Single Oocyte Research Articles

USING AUTOMATED CAPILLARY GEL ELECTROPHORESIS TO DETECT MULTIPLE PROTEINS FROM SINGLE OOCYTE AND BLASTOCYST

Traditional protein analysis by Western blot often requires a pool of large amount of samples that limits its utilization for human reproductive research. The objective of this study was to develop a highly sensitive and reproducible protocol to analyze metabolic proteins from single oocyte and embryo from humans or animals.

Effect of a dietary and exercise intervention in women with overweight and obesity undergoing fertility treatments: protocol for a randomized controlled trial

BackgroundDistinct molecular, inflammatory, and metabolic signatures are present in oocytes and follicular fluid derived from women with obesity when compared to those derived from normal weight women, which suggest existing signals that may program future offspring for metabolic diseases. This study aims to assess the feasibility and efficacy of a peri-conception nutrition and exercise intervention on mitigating obesity-associated changes in oocyte gene expression profiles and follicular fluid metabolites.MethodsThis single blinded randomized control trial will include 120 women with a BMI of 25–45 kg/m2, ≥21 years of age, and undergoing in vitro fertilization (IVF) treatments. Participants will be randomized to standard of care (N = 60) or an intervention group (N = 60) in a block design by polycystic ovary syndrome status. The intervention will combine a dietary component (Mediterranean meal plan) with exercise prescription following the Physical Activity Guidelines for Americans. Participants will be assessed pre- and post-intervention. The standard of care group will be offered to join the intervention group if the IVF treatments are unsuccessful as a cross over design. Recruitment is anticipated to start in July of 2021. Primary outcomes will include single oocyte gene expression profiles and follicular fluid metabolites. Mann-Whitney U nonparametric tests will be used to assess potential differences for each stratum. Follicular fluid and serum metabolites will be analyzed using a one-factor Analysis of Covariance (ANCOVA) at four levels, pair-wise comparisons using Tukey-Kramer post-hoc tests will be used to identify groups whose means differ significantly while retaining the family-wise error rate at 5%. When the design is balanced, two-way Analysis of Variance (ANOVA), or non-parametric Friedman test will be used in data analysis. Additionally, general linear models and ANCOVA may be used to control for covariates. Significance will be set at p < 0.05. Findings will be disseminated via peer-reviewed manuscripts and presentations at scientific conferences.DiscussionThis study will provide novel data and key information on the impact of a dietary and exercise intervention on oocyte gene expression and follicular fluid content. Results will demonstrate the potential of such intervention in mitigating obesity-induced changes in oocyte gene expression and follicular fluid metabolites.Trial registrationClinicalTrials.gov (NCT04273048): submitted November 13, 2019; posted February 17, 2020.

P-186 Volumetric imaging provides insight into the 3D ultrastructural organization of maturing human oocytes

Abstract Study question Is volume electron microscopy suitable for high-resolution oocyte imaging in three dimensions (3D)? Summary answer Focused Ion Bean Scanning Electron Microscopy (FIB-SEM) allows 3D visualization and quantitative analysis of ultrastructural features in the large human oocyte volume. What is known already Transmission electron microscopy (TEM) has been traditionally used to study the fine morphology of female gametes. However, 2D micrographs provide only limited information about the topology of subcellular structures. Volumetric studies are needed to elucidate the 3D organization of complex oocyte cytoplasm. Study design, size, duration An academic study conducted in collaboration with an IVF clinic. Advanced 3D - ultrastructural analysis was performed on 9 human oocytes representing 3 stages of maturation (3 germinal vesicle (GV), 3 metaphase I (MI), 3 metaphase (MII) oocytes) collected from February 2018 to November 2019. Participants/materials, setting, methods Spare IVF oocytes, donated by 9 young egg donors (22-29 years), were cultured in vitro until they reached a defined developmental stage. Each oocytés meiotic status was determined based on the presence/absence of a prophase nucleus, a polar body, and a non-invasively detectable MI/MII spindle. Following standard TEM preparation, individual oocyte-containing resin blocks were coated with a thin carbon layer[JJ1] , mounted on the microscope stage, and subjected to FIB-SEM imaging. Main results and the role of chance FIB-SEM tomography provided an unprecedented view of the oocytés intracellular morphology. Automated serial scanning of newly exposed sample surface generated large stacks (120-1294 slices) of ultrastructural images with 40-100 nm z-resolution. The tomographic reconstruction of acquired datasets revealed the spatial arrangement of inner oocyteś structures. The imaging protocol was optimized to ensure sufficient image detail, minimal noisiness, and time-efficiency of large volume scanning. Comparison of oocytes fixed at different maturation stages confirmed previous TEM observations that the cortical region of GV oocytes is deprived of membraneous structures, and major organelle redistribution occurs during the MI phase. Semi-automated 3D image segmentation was employed to distinguish distinct organelle populations and evaluated their abundance. Subsequent quantitative analysis of volumetric data showed that the mitochondrion occupies ∼ 5.27 % of MII oocyte volume. In conclusion, the volumetric imaging, followed by advanced image analysis, maximizes the amount of morphological data obtained from a single human oocyte. Limitations, reasons for caution The imaging procedure was pioneered on a small number of hormonally-primed oocytes, which failed to complete development in vivo. There is a trade-off between resolution, the size of the 3D volume, and imaging time. Block-face ion milling during FIB-SEM imaging inevitably results in sample destruction. Wider implications of the findings This proof-of-concept study opens up new possibilities to study the delicate architecture of scarce human oocytes. Enhancing our knowledge of the spatial organization of ooplasm is pivotal for developing experimental and therapeutical strategies involving oocyte microsurgery. Trial registration number not applicable

P–186 Volumetric imaging provides insight into the 3D ultrastructural organization of maturing human oocytes

Abstract Study question Is volume electron microscopy suitable for high-resolution oocyte imaging in three dimensions (3D)? Summary answer Focused Ion Bean Scanning Electron Microscopy (FIB-SEM) allows 3D visualization and quantitative analysis of ultrastructural features in the large human oocyte volume. What is known already Transmission electron microscopy (TEM) has been traditionally used to study the fine morphology of female gametes. However, 2D micrographs provide only limited information about the topology of subcellular structures. Volumetric studies are needed to elucidate the 3D organization of complex oocyte cytoplasm. Study design, size, duration An academic study conducted in collaboration with an IVF clinic. Advanced 3D - ultrastructural analysis was performed on 9 human oocytes representing 3 stages of maturation (3 germinal vesicle (GV), 3 metaphase I (MI), 3 metaphase (MII) oocytes) collected from February 2018 to November 2019. Participants/materials, setting, methods Spare IVF oocytes, donated by 9 young egg donors (22–29 years), were cultured in vitro until they reached a defined developmental stage. Each oocytés meiotic status was determined based on the presence/absence of a prophase nucleus, a polar body, and a non-invasively detectable MI/MII spindle. Following standard TEM preparation, individual oocyte-containing resin blocks were coated with a thin carbon layer[JJ1] , mounted on the microscope stage, and subjected to FIB-SEM imaging. Main results and the role of chance FIB-SEM tomography provided an unprecedented view of the oocytés intracellular morphology. Automated serial scanning of newly exposed sample surface generated large stacks (120–1294 slices) of ultrastructural images with 40–100 nm z-resolution. The tomographic reconstruction of acquired datasets revealed the spatial arrangement of inner oocyteś structures. The imaging protocol was optimized to ensure sufficient image detail, minimal noisiness, and time-efficiency of large volume scanning. Comparison of oocytes fixed at different maturation stages confirmed previous TEM observations that the cortical region of GV oocytes is deprived of membraneous structures, and major organelle redistribution occurs during the MI phase. Semi-automated 3D image segmentation was employed to distinguish distinct organelle populations and evaluated their abundance. Subsequent quantitative analysis of volumetric data showed that the mitochondrion occupies ∼ 5.27% of MII oocyte volume. In conclusion, the volumetric imaging, followed by advanced image analysis, maximizes the amount of morphological data obtained from a single human oocyte. Limitations, reasons for caution The imaging procedure was pioneered on a small number of hormonally-primed oocytes, which failed to complete development in vivo. There is a trade-off between resolution, the size of the 3D volume, and imaging time. Block-face ion milling during FIB-SEM imaging inevitably results in sample destruction. Wider implications of the findings: This proof-of-concept study opens up new possibilities to study the delicate architecture of scarce human oocytes. Enhancing our knowledge of the spatial organization of ooplasm is pivotal for developing experimental and therapeutical strategies involving oocyte microsurgery. Trial registration number Not applicable

P–528 rDNA methylation of human oocytes of women undergoing intracytoplasmic sperm injection (ICSI) increases with maternal age

Abstract Study question Is there a correlation between the age of women undergoing ICSI and methylation pattern of rDNA core promoter and upstream control element in immature human oocytes? Summary answer Methylation levels of the upstream control element and the rDNA core promoter in immature human oocytes increase with age of women undergoing ICSI. What is known already Methylation of ribosomal DNA (rDNA) in germ cells regulates temporary and spatially highly coordinated nucleolar activity, cellular metabolism, and thus developmental potential of the early embryo. Alterations of methylation pattern may therefore cause dysregulation of genes and signal cascades resulting in limited fertility. It has been shown that the methylation of sperm rDNA increases with the donor’s age. The positive correlation between sperm rDNA methylation and age has been conserved among mammals during evolution including humans and mice. In contrast to sperm, little is known about the methylome of human oocytes and its role in human reproduction. Study design, size, duration Consecutive women undergoing ICSI because of male subfertility were included. Patients with endometriosis, polycystic ovary syndrome, ovarian, uterine or breast cancer, as well as patients with an anti-Mullerian hormone level &amp;lt;1ng/ml were excluded. Immature oocytes (germinal vesicle; GV) collected during oocyte pick-up at the Fertility Centre Dortmund between 2018 and 2020 were examined. Participants/materials, setting, methods Cumulus-free GV oocytes which were not usable for ICSI were rinsed in phosphate buffer and stored at –20 °C until further investigation. Multiplex-PCR followed by singleplex-PCRs were carried out on the rDNA core promoter and upstream control element. Methylation levels were quantified by bisulphite pyrosequencing. Two oppositely imprinted genes (hPEG3 and hGTL2) were used as controls to ensure correct amplification and bisulphite conversion. Spearman’s-rank-order-correlation and Mann-Whitney-U-Test were used for statistical analysis. Main results and the role of chance For each GV oocyte, nine different Cytosine-phosphate-Guanine dinucleotides (CpGs) were quantified by bisulphite pyrosequencing for the rDNA core promoter and 26 different CpGs for the upstream control element (UCE). 120 human single oocytes from 60 women were analyzed. Connected statistical analysis was used if one patient had more than one oocyte. The age of the included women ranged from 26 to 40 years (mean±SD 33.5±3.2). Only oocytes which showed a correct methylation pattern for at least one imprinting control gene (hPEG3 and hGTL2) were considered for analysis. Mean methylation level ranged from 2–31% (mean±SD 8.7±5.5) of the analyzed CpGs for the rDNA core promoter and from 3–36% (mean±SD 11.4±7.1) CpGs for UCE. Spearman’s correlation analysis revealed that the methylation levels of the human oocyte rDNA core promoter and rDNA UCE significantly increased with the age of the donor (p &amp;lt; 0.05). Correlation coefficient for rDNA core promoter was r = 0.22 and for upstream control element r = 0.21. It is also interesting to note that different oocytes from the same donors can display enormous methylation variation. Regarding clinical parameters, no correlation was observed between the methylation pattern of the rDNA core promoter or UCE and the body mass index or smoking status, respectively. Limitations, reasons for caution Limitations of this study include difficulties in extrapolating the findings to the general population, because no data of women not undergoing ICSI are available. Only GV-oocytes were analyzed. Additional research is needed to clarify the effect of different methylation pattern with increasing female age and its role in human reproduction. Wider implications of the findings: We propose that the increase of rDNA methylation in male and female germ cells with advanced age directly or indirectly influences the regulation of nucleolar activity, cellular metabolism, and thus the developmental potential of the early embryo. This age-dependent epigenetic effect may result in decreased human fertility. Trial registration number NCT03565107

O-184 Maternally inherited differences in mitochondrial DNA genotype between ART and spontaneously conceived individuals associate with low birthweight

Abstract Study question Can mitochondrial DNA (mtDNA) variants explain the differences in birthweight between ART and spontaneously conceived (SC) individuals and how do they originate? Summary answer Children born after ART carry more frequently a different mtDNA variant composition, both maternally inherited and de novo, which are predictive of their birthweight percentile. What is known already Children born after ART show an increased risk of lower birthweight and of developing a mild abnormal cardio-metabolic profile later in life. Variation in the mtDNA associates with overall health in the general population, including cardio-metabolic fitness, and can result in changes in mitochondrial function. We hypothesized that mitochondrial DNA variants could explain the differences in birthweight between ART and SC individuals and that these differences may result from maternal transmission and/or from the ovarian stimulation (OS) used in ART. Study design, size, duration We deep-sequenced the mtDNA of 472 individuals of who 283 ART and 189 SC, 182 mother-child pairs and 113 single oocytes from both natural menstrual cycles and OS cycles. The mtDNA was compared between groups and Fisher linear discriminant analysis was used as predictive model for the birthweight percentile. Participants/materials, setting, methods Mitochondrial DNA was enriched by long-range PCR and subsequently sequenced on an Illumina platform. mtDNA server and MuTect were used for variant calling for variants with a load higher than 1.5%, versus the reference NC_012920.1. An orthogonally rotated factor analysis was used to reduce the dimensionality of the studied dependent variables in the complex data of the heteroplasmic variants. Main results and the role of chance ART individuals carried more frequently haplogroup U4 (p = 0.004) and component analysis indicated that they carry a different mtDNA heteroplasmic variant composition than SC individuals (p = 0.01), driven by non-synonymous protein-coding and rRNA-coding variants. These differences were also predictive of the risk of a lower birthweight percentile, especially for the SC children, together with the absence of haplogroup T, the presence of homoplasmic tRNA-variants, pregnancy-induced hypertension and the embryo culture medium used. The differences in heteroplasmic variation observed in the ART children resulted from both maternal transmission (p = 0.03) and de novo mutagenesis (p = 0.02). Mothers of ART children showed a similar mtDNA genotype as their children and differed in the same variant composition when compared to the mothers of SC children (p = 0.03). Furthermore, the comparison of oocytes from the same donors retrieved in natural menstrual cycles and after one OS cycle showed that OS does not increase de novo mutagenesis. Additionally, clinical parameters such as the total dosage of FSH units, the number of oocytes retrieved, and maternal age did not show any correlation with the differences observed in ART individuals. Limitations, reasons for caution This study is observational with no functional tests being performed. Wider implications of the findings We demonstrate an association between a lower birthweight percentile and a mtDNA variant composition which is more frequently carried by ART children. These non-disease associated mtDNA variants could cause a suboptimal mitochondrial function affecting the birthweight. Long-term health consequences of these differences remain to be further elucidated. Trial registration number Not applicable

P–676 Mild stimulation followed by embryo accumulation via vitrification appears to be beneficial for managing poor ovarian response: A retrospective cohort study including 610 patients

Abstract Study question Could embryo accumulation employing mild stimulation cycles prove beneficial for managing patients presenting with poor ovarian response (POR)? Summary answer Embryo accumulation may be an efficient POR management strategy, enabling a higher number and quality cohort of embryos, ultimately improving success results. What is known already It is widely accepted that POR constitutes a challenging condition. The limited oocyte yield associated with POR detrimentally impacts in vitro fertilization (IVF) success rates. Moreover, the documented heterogeneity among POR patients compromises our efforts to successfully address POR, despite the advances noted regarding stimulation protocols employed today. Considering the aforementioned, embryo accumulation following consecutive stimulation cycles has emerged as an alternative management strategy towards increasing the number of available embryos prior to embryo transfer (ET), mimicking normoresponding conditions. However, only few studies have been so far conducted and the need for further data is underlined. Study design, size, duration A single-center retrospective study was conducted in the Centre of Human Reproduction, Genesis-Athens Clinic from January 2015-December 2019. Only patients presenting with POR according to Bologna criteria were included. In total, 610 POR patients were considered eligible and were divided in three groups namely, mild stimulation-fresh ET (150 IUs of gonadotropins) (MILDF), mild stimulation employing embryo accumulation (MILDA), and natural cycle employing embryo accumulation (NATA). Respective comparisons on embryology and pregnancy data are provided. Participants/materials, setting, methods Resulting embryos from the MILDF, MILDA, and NATA groups were cultured up to the cleavage stage and categorized into three groups according to quality, namely top (grade 1), good (grade 2–3) and poor (grade 4–5) (Veeck, 1999). Top and good quality embryos were considered eligible for ET/vitrification. The banking scenario entailed accumulation of at least three embryos, including at least one top quality embryo. Embryo transfers included up to two cleavage stage embryos. Main results and the role of chance Comparing MILDF and MILDA groups, a higher number of available oocytes and embryos was observed in MILDA (2.36±1.15 vs 6.58±1.11; 1.72±1.02 vs 3.51±0.61, P-value&amp;lt;0.001). However, a mean number of 3.90±1.56 oocyte retrievals were required to conclude MILDA compared to MILDF which was concluded following a single oocyte retrieval (P-value&amp;lt;0.001). Cancellation-rate was significantly lower in the MILDA compared to MILDF group (0% vs 18.93%, P-value &amp;lt;0.001). A higher proportion of top quality embryos were transferred in the MILDA group (66.58% vs 43.67%, P-value&amp;lt;0.001). The MILDA group presented with higher positive-HCG (27.89% vs 23.30%, P-value=0.302), clinical-pregnancy (22.11% vs 17.96%, P-value=0.316) and live-birth rates (16.84% vs 14.08%, P-value=0.487). However, these differences were not significant. Comparing MILDA and NATA groups, the MILDA presented with a lower number of required oocyte retrievals and a higher number of oocytes per oocyte retrieval compared with NATA (3.90±1.56 vs 7.15±1.80; 1.95±0.74 vs 0.89±0.20, P-value&amp;lt;0.001). Moreover, the MILDA presented with a higher mean number of resulting embryos (5.20±0.78 vs 4.82±0.88, P-value&amp;lt;0.001). No difference was observed regarding the proportion of the resulting top quality embryos. The MILDA group presented with slightly higher clinical-pregnancy (22.11% vs 20.09%, P-value=0.628) and live-birth (16.84% vs 14.02%, P-value=0.490) rates, however these differences were not significant. Limitations, reasons for caution The retrospective nature of the study constitutes a major limitation. Considering that numerous confounders are inevitable when retrospective data is analyzed, authors employed strict eligibility criteria in an effort to reduce bias. Statistical analysis revealed a well-controlled population, considering that general patients’ characteristics did not differ between the three groups. Wider implications of the findings: Embryo accumulation may constitute an efficient management strategy for POR, as more embryos of better quality are available for ET compared to fresh-IVF-ET. Mild stimulation should be preferred for embryo accumulation instead of natural cycles, as less oocyte retrievals are required. Future studies should be conducted to verify these conclusions. Trial registration number Not applicable

Transcriptome Profiling of Oocytes at the Germinal Vesicle Stage from Women from Mongolia with Polycystic Ovary Syndrome.

BackgroundPolycystic ovary syndrome (PCOS) is one of the most common endocrine disorders. Evidence indicates that genetic and environmental factors contribute to the pathogenesis of PCOS. The molecular basis of PCOS is not well understood.MethodsWhole-genome RNA sequencing was performed on single oocyte at the germinal vesicle (GV) stage from females with normal ovulation and females with PCOS. All subjects were women from Mongolia undergoing intracytoplasmic sperm injection in vitro fertilization (ICSI-IVF) who met the Rotterdam criteria for PCOS. Women with normal ovulation who were undergoing ICSI-IVF owing to male factor infertility were recruited as control subjects.ResultsA total of 1313 differentially expressed genes were found by bio-informatics software in the GV oocytes of PCOS patients and compared with the control group. There were 367 upregulated and 946 downregulated genes (fold change > 2, false discovery rate < 0.01). When compared with the healthy controls, it was shown that the DEGs like VEGF, IGF, FADS1 et al were investigated as potential causes of PCOS oocytes. The DEGs were related to kinase activity, cell proliferation, gene regulation, and the signaling pathways of phosphatidylinositol 3-kinase, Hippo, and ECM-receptor pathway in patients with PCOS. In addition, the interconnected gene co-expression network was constructed by gene bionetwork analysis, indicating that ITGB5, ITGB3, and CAV2 were the core genes in regulating the module expression of DEGs in PCOS.ConclusionRNA sequencing analysis demonstrated DEGs were linked to inflammation, cardiovascular disease, and lipid metabolism in the GV oocytes of women with PCOS. We hypothesize that ITGB5, ITGB3, and CAV2 may be involved in metabolic disorders associated with the different phenotypes of PCOS.

The reproductive system of Ceracis cornifer (Mellié) and first description of sperm structure in a minute tree-fungus beetle (Tenebrionoidea: Ciidae)

Studies on the spermatogenesis of Tenebrionidae beetles (Tenebrionoidea) have shown an unusual organization of spermatozoa, in which they are arranged antiparallelly within the testicular cysts. Despite such works, many taxa of Tenebrionoidea remain to be studied, including the minute tree-fungus beetles (Ciidae). Among the challenges in the study of the internal morphology of ciids is their small body size (about 2 mm or less), which makes dissections and comparisons extremely arduous. Here, we provide, for the first time, an anatomical and histological study of the reproductive system (female and male) and a description of sperm structure of Ceracis cornifer Mellié (Ciidae), under light and transmission electron microscopes. We pointed out the growth of a single oocyte at a time in females, aside of evidences toward a constant copulatory activity that can provide a continuous supply of sperm in their small spermatheca. In males, sperm have an antiparallel organization within the cysts, a condition observed so far only in members of Tenebrionoidea. Furthermore, we observed sperm dimorphism in C. cornifer. We described, for the first time, this characteristic in a Tenebrionoidea taxon, and discussed the possible relationship between sperm dimorphism and antiparallel organization of sperm.

Single-Oocyte Gene Expression Suggests That Curcumin Can Protect the Ovarian Reserve by Regulating the PTEN-AKT-FOXO3a Pathway.

A better understanding of the mechanism of primordial follicle activation will help us better understand the causes of premature ovarian insufficiency (POI), and will help us identify new drugs that can be applied to the clinical treatment of infertility. In this study, single oocytes were isolated from primordial and primary follicles, and were used for gene profiling with TaqMan array cards. Bioinformatics analysis was performed on the gene expression data, and Ingenuity Pathway Analysis was used to analyze and predict drugs that affect follicle activation. An ovarian in vitro culture system was used to verify the function of the drug candidates, and we found that curcumin maintains the ovarian reserve. Long-term treatment with 100 mg/kg curcumin improved the ovarian reserve indicators of AMH, FSH, and estradiol in aging mice. Mechanistic studies show that curcumin can affect the translocation of FOXO3, thereby inhibiting the PTEN-AKT-FOXO3a pathway and protecting primordial follicles from overactivation. These results suggest that curcumin is a potential drug for the treatment of POI patients and for fertility preservation.

Defining the Program of Maternal mRNA Translation during In vitro Maturation using a Single Oocyte Reporter Assay.

Events associated with oocyte nuclear maturation have been well described. However, much less is known about the molecular pathways and processes that take place in the cytoplasm in preparation for fertilization and acquisition of totipotency. During oocyte maturation, changes in gene expression depend exclusively on the translation and degradation of maternal messenger RNAs (mRNAs) rather than on transcription. Execution of the translational program, therefore, plays a key role in establishing oocyte developmental competence to sustain embryo development. This paper is part of a focus on defining the program of maternal mRNA translation that takes place during meiotic maturation and at the oocyte-to-zygote transition. In this method paper, a strategy is presented to study the regulation of translation of target mRNAs during in vitro oocyte maturation. Here, a Ypet reporter is fused to the 3' untranslated region (UTR) of the gene of interest and then micro-injected into oocytes together with polyadenylated mRNA encoding for mCherry to control for injected volume. By using time-lapse microscopy to measure reporter accumulation, translation rates are calculated at different transitions during oocyte meiotic maturation. Here, the protocols for oocyte isolation and injection, time-lapse recording, and data analysis have been described, using the Ypet/interleukin-7 (IL-7)-3' UTR reporter as an example.

Defining the Program of Maternal mRNA Translation during In vitro Maturation using a Single Oocyte Reporter Assay

Defining the Program of Maternal mRNA Translation during In vitro Maturation using a Single Oocyte Reporter Assay

Comparison of Ovarian Morphology and Follicular Disturbances between Two Inbred Strains of Cotton Rats (Sigmodon hispidus).

Simple SummaryMulti-oocyte follicles have been reported in several mammals, especially in rabbits and hamsters, although their significance remains unclear. The present study compared ovarian histology, focusing on folliculogenesis, between two inbred cotton rat strains maintained at Hokkaido Institute of Public Health and the University of Miyazaki. Abundant multi-oocyte follicles and double-nucleated oocytes were observed in the Hokkaido strain, whereas Miyazaki had fewer multi-oocyte follicles and lacked double-nucleated oocytes. These findings indicate that early folliculogenesis events such as oocyte nest breakdown and oocyte vitality, rather than proliferation and cell death in each oocyte, affect the unique ovarian phenotypes found in cotton rats, including multi-oocyte follicles or double-nucleated oocytes, and their differences between strains. Therefore, these results can clarify mammalian folliculogenesis and its abnormal processes.Most mammalian ovarian follicles contain only a single oocyte having a single nucleus. However, two or more oocytes and nuclei are observed within one follicle and one oocyte, respectively, in several species, including cotton rat (CR, Sigmodon hispidus). The present study compared ovarian histology, focusing on folliculogenesis, between two inbred CR strains, HIS/Hiph and HIS/Mz. At 4 weeks of age, ovarian sections from both the strains were analyzed histologically. Multi-oocyte follicles (MOFs) and double-nucleated oocytes (DNOs) were observed in all stages of developing follicles in HIS/Hiph, whereas HIS/Mz had MOFs up to secondary stages and lacked DNOs. The estimated total follicles in HIS/Mz were almost half that of HIS/Hiph, but interstitial cells were well developed in HIS/Mz. Furthermore, immunostaining revealed no clear strain differences in the appearance of oocytes positive for Ki67, PCNA, and p63 in MOF or DNOs; no cell death was observed in these oocytes. Ultrastructural analysis revealed more abundant mitochondrial clouds in oocytes of HIS/Hiph than HIS/Mz. Thus, we clarified the strain differences in the CR ovary. These findings indicate that early events during folliculogenesis affect the unique ovarian phenotypes found in CRs, including MOFs or DNOs, and their strain differences.

Day 5 vitrified blastocyst transfer versus day 6 vitrified blastocyst transfer in oocyte donation program

The superiority of day 5 blastocysts compared to day 6 blastocysts in fresh cycle transfers was previously demonstrated and attributed mainly to endometrial asynchrony. Data from frozen blastocysts transfers showed conflicting results, possibly due to heterogeneous patient population and embryo quality. The aim of this study was to compare clinical pregnancy rate (CPR) and live birth rate (LBR) between transfers of vitrified day 5 blastocysts and day 6 blastocysts in oocyte donation, blastocyst-only cycles. In a retrospective, multi-center study, with a single oocyte donation program, a total of 1840 frozen embryo transfers (FET’s) were analyzed, including 1180 day 5 blastocysts and 660 day 6 blastocysts transfers. Day 5 blastocyst transfers had better embryonic development and significantly higher CPRs (34.24% vs. 20.15%, P < 0.0001), higher LBRs (26.89% vs. 14.77%, P < 0.0001), less cycles to LBR (1.83 ± 0.08 vs. 2.39 ± 0.18, P = 0.003) and shorter time to LBRs (76.32 ± 8.7 vs. 123.24 ± 19.1 days, P = 0.01), compared to day 6 transfers, respectively. A multivariate stepwise logistic regression indicated, that day 5 transfer was an independent factor for CPRs (OR 1.91; 95% CI 1.43–2.54, P < 0.001) and LBRs (OR 2.26; 95% CI 1.19–4.28, P = 0.01), regardless of embryo quality, compared to day 6. In conclusion, day 5 blastocysts in oocyte donation program have significantly higher CPRs and LBRs, and present shorter time to delivery, compared to day 6 blastocysts, regardless of embryo quality.

Decoding dynamic epigenetic landscapes in human oocytes using single-cell multi-omics sequencing

Developing female human germ cells undergo genome-wide epigenetic reprogramming, but de novo DNA methylation dynamics and their interplay with chromatin states and transcriptional activation in developing oocytes is poorly understood. Here, we developed a single-cell multi-omics sequencing method, scChaRM-seq, that enables simultaneous profiling of the DNA methylome, transcriptome, and chromatin accessibility in single human oocytes and ovarian somatic cells. We observed a global increase in DNA methylation during human oocyte growth that correlates with chromatin accessibility, whereas increases of DNA methylation at specific features were associated with active transcription. Integrated analyses of multi-omics data from humans and mice revealed species-specific gene expression, and promoter accessibility contributes to gene body methylation programs. Alu elements retained low DNA methylation levels and high accessibility in early growing oocytes and were located near developmental genes in humans and mice. Together, these findings show how scChaRM-seq can provide insight into DNA methylation pattern establishment.

Single human oocyte transcriptome analysis reveals distinct maturation stage-dependent pathways impacted by age.

Female fertility is inversely correlated with maternal age due to a depletion of the oocyte pool and a reduction in oocyte developmental competence. Few studies have addressed the effect of maternal age on the human mature oocyte (MII) transcriptome, which is established during oocyte growth and maturation, however, the pathways involved remain unclear. Here, we characterize and compare the transcriptomes of a large cohort of fully grown germinal vesicle stage (GV) and in vitro matured (IVM‐MII) oocytes from women of varying reproductive age. First, we identified two clusters of cells reflecting the oocyte maturation stage (GV and IVM‐MII) with 4445 and 324 putative marker genes, respectively. Furthermore, we identified genes for which transcript representation either progressively increased or decreased with age. Our results indicate that the transcriptome is more affected by age in IVM‐MII oocytes (1219 genes) than in GV oocytes (596 genes). In particular, we found that transcripts of genes involved in chromosome segregation and RNA splicing significantly increased representation with age, while genes related to mitochondrial activity showed a lower representation. Gene regulatory network analysis facilitated the identification of potential upstream master regulators of the genes involved in those biological functions. Our analysis suggests that advanced maternal age does not globally affect the oocyte transcriptome at GV or IVM‐MII stages. Nonetheless, hundreds of genes displayed altered transcript representation, particularly in IVM‐MII oocytes, which might contribute to the age‐related quality decline in human oocytes.

Reproduction, recruitment, and growth of the Arctic deep‐sea hydroid Bouillonia cornucopia

AbstractInvertebrates in polar and deep‐sea environments that have complex life histories are exposed to unique environmental conditions that may favor non‐pelagic development and K‐strategist reproduction. Although many polar species follow this strategy, the numerically most abundant species tend to have more r‐strategist life‐history characteristics. We deployed artificial substrata over 3 years in the Arctic deep sea and collected hundreds of specimens of the athecate hydroid Bouillonia cornucopia. While this species has previously been described as rare, we report dense, patchy recruitment on artificial substrata, suggesting that B. cornucopia is highly opportunistic. This species has rapid growth compared to other sessile invertebrates in the study area, high fecundity, and continuous reproduction—all characteristics of an r‐selected life history. The species’ gonophores are simple, lacking an obvious spadix or radial canals. We observed nurse cells in histological sections of female gonophores, but no male gonophores were observed. Gonophores break away from the blastostyles in mature specimens and appear to have fertilization envelopes, suggesting that each gonophore is composed of a single oocyte and that embryological development occurs in the water column. Hydroids are typically the first invertebrates to recruit to substrata in the Arctic but are easily overgrown. The opportunistic life histories of B. cornucopia and other hydroids may be adaptive for maintaining populations in the face of high mortality. Our study demonstrates the utility of artificial substrata for collections of otherwise rare opportunistic species.

Fibrillarin-GFP Facilitates the Identification of Meiotic Competent Oocytes.

The nucleolus undergoes significant functional changes and plays important roles during mammalian oocyte meiotic maturation. Fibrillarin (FBL) is the component of nucleolar small nuclear ribonucleoprotein (snRNP) particle and localizes to the dense fibrillar component (DFC) of the nucleolus. We found that FBL–GFP displays an uneven and cloudy localization in the nucleolus of non-surrounded nucleolus (NSN) oocytes, while it distributes evenly and to a few bright dots in the surrounded nucleolus (SN) oocytes. Accordingly, NSN oocytes showed active nascent RNA transcription, while the SN group was transcriptionally quiescent. NSN geminal vesicles also contained more DNA damage marker γH2AX foci. Based on different FBL–GFP patterns in live oocytes, the ones with superior meiotic maturation potential can be identified. Global transcriptome profiling revealed a significant difference in single SN and NSN oocytes. Thus, FBL–GFP can serve as a marker for nucleolus activity, which also correlates with transcription activity and the quality of oocytes.

Decoding the transcriptome of pre-granulosa cells during the formation of primordial follicles in the mouse

Primordial follicles, a finite reservoir of eggs in mammalian ovaries, are composed of a single oocyte and its supporting somatic cells, termed granulosa cells. Although their formation may require reciprocal interplay between oocytes and pre-granulosa cells, precursors of granulosa cells, little is known about the underlying mechanisms. We addressed this issue by decoding the transcriptome of pre-granulosa cells during the formation of primordial follicles. We found that marked gene expression changes, including extracellular matrix, cell adhesion, and several signaling pathways, occur along with primordial follicle formation. Importantly, differentiation of Lgr5-EGFP-positive pre-granulosa cells to FOXL2-positive granulosa cells was delayed in mutant ovaries of the germ cell-specific genes Nanos3 and Figla, accompanied by perturbed gene expression in mutant pre-granulosa cells. These results suggest that proper development of oocytes is required for the differentiation of pre-granulosa cells. Our data provide a valuable resource for understanding the gene regulatory networks involved in the formation of primordial follicles.

Melatonin Modulates Lipid Metabolism in Porcine Cumulus-Oocyte Complex via Its Receptors.

Lipid is a crucial energy resource for mammalian oocyte. Melatonin could benefit the maturation of porcine oocyte in vitro, but the related mechanism is not elucidated yet. In the current study, methods to monitor lipid metabolism in single live oocytes were firstly established using probes (Lipi-Blue and Lipi-Green). It was observed that both lipid biogenesis and lipolysis occurred in maturing oocyte, but the general level of lipids dropped. Then maturing oocytes stained with probes were treated with melatonin or lipid metabolic-related inhibitors (triacsin C, rotenone, or etomoxir). The results showed that the lipid metabolism and maturation of porcine oocytes were all disrupted and that melatonin rescued the oocytes treated with triacsin C or rotenone, but not those treated with etomoxir. Further investigation demonstrated that cumulus cells are able to transfer lipids to oocytes via gap junctions. It was also observed that melatonin receptors exist in cumulus cells and are required for oocytes to maintain lipid metabolism. Meanwhile, the global gene expressing in cumulus cells was also modulated by melatonin, especially the genes related to antioxidants (SOD1, GPX1, GPX3, GPX4, PRDX2, and PRDX5), lipid metabolism (FABP3, FABP5, ACACB, TECR, etc.), and mitochondrial respiration (GPD1, ETFB, CYC1, and the genes of ATP synthase). Altogether the current research demonstrates that melatonin modulates lipid metabolism in maturing oocytes through its receptors in cumulus cells and benefits the developmental competence of oocytes.