone (AMH), and GDF-15 levels were measured in their serum. The antagonist protocol patients' total oocyte, meiosis II (MII) oocytes, embryo count, and clinical pregnancy rates were documented.
 Results: In terms of serum GDF-15 concentrations, there was no statistically significant difference among the mean values of the three study groups. The mean FSH level at baseline was substantially higher in the DOR group compared to the PCOS group. The median serum AMH levels of all three groups were found to be statistically different. The antagonist protocol patients' total oocytes, meiosis II (MII) oocytes, embryo count, and clinical pregnancy rates were documented.
 Conclusion: In the present study, a significant and strong correlation between serum GDF-15 level and consequent embryo number was detected. Thereby, serum GDF-15 level may be considered to be a biomarker for predicting IVF clinical outcomes.

Aneuploidy in oocytes from women of advanced maternal age: analysis of the causal meiotic errors and impact on embryo development.

In oocytes of advanced maternal age (AMA) women, what are the mechanisms leading to aneuploidy and what is the association of aneuploidy with embryo development? Known chromosome segregation errors such as precocious separation of sister chromatids explained 90.4% of abnormal chromosome copy numbers in polar bodies (PBs), underlying impaired embryo development. Meiotic chromosomal aneuploidies in oocytes correlate with AMA (>35 years) and can affect over half of oocytes in this age group. This underlies the rationale for PB biopsy as a form of early preimplantation genetic testing for aneuploidy (PGT-A), as performed in the 'ESHRE STudy into the Evaluation of oocyte Euploidy by Microarray analysis' (ESTEEM) randomized controlled trial (RCT). So far, chromosome analysis of oocytes and PBs has shown that precocious separation of sister chromatids (PSSC), Meiosis II (MII) non-disjunction (ND), and reverse segregation (RS) are the main mechanisms leading to aneuploidy in oocytes. Data were sourced from the ESTEEM study, a multicentre RCT from seven European centres to assess the clinical utility of PGT-A on PBs using array comparative genomic hybridization (aCGH) in patients of AMA (36-40 years). This included data on the chromosome complement in PB pairs (PGT-A group), and on embryo morphology in a subset of embryos, up to Day 6 post-insemination, from both the intervention (PB biopsy and PGT-A) and control groups. ESTEEM recruited 396 AMA patients: 205 in the intervention group and 191 in the control group. Complete genetic data from 693 PB pairs were analysed. Additionally, the morphology from 1034 embryos generated from fertilized oocytes (two pronuclei) in the PB biopsy group and 1082 in the control group were used for statistical analysis. Overall, 461/693 PB pairs showed abnormal segregation in 1162/10 810 chromosomes. The main observed abnormal segregations were compatible with PSSC in Meiosis I (MI) (n = 568/1162; 48.9%), ND of chromatids in MII or RS (n = 417/1162; 35.9%), and less frequently ND in MI (n = 65/1162; 5.6%). For 112 chromosomes (112/1162; 9.6%), we observed a chromosome copy number in the first PB (PB1) and second PB (PB2) that is not explained by any of the known mechanisms causing aneuploidy in oocytes. We observed that embryos in the PGT-A arm of the RCT did not have a significantly different morphology between 2 and 6 days post-insemination compared to the control group, indicating that PB biopsy did not affect embryo quality. Following age-adjusted multilevel mixed-effect ordinal logistic regression models performed for each embryo evaluation day, aneuploidy was associated with a decrease in embryo quality on Day 3 (adjusted odds ratio (aOR) 0.62, 95% CI 0.43-0.90), Day 4 (aOR 0.15, 95% CI 0.06-0.39), and Day 5 (aOR 0.28, 95% CI 0.14-0.58). RS cannot be distinguished from normal segregation or MII ND using aCGH. The observed segregations were based on the detected copy number of PB1 and PB2 only and were not confirmed by the analysis of embryos. The embryo morphology assessment was static and single observer. Our finding of frequent unexplained chromosome copy numbers in PBs indicates that our knowledge of the mechanisms causing aneuploidy in oocytes is incomplete. It challenges the dogma that aneuploidy in oocytes is exclusively caused by mis-segregation of chromosomes during MI and MII. Data were mined from a study funded by ESHRE. Illumina provided microarrays and other consumables necessary for aCGH testing of PBs. None of the authors have competing interests. Data were mined from the ESTEEM study (ClinicalTrials.gov Identifier NCT01532284).

O-307 Aneuploidy in oocytes of women of advanced maternal age: impact on embryo development and evidence for a novel mechanism of meiotic error

Abstract Study question In oocytes of advanced maternal age (AMA) women, what is the association of aneuploidy with embryo development and what are the mechanisms leading to aneuploidy? Summary answer We confirmed precocious separation of sister chromatids as the main cause of aneuploidy. Known chromosome segregation errors explained 90.4% of the observed abnormal chromosome copy numbers in polar bodies. What is known already Meiotic chromosomal aneuploidies in oocytes correlate with AMA (>35 years), and can affect over half of oocytes in this age group. This underlies the rationale for polar body (PB) biopsy as a form of early PGT-A, as performed in the ‘ESHRE STudy into the Evaluation of oocyte Euploidy by Microarray analysis’ (ESTEEM) randomised controlled trial (RCT). So far, chromosome analysis of oocytes and PBs has shown that precocious separation of sister chromatids (PSSC), meiosis II (MII) nondisjunction (ND) and reverse segregation (RS) are the main mechanisms leading to aneuploidy in oocytes. Study design, size, duration Data was sourced from the ESTEEM study, a multicentre RCT from seven European centres to assess the clinical utility of PGT-A on PBs using array comparative genomic hybridisation (aCGH) in patients of AMA (36-40 years). This included data on the chromosome complement in PB pairs, and on embryo morphology in a subset of embryos, up to day 6 post insemination (dpi), from both the intervention (PB biopsy for PGT-A) and from control (no biopsy) arms. Participants/materials, setting, methods ESTEEM recruited 396 AMA patients: 205 in the intervention group and 191 in the control group. Complete genetic data from 693 PB pairs were analysed. Additionally, the morphology from 1034 embryos generated from fertilized oocytes (2 pronuclei) in the PB biopsy group and 1082 in the control group were compared using age-adjusted multilevel mixed-effect ordinal logistic regression for each day of embryo scoring and summarized with adjusted odds ratios (aOR) and 95% confidence intervals (CI). Main results and the role of chance Overall, 461/693 PB pairs showed abnormal segregation in 1162/10810 chromosomes. The main observed abnormal segregations were compatible with PSSC in meiosis I (MI) (n = 568/1162; 48,9%), ND of chromatids in MII or RS (n = 417/1162; 35.9%) and less frequently ND in MI (n = 65/1162; 5.6%). For 112 chromosomes (112/1162; 9,6%), we observed a chromosome copy number in PB1 and PB2 that is not explained by any of the known mechanisms causing aneuploidy in oocytes. We observed that embryos in the PGT-A arm of the RCT did not have a significantly different morphology between 2 and 6 dpi compared to the control group, indicating that polar body biopsy did not affect embryo quality. Following age-adjusted multilevel mixed-effect ordinal logistic regression models performed for each embryo evaluation day (using the before-mentioned embryo scoring subcategories), aneuploidy was associated with a decrease in embryo quality on day 3 (aOR 0.62, 95% CI 0.43-0.90), day 4 (aOR 0.15, 95% CI 0.06-0.39) and day 5 (aOR 0.28, 95% CI 0.14-0.58). Limitations, reason for caution RS cannot be distinguished from normal segregation or MII ND using aCGH. The observed segregations were based on the detected copy number of PB1 and PB2 only and were not confirmed by the analysis of embryos. The embryo morphology assessment was static and single observer. Wider implications of the findings Our finding of frequent unexplained chromosome copy numbers in polar bodies indicates that our knowledge of the mechanisms causing aneuploidy in oocytes is incomplete. It challenges the dogma that aneuploidy in oocytes is exclusively caused by missegregation of chromosomes during meiosis I and II. Study funding/competing interest(s) Data was mined from a study funded by ESHRE. Illumina provided microarrays and other consumables necessary for aCGH testing of polar bodies. None of the authors have competing interests. This work is dedicated to the memory of Professor Joep Geraedts. Trial registration number NCT01532284 Study funding Yes Funding source Funding by national/international organization(s)

Loss of the Maternal Effect Gene Nlrp2 Alters the Transcriptome of Ovulated Mouse Oocytes and Impacts Expression of Histone Demethylase KDM1B.

The subcortical maternal complex (SCMC) is a multiprotein complex in oocytes and preimplantation embryos that is encoded by maternal effect genes. The SCMC is essential for zygote-to-embryo transition, early embryogenesis, and critical zygotic cellular processes, including spindle positioning and symmetric division. Maternal deletion of Nlrp2, which encodes an SCMC protein, results in increased early embryonic loss and abnormal DNA methylation in embryos. We performed RNA sequencing on pools of meiosis II (MII) oocytes from wild-type and Nlrp2-null female mice that were isolated from cumulus-oocyte complexes (COCs) after ovarian stimulation. Using a mouse reference genome-based analysis, we found 231 differentially expressed genes (DEGs) in Nlrp2-null compared to WT oocytes (123 up- and 108 downregulated; adjusted p < 0.05). The upregulated genes include Kdm1b, a H3K4 histone demethylase required during oocyte development for the establishment of DNA methylation marks at CpG islands, including those at imprinted genes. The identified DEGs are enriched for processes involved in neurogenesis, gland morphogenesis, and protein metabolism and for post-translationally methylated proteins. When we compared our RNA sequencing data to an oocyte-specific reference transcriptome that contains many previously unannotated transcripts, we found 228 DEGs, including genes not identified with the first analysis. Interestingly, 68% and 56% of DEGs from the first and second analyses, respectively, overlap with oocyte-specific hyper- and hypomethylated domains. This study shows that there are substantial changes in the transcriptome of mouse MII oocytes from female mice with loss of function of Nlrp2, a maternal effect gene that encodes a member of the SCMC.

Effect of three-dimensional collagen membrane culture and presence of cumulus cells on maturation of bovine oocytes.

The use of animals as experimental models has been proposed to improve the techniques applied in human reproduction clinics. This prospective and observational study evaluates the effects of the use of cumulus cells and collagen membrane on the maturation process of bovine oocytes. Design and Setting: Bovine oocytes with or without cumulus cells were cultured in maturation medium for 24 hours in the conventional system (2D), central well plates and in the three-dimensional (3D) system. Intervention: The oocytes were positioned in the collagen membrane and matured for the same period. The morphological evaluation was carried out with the parameters of maturation. Main Outcome Measure: Presence or absence of the first polar corpuscle, which were observed and classified as germinal vesicle (GV), meiosis I (MI) and meiosis II (MII). The percentage of oocytes in GV was higher (p<0.05) in treatments without cumulus cells than those with cells. The rates of MII were higher (p<0.05) in the treatments with cumulus cells, independent of the culture system. In general, oocytes with presence of cumulus cells have approximately 1.7 times more chances (p<0.001) of reaching MII after MIV than those matured without cells. The presence of the cells in the cumulus is essential for the maturation process of bovine oocytes; the three-dimensional collagen membrane culture system is favorable for the maturation process of bovine oocytes.

Sperm-specific glycogen synthase kinase 3 is required for sperm motility and the post-fertilization signal for female meiosis II in Caenorhabditis elegans.

In most sexually reproducing animals, sperm entry provides the signal to initiate the final stages of female meiosis. In Caenorhabditis elegans, this signal is required for completion of female anaphase I and entry into meiosis II (MII). memi-1/2/3 (meiosis-to-mitosis) encode maternal components that facilitate this process; memi-1/2/3(RNAi) results in a skipped-MII phenotype. Previously, we used a gain-of-function mutation, memi-1(sb41), to identify genetic suppressors that represent candidates for the sperm-delivered signal. Herein, we characterize two suppressors of memi-1(sb41): gskl-1 and gskl-2. Both genes encode functionally redundant sperm glycogen synthase kinase, type 3 (GSK3) protein kinases. Loss of both genes causes defects in male spermatogenesis, sperm pseudopod treadmilling and paternal-effect embryonic lethality. The two kinases locate within the pseudopod of activated sperm, suggesting that they directly or indirectly regulate the sperm cytoskeletal polymer major sperm protein (MSP). The GSK3 genes genetically interact with another memi-1(sb41) suppressor, gsp-4, which encodes a sperm-specific PP1 phosphatase, previously proposed to regulate MSP dynamics. Moreover, gskl-2 gsp-4; gskl-1 triple mutants often skip female MII, similar to memi-1/2/3(RNAi). The GSK3 kinases and PP1 phosphatases perform similar sperm-related functions and work together for post-fertilization functions in the oocyte that involve MEMI.

Heat shock-induced failure of meiosis I to meiosis II transition leads to 2n pollen formation in a woody plant.

The formation of diploid gametes through chromosome doubling is a major mechanism of polyploidization, diversification, and speciation in plants. Unfavorable climate conditions can induce or stimulate the production of diploid gametes during meiosis. Here, we demonstrated that heat shock stress (38°C for 3 or 6 h) induced 2n pollen formation, and we generated 42 triploids derived from heat shock-induced 2n pollen of Populus canescens. Meiotic analysis of treated pollen mother cells revealed that induced 2n pollen originated from the complete loss of meiosis II (MII). Among the 42 triploids, 38 triploids derived from second division restitution (SDR)-type 2n pollen and 4 triploids derived from first division restitution-type 2n pollen were verified using simple sequence repeats(SSR) molecular markers. Twenty-two differentially expressed genes related to the cell cycle were identified and characterized by expression profile analysis. Among them was POPTR_0002s08020g (PtCYCA1;2), which encodes a type A Cyclin CYCA1;2 that is required for the meiosis I (MI) to MII transition. After male flower buds were exposed to heat shock, a significant reduction was detected in PtCYCA1;2 expression. We inferred that the failure of MI-to-MII transitions might be associated with downregulated expression of PtCYCA1;2, leading to the formation of SDR-type 2n pollen. Our findings provide insights into mechanisms of heat shock-induced 2n pollen formation in a woody plant and verify that sensitivity to environmental stress has evolutionary importance in terms of polyploidization.

Maternal DDB1 regulates apoptosis and lineage differentiation in porcine preimplantation embryos.

Maternal-effect genes (MEGs) play a critical role in modulating both cellular and molecular biology events in preimplantation embryonic development. Damage-specific DNA binding protein 1 (DDB1) is a gene that participates in meiotic resumption, ovulation, and embryonic stem cell maintenance. Its function in preimplantation development is not well-studied. We aimed to explore the expression pattern, genomic heritage, and potential molecular mechanisms of DDB1 in preimplantation embryos in porcine. In this study, RNA interference, microinjection, RT-qPCR, immunofluorescence staining and single-cell RNA sequencing were used to explore the molecular function of DDB1 in porcine preimplantation embryos. DDB1 was found to be expressed in germinal vesicle (GV) and Meiosis II (MII) oocytes and in preimplantation embryos. We confirmed it is a MEG. DDB1 -deficient blastocysts had a significantly reduced number of trophectoderm cells, an increased apoptotic cell number and increased apoptosis index. According to a next-generation sequencing (NGS) analysis, 236 genes (131 upregulated and 105 downregulated) significantly changed in the DDB1 -deficient morula. The myeloid leukaemia factor 1 (MLF1 ) and yes-associated protein 1 (YAP1 ) expressions were significantly upregulated and downregulated respectively, in the DDB1 -deficient morula. In combination with the decreased expression of TEAD4 , CDX2 , GATA3 , OCT4 , and NANOG and the increased expression of SOX2 in the blastocyst, DDB1 may play a role in determining lineage differentiation and pluripotency maintenance. DDB1 is a MEG and it plays a crucial role in porcine preimplantation embryonic development. This study provides a theoretical basis for further understanding the molecular mechanisms of preimplantation embryo development.

Effects of prematuration culture with a phosphodiesterase-3 inhibitor on oocyte morphology and embryo quality in in vitro maturation.

ObjectiveThe study assessed the developmental potential of germinal vesicle (GV) oocytes subjected to in vitro maturation (IVM) after prematuration culture with cilostamide (a phosphodiesterase-3 inhibitor) and the impact of cilostamide exposure on the morphology of meiosis II (MII) oocytes and subsequent embryo quality. MethodsIn total, 994 oocytes were collected from 63 patients. Among 307 GV oocytes, 140 oocytes were selected for the experimental group and 130 oocytes for the control group. The denuded GV-stage oocytes were cultured for 6 hours with cilostamide in the experimental group and without cilostamide in the control group. After 6 hours, the oocytes in the experimental group were washed and transferred to fresh IVM medium. The maturational status of the oocytes in both groups was examined at 26, 36, and 48 hours. Fertilization was assessed at 18 hours post-intracytoplasmic sperm injection. Embryo quality was assessed on days 3 and 5.ResultsIn total, 92.1% of the oocytes remained in the GV stage, while 6.4% converted to the MI stage (p<0.01) after cilostamide exposure. In both groups, more MII oocytes were observed at 36 hours (25.8% vs. 21.5%) than at 26 hours (10.8% vs. 14.6%) and 48 hours (13% vs. 7.9%) (p>0.05). With the advent of cilostamide, blastocyst quality was better in the experimental group than in the control group (p<0.05). ConclusionsCilostamide effectively blocked nuclear maturation and promoted cytoplasmic growth. Prematuration culture with cilostamide enabled synchronization between cytoplasmic and nuclear maturity, resulting in better blastocyst outcomes.

GAS6 ameliorates advanced age-associated meiotic defects in mouse oocytes by modulating mitochondrial function.

Previously, we reported that the silencing of growth arrest-specific gene 6 (Gas6) expression in oocytes impairs cytoplasmic maturation by suppressing mitophagy and inducing mitochondrial dysfunction, resulting in fertilization failure. Here, we show that oocyte aging is accompanied by an increase in meiotic defects associated with chromosome misalignment and abnormal spindle organization. Intriguingly, decreased Gas6 mRNA and protein expression were observed in aged oocytes from older females. We further explored the effect of GAS6 on the quality and fertility of aged mouse oocytes using a GAS6 rescue analysis. After treatment with the GAS6 protein, aged oocytes matured normally to the meiosis II (MII) stage. Additionally, maternal age-related meiotic defects were reduced by GAS6 protein microinjection. Restoring GAS6 ameliorated the mitochondrial dysfunction induced by maternal aging. Ultimately, GAS6-rescued MII oocytes exhibited increased ATP levels, reduced ROS levels and elevated glutathione (GSH) levels, collectively indicating improved mitochondrial function in aged oocytes. Thus, the age-associated decrease in oocyte quality was prevented by restoring GAS6. Importantly, GAS6 protein microinjection in aged oocytes also rescued fertility. We conclude that GAS6 improves mitochondrial function to achieve sufficient cytoplasmic maturation and attenuates maternal age-related meiotic errors, thereby efficiently safeguarding oocyte quality and fertility.

Immature Follicular Origins and Disrupted Oocyte Growth Pathways Contribute to Decreased Gamete Quality During Reproductive Juvenescence in Mice.

Egg quality dictates fertility outcomes, and although there is a well-documented decline with advanced reproductive age, how it changes during puberty is less understood. Such knowledge is critical, since advances in Assisted Reproductive Technologies are enabling pre- and peri-pubertal patients to preserve fertility in the medical setting. Therefore, we investigated egg quality parameters in a mouse model of the pubertal transition or juvenescence (postnatal day; PND 11–40). Animal weight, vaginal opening, serum inhibin B levels, oocyte yield, oocyte diameter, and zona pellucida thickness increased with age. After PND 15, there was an age-associated ability of oocytes to resume meiosis and reach metaphase of meiosis II (MII) following in vitro maturation (IVM). However, eggs from the younger cohort (PND 16–20) had significantly more chromosome configuration abnormalities relative to the older cohorts and many were at telophase I instead of MII, indicative of a cell cycle delay. Oocytes from the youngest mouse cohorts originated from the smallest antral follicles with the fewest cumulus layers per oocyte, suggesting a more developmentally immature state. RNA Seq analysis of oocytes from mice at distinct ages revealed that the genes involved in cellular growth signaling pathways (PI3K, mTOR, and Hippo) were consistently repressed with meiotic competence, whereas genes involved in cellular communication were upregulated in oocytes with age. Taken together, these data demonstrate that gametes harvested during the pubertal transition have low meiotic maturation potential and derive from immature follicular origins.

An exploration of contributing factors to the oocyte utility rate of high responders in an in vitro fertilization cycle

Objective: To figure out the clinical factors contributing to the oocytes utility rate (OUR) of high responders in in vitro fertilization (IVF)-embryo transfer treatment. Methods: OUR was defined by the number of usable embryos for transfer and (or) cryopreservation divided by the number of oocytes retrieved in a freeze-all cycle. The cycles with ≥15 eggs were included from January 2013 to December 2019. Those with OUR at the top 10% (Group A) and the bottom 10% (Group B) were picked and compared for patients' characteristics, parameters relating to ovary stimulation and pregnant outcomes. Multifactorial logistic regression was applied to reveal the risk factors affecting OUR in them. Results: A total of 43 patients were included in Group A (OUR: 77.4%, 601/776) and 47 for Group B (OUR: 11.9%, 104/874). Previous IVF/intracytoplasmic sperm injection (ICSI; OR=0.10, 95%CI: 0.01-0.81) and endometriosis (OR=0.16, 95%CI: 0.03-0.84) were negative factors for OUR (P<0.05); dual suppression protocol (OR=3.74, 95%CI: 1.06-26.86) and longer days of stimulation (OR=3.24, 95%CI: 1.25-8.42) were protective factors in terms of ovarian stimulation (P<0.05), on contrary to that, any decline of estradiol during the stimulation led to poorer OUR (OR=0.16, 95%CI: 0.04-0.64). Although two groups had similar quantities of eggs and metaphase of meiosis Ⅱ (MⅡ) oocytes, distinguished cumulative clinical pregnancy rate and cumulative live birth rate were seen in Group A and group B respectively [95.3% (41/43) vs 40.4% (19/47) and 90.7% (39/43) vs 31.9% (15/47), all P<0.01]. Conclusions: High responders with attempted IVF/ICSI and endometriosis should be considered as risk factors for OUR. Over ovarian stimulation, dual suppression and a slightly longer stimulating duration could be tried; besides, estradiol decline should be prevented for a better OUR.

OOCYTE STIMULATION/TRIGGER PROTOCOL CORRELATES WITH THE PROPORTION OF IMMATURE OOCYTES RETRIEVED IN ASSISTED REPRODUCTIVE TECHNOLOGY CYCLES

The goal of oocyte retrieval in cycles of oocyte cyropreservation and intracytoplasmic sperm injection (ICSI) is to obtain meiosis II (MII) mature oocytes that can be immediately fertilized or frozen without the need for in vitro maturation. The fraction of oocytes that are immature following controlled ovarian hyperstimulation (COH), in other words, arrested in germinal vesicle (GV) or meiosis I (MI) phases of the cell cycle, varies from patient to patient. In this study, we identify parameters that are associated with the proportion of immaturity (Pim = number of immature oocytes [GV and MI] / total number of oocytes retrieved [GV, MI, and MII]).

Modeling the Control of Meiotic Cell Divisions: Entry, Progression, and Exit

Upon nitrogen starvation, Schizosaccharomyces pombe exit the mitotic cell cycle and become irreversibly committed to the completion of meiosis program. Meiotic cell divisions are coordinated with sporulation events to produce haploid spores. In the last few decades, experiments on fission yeast have revealed different molecular players involved in two meiotic cell divisions, meiosis I (MI) and meiosis II (MII). How the MI entry, MI-to-MII transition, and MII exit occur because of the dynamics of the regulatory network is not well understood. In this work, we developed a comprehensive mathematical model of the network that describes the temporal dynamics of meiotic progression. The model accounts for the phenotypes of several experimental data (single and multiple mutations). We demonstrate the control strategy involving multiple feedback loops to yield two successive division cycles. The differential regulation of anaphase-promoting complex/cyclosome (APC/C) coactivators and its inhibitors is crucial for the dynamics of both MI-to-MII transition and MII exit. This model generates mechanistic insights that help in further experiments and modeling.

The association between maternal occupation and down syndrome: A report from the national Down syndrome project.

Among live births, Down syndrome (DS) due to trisomy 21 is the most commonly occurring autosomal trisomy, typically resulting from meiotic nondisjunction. Currently, advanced maternal age and altered recombination patterns are the only well-known risk factors for nondisjunction. Maternal occupation has not been investigated as a risk factor for maternally-derived cases of trisomy 21. This study explored the association between maternal occupation and chromosome 21 nondisjunction, stratified by the stage of maternal error - either Meiosis I (MI) or Meiosis II (MII). Additionally, we investigated specific toxic agents associated with occupation classes. Using narrative job descriptions from the National Down Syndrome Project (NDSP), a population-based case-control study, occupation was coded using the 2010 Standard Occupational Classification (SOC). Odds ratios were calculated for the association between occupation class and having a child with DS, stratified by meiotic stage. An exposure analysis was performed within occupational classes that were statistically significant predictors of having a child with DS. Odds ratios were calculated to analyze associations between individual exposures and having a child with DS. The odds of MII nondisjunction were increased among Production Workers (OR = 3.15; 95%CI = 1.52,6.55). Women who worked as Life, Physical and Social Scientists or in Food Preparation and Serving-Related Occupations experienced greater likelihood of MI errors (OR = 5.72(1.80,18.20), and OR = 1.87(1.08,3.24), respectively). Exposure to solvents within the Production Worker group was a significant predictor (p < 0.05) for MI nondisjunction. No other environmental agents had a significant association with nondisjunction. Specific maternal occupation classes were associated with MI and MII chromosome 21 nondisjunction. These occupation classes were selected for an exposure analysis, which determined solvents as highly predictive of MI nondisjunction among Production Workers. Findings from this analysis will serve to further explore the relationship between maternal occupation and chromosome 21 nondisjunction.

Molecular aspects of aneuploidy in preimplantation human embryos: a mini-review

Aneuploidy is a major obstacle in achieving successful pregnancies in in vitro fertilization (IVF) procedures. In practice, more than 50% of preimplantation embryos are aneuploid and unable to achieve viable pregnancy. Analysis of human sperm and oocytes has showed that aneuploidy in preimplantation embryos is predominantly caused by an error-prone meiotic chromosome segregation mechanism in oocytes. In reproductive aged women, aneuploidy rate in human oocytes is reaching 20–30%, in contrast to human sperm, that only 1–8% have an abnormal chromosomal content. Meiotic aneuploidy can occur from meiosis I (MI) non-disjunction, meiosis II (MII) non-disjunction and MI or MII pre-division. Multiple factors have been suggested to contribute to meiotic aneuploidy including maternal age and impaired cohesin complex formation. Maternal age is the major critical factor related to aneuploidy; 50% of the oocytes from advanced age women (≥40 years old) are aneuploid due to meiotic errors. The relationship between maternal age and altered recombination, which is entirely dependent on the context of chromosomes, can also contribute to development of aneuploidy. Genomic errors can also arise during post-fertilization mitotic divisions, resulting in embryonic mosaicism. The mechanisms leading to embryonic mosaicism during embryonic mitosis are; non-disjunction, anaphase lagging and endoreplication. Mosaicism exists in ~15–90% of all cleavage stage human embryos and in general, mitotic aneuploidies rise from 63% at the cleavage stage to 95% in blastocyst stage. Relaxation or absence of cell cycle checkpoints are possible mechanisms that have been hypothesized to account for mitotic aneuploidy in early human preimplantation embryos. Specifically, defects in spindle assembly checkpoint (SAC) and malfunction of cohesion complexes, can lead to premature chromosome separation, while delay in their removal may result in non-disjunction. Furthermore, extra-nuclear DNA formation generated from lagging chromosomes with severe DNA damage (i.e., micronucleation), may be a major mechanism of mitotic aneuploidy. Once source of aneuploidy involves paternal factor is related to mitotic aneuploidy, as severe sperm defects and advanced paternal age can increase the percentage of mitotic abnormalities in embryos. Further work and analysis into the molecular mechanisms that lead to meiotic and mitotic aneuploidy in preimplantation embryos, could inspire clinical strategies to reduce the occurrence of aneuploidy and consequently improve the success rates of IVF.

Role of Type 1 Inositol 1,4,5-triphosphate Receptors in Mammalian Oocytes.

The ability of oocytes to undergo normal fertilization and embryo development is acquired during oocyte maturation which is transition from the germinal vesicle stage (GV), germinal vesicle breakdown (GVBD) to metaphase of meiosis II (MII). Part of this process includes redistribution of inositol 1,4, 5-triphosphate receptor (IP3R), a predominant Ca2+ channel on the endoplasmic reticulum membrane. Type 1 IP3R (IP3R1) is expressed in mouse oocytes dominantly. At GV stage, IP3R1 are arranged as a network throughout the cytoplasm with minute accumulation around the nucleus. At MII stage, IP3R1 diffuses to the entire cytoplasm in a more reticular manner, and obvious clusters of IP3R1 are observed at the cortex of the egg. This structural reorganization provides acquisition of [Ca2+]i oscillatory activity during fertilization. In this review, general properties of IP3R1 in somatic cells and mammalian oocyte are introduced.

Incidence and origin of meiotic whole and segmental chromosomal aneuploidies detected by karyomapping.

What is the incidence and origin of meiotic whole and segmental aneuploidies detected by karyomapping at a blastocyst stage in human-derived IVF embryos? What is the distribution of various types of errors, including rare chromosomal abnormalities? The incidence of chromosomal aneuploidies was assessed in 967 trophectoderm biopsies from 180 couples who underwent 215 cycles of IVF with preimplantation genetic testing for monogenetic disease with a known causal mutation with a mean maternal age of 32.7 years. DNA from both parents and a reference sample was genotyped together with the analysed trophectoderm samples by karyomapping (single-nucleotide-polymorphism-based array). Chromosomal abnormalities were detected in 31% of the analysed samples. At least one whole chromosomal aneuploidy was detected in 27.1% of the trophectoderm biopsies, whereas a segmental aneuploidy was detected in 5.1% of the trophectoderm biopsies. Our results reveal that segmental aneuploidies predominantly affect paternally derived chromosomes (70.4%; P < 0.01) compared with whole chromosomal aneuploidies that more frequently affect maternally derived chromosomes (90.1%; P < 0.0001). Also, the frequency of meiosis I (MI) and meiosis II (MII) errors was established in meiotic trisomies; MI errors were observed to be more frequent (n = 102/147 [69.4%]) than MII errors (n = 45/147 [30.6%]). Karyomapping is a robust method that is suitable for preimplantation genetic testing for monogenetic disease and for detecting meiotic aneuploidies, including meiotic segmental aneuploidies, and provides complex information about their parental origin. Our results revealed that segmental aneuploidy more frequently affects paternal chromosomes compared with whole chromosomal aneuploidy in human IVF embryos at the blastocyst stage.

GnRH antagonist versus follicular-phase single-dose GnRH agonist protocol in patients of normal ovarian responses during controlled ovarian stimulation

Objective: This study aims to explore the differences of the ovarian stimulation (OS) characteristics, laboratory, and clinical outcomes between follicular-phase single-dose gonadotropin-releasing hormone (GnRH) agonist protocol and GnRH antagonist protocol during controlled ovarian hyperstimulation (COH).Methods: About 1883 consecutive IVF/ICSI fresh cycles of normal ovarian responders were retrospectively analyzed, with 1229 in the single-dose GnRH agonist protocol group and 654 in the GnRH antagonist protocol group at Reproductive Medical Center of Tongji Hospital from 1 January 2014 to 31 December 2017.Results: The follicular-phase single-dose GnRH agonist group showed significantly more oocytes obtained, higher implantation rate and pregnancy rate, as well as lower luteinizing hormone (LH) level and estradiol (E2)/oocyte ratio on the day of human chorionic gonadotropin (hCG) administration. However, differences were not significant in meiosis II (MII) oocyte rate, two pronuclear zygote (2PN) embryo rate, viable embryo rate or high-quality embryo rate, compared with the GnRH antagonist group. Further comparison of clinical outcomes in the first frozen-thawed cycles did not show significant difference in either implantation or clinical pregnancy rate between the two protocol groups.Conclusions: Follicular-phase single-dose GnRH agonist protocol may achieve better clinical outcomes in normal ovarian responders, which could be explained more by positive effect on endometrial receptivity rather than embryo quality.