Single-cell proteomics reveals decreased abundance of proteostasis and meiosis proteins in advanced maternal age oocytes.

Advanced maternal age is associated with a decline in oocyte quality, which often leads to reproductive failure in humans. However, the mechanisms behind this age-related decline remain unclear. To gain insights into this phenomenon, we applied plexDIA, a multiplexed data-independent acquisition, single-cell mass spectrometry method, to analyze the proteome of oocytes from both young women and women of advanced maternal age. Our findings primarily revealed distinct proteomic profiles between immature fully grown germinal vesicle and mature metaphase II oocytes. Importantly, we further show that a woman's age is associated with changes in her oocyte proteome. Specifically, when compared to oocytes obtained from young women, advanced maternal age oocytes exhibited lower levels of the proteasome and TRiC complex, as well as other key regulators of proteostasis and meiosis. This suggests that aging adversely affects the proteostasis and meiosis networks in human oocytes. The proteins identified in this study hold potential as targets for improving oocyte quality and may guide future studies into the molecular processes underlying oocyte aging.

Phospholipase C Zeta in Human Spermatozoa: A Systematic Review on Current Development and Clinical Application.

During fertilization, the fusion of the spermatozoa with the oocytes causes the release of calcium from the oocyte endoplasmatic reticulum. This, in turn, triggers a series of calcium ion (Ca2+) oscillations, a process known as oocyte activation. The sperm-specific factor responsible for oocyte activation is phospholipase C zeta (PLCζ). Men undergoing intracytoplasmic sperm injection (ICSI) with their spermatozoa lacking PLCζ are incapable of generating Ca2+ oscillation, leading to fertilization failure. The immunofluorescence assay is the most used technique to assess the expression and localization of PLCζ and to diagnose patients with reduced/absent ability to activate the oocytes. In these patients, the use of assisted oocyte activation (AOA) technique can help to yield successful ICSI results and shorten the time of pregnancy. However, the production of a stable PLCζ recombinant protein represents a new powerful therapeutic approach to treating individuals with this condition. We aim to conduct a systematic review focusing on the expression, level, and localization of PLCζ, discussing the novel genetic mutation associated with its impairment. In addition, we highlight the benefits of AOA, looking at new and less invasive methods to diagnose and treat cases with PLCζ dysfunction.

Genome diversity and instability in human germ cells and preimplantation embryos.

Genome diversity is essential for evolution and is of fundamental importance to human health. Generating genome diversity requires phases of DNA damage and repair that can cause genome instability. Humans have a high incidence of de novo congenital disorders compared to other organisms. Recent access to eggs, sperm and preimplantation embryos is revealing unprecedented rates of genome instability that may result in infertility and de novo mutations that cause genomic imbalance in at least 70% of conceptions. The error type and incidence of de novo mutations differ during developmental stages and are influenced by differences in male and female meiosis. In females, DNA repair is a critical factor that determines fertility and reproductive lifespan. In males, aberrant meiotic recombination causes infertility, embryonic failure and pregnancy loss. Evidence suggest germ cells are remarkably diverse in the type of genome instability that they display and the DNA damage responses they deploy. Additionally, the initial embryonic cell cycles are characterized by a high degree of genome instability that cause congenital disorders and may limit the use of CRISPR-Cas9 for heritable genome editing.

Specific processing of meiosis-related transcript is linked to final maturation in human oocytes.

Human meiosis in oocytes entails an intricate regulation of the transcriptome to support late oocyte growth and early embryo development, both crucial to reproductive success. Currently, little is known about the co- and post-transcriptional mRNA processing mechanisms regulating the last meiotic phases, which contribute to transcriptome complexity and influence translation rates. We analyzed gene expression changes, splicing and pre-mRNA processing in an RNA sequencing set of 40 human oocytes at different meiotic maturation stages, matured both in vivo and in vitro. We found abundant untranslated region (UTR) processing, mostly at the 3' end, of meiosis-related genes between the germinal vesicle (GV) and metaphase II (MII) stages, supported by the differential expression of spliceosome and pre-mRNA processing related genes. Importantly, we found very few differences among GV oocytes across several durations of IVM, as long as they did not reach MII, suggesting an association of RNA processing and successful meiosis transit. Changes in protein isoforms are minor, although specific and consistent for genes involved in chromosome organization and spindle assembly. In conclusion, we reveal a dynamic transcript remodeling during human female meiosis, and show how pre-mRNA processing, specifically 3'UTR shortening, drives a selective translational regulation of transcripts necessary to reach final meiotic maturation.

All that glitters is not gold: a stereological study of human donor oocytes.

Here we report a quantitative analysis of human metaphase II (MII) oocytes from a 22-year-old oocyte donor, retrieved after ovarian-controlled hyperstimulation. Five surplus donor oocytes were processed for transmission electron microscopy (TEM), and a stereological analysis was used to quantify the distribution of organelles, using the point-counting technique with an adequate stereological grid. Comparisons between means of the relative volumes (Vv) occupied by organelles in the three oocyte regions, cortex (C), subcortex (SC) and inner cytoplasm (IC), followed the Kruskal-Wallis test and Mann-Whitney U-test with Bonferroni correction. Life cell imaging and TEM analysis confirmed donor oocyte nuclear maturity. Results showed that the most abundant organelles were smooth endoplasmic reticulum (SER) elements (26.8%) and mitochondria (5.49%). Significant differences between oocyte regions were found for lysosomes (P = 0.003), cortical vesicles (P = 0.002) and large SER vesicles (P = 0.009). These results were quantitatively compared with previous results using prophase I (GV) and metaphase I (MI) immature oocytes. In donor MII oocytes there was a normal presence of cortical vesicles, SER tubules, SER small, medium and large vesicles, lysosomes and mitochondria. However, donor MII oocytes displayed signs of cytoplasmic immaturity, namely the presence of dictyosomes, present in GV oocytes and rare in MI oocytes, of SER very large vesicles, characteristic of GV oocytes, and the rarity of SER tubular aggregates. Results therefore indicate that the criterion of nuclear maturity used for donor oocyte selection does not always correspond to cytoplasmic maturity, which can partially explain implantation failures with the use of donor oocytes.

Expression of genes in the AKT signalling pathway in human oocytes from patients with polycystic ovaries.

Polycystic ovary syndrome is an endocrine disorder commonly found among females of reproductive age. Different factors have been correlated with this syndrome, although the aetiology of the disease is still unrecognized with both environmental and hereditary factors leading to the progression. Hormonal effects of the AKT pathway have made it an interesting study unit for PCOS cases. The aim of this study was to investigate the expression patterns of genes involved in the AKT pathway, including IRS1, IRS2, AKT1 and AKT2. In total, 13 human oocytes were collected for this study at the meiosis II stage, in which seven of them were collected from individuals with polycystic ovaries and the rest formed the control group of individuals with no signs of polycystic ovaries. RNA was extracted from oocytes and then the RNA was converted into cDNA for the real-time PCR process. Expression levels of four genes in the AKT pathway, in addition to housekeeping gene (ACTB), were evaluated. Expression levels of each gene were quantified using real-time PCR and statistical analysis was performed. The results of this study showed that there was no significant correlation between the expression of genes in oocyte samples obtained from patients with polycystic ovaries and the control group. This study is the first to evaluate the expression levels of genes involved in the AKT pathway in human oocyte samples. Therefore, it provides crucial information to form the basis of further studies.

Chromatin Configuration in Diplotene Mouse and Human Oocytes during the Period of Transcriptional Activity Extinction.

In the oocyte nucleus, called the germinal vesicle (GV) at the prolonged diplotene stage of the meiotic prophase, chromatin undergoes a global rearrangement, which is often accompanied by the cessation of its transcriptional activity. In many mammals, including mice and humans, chromatin condenses around a special nuclear organelle called the atypical nucleolus or formerly nucleolus-like body. Chromatin configuration is an important indicator of the quality of GV oocytes and largely predicts their ability to resume meiosis and successful embryonic development. In mice, GV oocytes are traditionally divided into the NSN (non-surrounded nucleolus) and SN (surrounded nucleolus) based on the specific chromatin configuration. The NSN-SN transition is a key event in mouse oogenesis and the main prerequisite for the normal development of the embryo. As for humans, there is no single nomenclature for the chromatin configuration at the GV stage. This often leads to discrepancies and misunderstandings, the overcoming of which should expand the scope of the application of mouse oocytes as a model for developing new methods for assessing and improving the quality of human oocytes. As a first approximation and with a certain proviso, the mouse NSN/SN classification can be used for the primary characterization of human GV oocytes. The task of this review is to analyze and discuss the existing classifications of chromatin configuration in mouse and human GV oocytes with an emphasis on transcriptional activity extinction at the end of oocyte growth.

Validating Reference Gene Expression Stability in Human Ovarian Follicles, Oocytes, Cumulus Cells, Ovarian Medulla, and Ovarian Cortex Tissue.

Human ovarian cells are phenotypically very different and are often only available in limited amounts. Despite the fact that reference gene (RG) expression stability has been validated in oocytes and other ovarian cells from several animal species, the suitability of a single universal RG in the different human ovarian cells and tissues has not been determined. The present study aimed to validate the expression stability of five of the most used RGs in human oocytes, cumulus cells, preantral follicles, ovarian medulla, and ovarian cortex tissue. The selected genes were glyceraldehyde 3-phosphate dehydrogenase (GAPDH), beta-2-microglobulin (B2M), large ribosomal protein P0 (RPLP0), beta-actin (ACTB), and peptidylprolyl isomerase A (PPIA). Overall, the stability of all RGs differed among ovarian cell types and tissues. NormFinder identified ACTB as the best RG for oocytes and cumulus cells, and B2M for medulla tissue and isolated follicles. The combination of two RGs only marginally increased the stability, indicating that using a single validated RG would be sufficient when the available testing material is limited. For the ovarian cortex, depending on culture conditions, GAPDH or ACTB were found to be the most stable genes. Our results highlight the importance of assessing RGs for each cell type or tissue when performing RT-qPCR analysis.

Next Generation Sequencing Detects Premeiotic Errors in Human Oocytes.

Autosomal aneuploidy is the leading cause of embryonic and foetal death in humans. This arises mainly from errors in meiosis I or II of oogenesis. A largely ignored source of error stems from germinal mosaicism, which leads to premeiotic aneuploidy. Molecular cytogenetic studies employing metaphase fluorescence in situ hybridization and comparative genomic hybridisation suggest that premeiotic aneuploidy may affect 10-20% of oocytes overall. Such studies have been criticised on technical grounds. We report here an independent study carried out on unmanipulated oocytes that have been analysed using next generation sequencing (NGS). This study confirms that the incidence of premeiotic aneuploidy in an unselected series of oocytes exceeds 10%. A total of 140 oocytes donated by 42 women gave conclusive results; of these, 124 (88.5%) were euploid. Sixteen out of 140 (11.4%) provided evidence of premeiotic aneuploidy. Of the 140, 112 oocytes were immature (germinal vesicle or metaphase I), of which 10 were aneuploid (8.93%); the remaining 28 were intact metaphase II - first polar body complexes, and six of these were aneuploid (21.4%). Of the 16 aneuploid cells, half contained simple errors (one or two abnormal chromosomes) and half contained complex errors. We conclude that germinal mosaicism leading to premeiotic aneuploidy is a consistent finding affecting at least 10% of unselected oocytes from women undergoing egg collection for a variety of reasons. The importance of premeiotic aneuploidy lies in the fact that, for individual oocytes, it greatly increases the risk of an aneuploid mature oocyte irrespective of maternal age. As such, this may account for some cases of aneuploid conceptions in very young women.

Clusters of smooth endoplasmic reticulum are absent in oocytes from unstimulated women.

RESEARCH QUESTION: What is the frequency of morphological dysmorphisms in immature human oocytes collected ex vivo from small antral follicles and matured in vitro? DESIGN: Human ovaries (n = 56) were excised for ovarian tissue cryopreservation (OTC). None of the patients had received exogenous gonadotrophins prior to the procedure. Immature oocytes released from small antral follicles were collected in connection with isolation of the cortex for OTC. The oocytes' maturation stage and the morphological characteristics of the cytoplasm, zona pellucida, perivitelline space and first polar body were assessed after in-vitro maturation (IVM). RESULTS: A total of 1649 immature oocytes were collected: 30% of oocytes matured to the metaphase II (MII) stage after IVM, while metaphase I (MI), germinal vesicle and degenerated oocytes accounted for 20%, 24% and 26%, respectively. The percentages of oocytes without any dysmorphisms were 53%, 92%, and 97% for the MII, MI and germinal vesicle stage oocytes, respectively. The most frequently observed dysmorphisms among the MII oocytes were first polar body fragmentation (22%), homogeneously distributed cytoplasmic granularity (16%) and an enlarged perivitelline space (14%). Interestingly, none of the oocytes at any stage had clusters of smooth endoplasmic reticulum (SER). CONCLUSIONS: Morphological dysmorphisms are present among in-vitro-matured oocytes at all maturation stages. The incidence of dysmorphisms increases as maturation progresses. The most frequent dysmorphism among MII oocytes after IVM was fragmentation of the first polar body. Clusters of SER were not observed in oocytes from unstimulated patients.

Melatonin improves the effect of cryopreservation on human oocytes by suppressing oxidative stress and maintaining the permeability of the oolemma.

Cryopreservation causes cryoinjury to oocytes and impairs their developmental competence. Melatonin (MLT) can improve the effect of cryopreservation in animal oocytes. However, no such studies on human oocytes have been reported. In this study, collected in vitro-matured human oocytes were randomly divided into the following groups: fresh group, MLT-treated cryopreservation (MC) group, and no-MLT-treated cryopreservation (NC) group. After vitrification and warming, viable oocytes from these three groups were assessed for their mitochondrial function, ultrastructure, permeability of oolemma, early apoptosis, developmental competence, and cryotolerance-related gene expression. First, fluorescence staining results revealed that oocytes from the 10-9  M subgroup showed the lowest intracellular reactive oxygen species and Ca2+ levels and highest mitochondrial membrane potential among the MC subgroups (10-11 , 10-9 , 10-7 , and 10-5  M). In subsequent experiments, oocytes from the 10-9  M-MC group were observed to maintain the normal ultrastructural features and the permeability of the oolemma. Compared with those of the oocytes in the NC group, the early apoptosis rate significantly decreased (P < .01), whereas both the high-quality cleavage embryo and blastocyst rates significantly increased (both P < .05) in the oocytes of the 10-9  M-MC group. Finally, single-cell RNA sequencing and immunofluorescence results revealed that aquaporin (AQP) 1/2/11 gene expression and AQP1 protein expression were upregulated in the MC group. Therefore, these results suggest that MLT can improve the effect of cryopreservation on human oocytes by suppressing oxidative stress and maintaining the permeability of the oolemma.

Stereological study of organelle distribution in human oocytes at metaphase I.

We have previously presented a stereological analysis of organelle distribution in human prophase I oocytes. In the present study, using a similar stereological approach, we quantified the distribution of organelles in human metaphase I (MI) oocytes also retrieved after ovarian stimulation. Five MI oocytes were processed for transmission electron microscopy and a classical manual stereological technique based on point-counting with an adequate stereological grid was used. Kruskal-Wallis and Mann-Whitney U-tests with Bonferroni correction were used to compare the means of relative volumes (Vv) occupied by organelles. In all oocyte regions, the most abundant organelles were mitochondria and smooth endoplasmic reticulum (SER) elements. No significant differences were observed in Vv of mitochondria, dictyosomes, lysosomes, or SER small and medium vesicles, tubular aggregates and tubules. Significant differences were observed in other organelle distributions: cortical vesicles presented a higher Vv (P = 0.004) in the cortex than in the subcortex (0.96% vs 0.1%) or inner cytoplasm (0.96% vs 0.1%), vesicles with dense granular contents had a higher Vv (P = 0.005) in the cortex than in the subcortex (0.1% vs 0%), and SER large vesicles exhibited a higher Vv (P = 0.011) in the inner cytoplasm than in the subcortex (0.2% vs 0%). Future stereological analysis of metaphase II oocytes and a combined quantitative data of mature and immature oocytes, will enable a better understanding of oocyte organelle distribution during in vivo maturation. Combined with molecular approaches, this may help improve stimulation protocols and in vitro maturation methods.

Voltage-gated proton channels exist in the plasma membrane of human oocytes.

STUDY QUESTION: Do human oocytes express voltage-gated proton channels? SUMMARY ANSWER: Human oocytes exhibit voltage-gated proton currents. WHAT IS KNOWN ALREADY: Voltage-gated proton currents have been reported in human sperm, where they contribute to capacitation and motility. No such studies of human oocytes exist. STUDY DESIGN, SIZE, DURATION: Voltage-clamp studies were undertaken using entire oocytes and vesicles derived from oocytes and in excised patches of membrane from oocytes. PARTICIPANTS/MATERIALS, SETTING, METHODS: Frozen, thawed human metaphase II oocytes were obtained from material donated to the gamete repository at the Rush Center for Advanced Reproductive Care. Prior to patch clamping, oocytes were warmed and equilibrated. Formation of an electrically tight seal requires exposing bare oolemma. Sections of the zona pellucida (ZP) were removed using a laser, followed by repeated pipetting, to further separate the oocyte from the ZP. Patch-clamp studies were performed using the whole-cell configuration on oocytes or vesicles derived from oocytes, and using inside-out patches of membrane, under conditions optimized to detect voltage-gated proton currents. MAIN RESULTS AND THE ROLE OF CHANCE: Proton currents are present at significant levels in human oocytes where they exhibit properties similar to those reported in other human cells, as well as those in heterologous expression systems transfected with the HVCN1 gene that codes for the voltage-gated proton channel. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: Human oocytes are large cells, which limits our ability to control the intracellular solution. Subtle effects of cryopreservation by vitrification and subsequent warming on properties of HVCN1, the HVCN1 gene product, cannot be ruled out. WIDER IMPLICATIONS OF THE FINDINGS: Possible functions for voltage-gated proton channels in human oocytes may now be contemplated. STUDY FUNDING/COMPETING INTEREST(S): NIH R35GM126902 (TED), Bears Care (DM). No competing interests. TRIAL REGISTRATION NUMBER: N/A.

Oocyte collection and in vitro maturation after train transportation of human follicular fluid aspirated from resected non-stimulated ovaries of patients with endometrial adenocarcinoma.

PURPOSE: Immature human oocytes from resected ovaries can be used for research and fertility preservation, though it is unknown whether it is feasible to transport oocytes for these purposes. This study examined in vitro maturation (IVM) outcomes after the transportation of human follicular fluid (HFF) containing oocytes. METHODS: Fourteen patients with endometrial adenocarcinoma were enrolled. Oocytes obtained from the resected ovaries of seven patients were transported with HFF by railway (transportation group). Samples of HFF from the other seven patients were not transported, and IVM was performed promptly (non-transportation group). The results of oocyte retrieval and IVM were compared. RESULTS: The average ages in the transportation and non-transportation groups were 40.1 ± 2.0 and 39.6 ± 1.8 years, respectively, and the average numbers of collected oocytes were 8.1 ± 8.4 and 5.1 ± 5.1, respectively. There was a significant negative correlation between the number of collected oocytes and age. The proportions of oocytes that reached meiosis II (maturation rate) after IVM were 38.6% and 69.2% in the transportation and non-transportation groups, respectively (P = 0.013). CONCLUSION: In this preliminary study, the usefulness of the transportation of HFF was limited. Further studies on maintaining oocyte normality during transportation are necessary for becoming the effective method for research and clinical use.

COMPARISON OF MORPHOMETRIC AND MORPHOLOGY OOCYTES AFTER IN VITRO MATURATION BETWEEN HEALTHY WOMEN AND PATIENTS WITH POLYCYSTIC OVARIAN SYNDROME.

OBJECTIVES: Polycystic ovarian syndrome (PCOS) occurs in 6-10% of all women in their reproductive age. In women with PCOS, controlled ovarian hyperstimulation (COH) often results in an increased risk of ovarian hyperstimulation syndrome (OHSS). In vitro maturation (IVM) of human oocyte is an alternative technique for in vitro fertilization (IVF). The aim of this study was to compare the morphometric analysis and morphology of oocytes after in- vitro maturation (IVM) between normal women and those suffering from polycystic ovary syndrome (PCOS). MATERIAL AND METHODS: Thirty two women of 20 to 35 years of age that were undergoing controlled ovarian stimulation by the ICSI/IVF protocol were chosen for the study. The immature oocytes (n=108) were divided into two groups: the first oocyte group was comprised of 16 normal women (n=54); and the second group included 16 women with PCOS (n=54); then the oocytes were matured in vitro. After 24-48h of incubation, the oocyte maturation rate and morphometric and morphological characteristics were assessed using an inverted microscope, and then the images were compared. RESULTS: There were significant differences in the maturity of oocytes between normal women and those with PCOS after IVM (P<0.05). Moreover, morphometric assessments revealed that there were no significant difference in the total diameter (µm) (zona thickness (ZPT) + perivitelline space width (PVS) + cytoplasm (CD) of oocytes between normal women and those with PCOS (156.3±6.8 and 137.7±9.9), respectively (P>0.05). Evaluation of morphological oocytes showed that morphological abnormalities, including ooplasmic vacuolization and granulation were higher in PCOS women compared to normal women (P<0.05). CONCLUSION: The increased quality of oocytes after IVM reflected a positive impact of IVM oocytes on normal women as compared to women with PCOS.

Endothelin-1 promotes human germinal vesicle-stage oocyte maturation by downregulating connexin-26 expression in cumulus cells.

STUDY QUESTION: Does endothelin-1 (ET-1) promote human oocyte maturation and by what mechanism? SUMMARY ANSWER: Addition of ET-1 to the medium in which human germinal vesicle (GV)-stage immature oocytes are cultured enhances the GV breakdown (GVBD) rate; the resumption of meiosis may be initiated by ET-1 downregulating the expression of connexin-26 (Cx26) in cumulus cells via endothelin receptor type B (ETRB), leading to decreased cAMP levels in the oocyte. WHAT IS KNOWN ALREADY: The paracrine factor ET-1 is secreted by ovarian somatic cells in pre-ovulatory follicles and regulates oocyte maturation in mice. Connexins, or gap junction proteins, form intercellular membrane channels that play important roles in the resumption of meiosis. STUDY DESIGN, SIZE, DURATION: This laboratory study was conducted over a 1-year period. The effects of ET-1 on meiotic resumption were evaluated in human GV-stage cumulus-oocyte complexes (COCs; 70 oocytes/group). The transcriptome profiles of ET-1-treated or untreated cumulus cells were compared to explore the possible mechanisms by which ET-1 may regulate oocyte maturation. PARTICIPANTS/MATERIALS, SETTING, METHODS: The ET-1, ETRA and ETRB expression levels in human cumulus cells from oocytes at different stages of maturation were evaluated using real-time quantitative PCR. Human GV-stage COCs collected from patients undergoing IVF at a university-affiliated infertility centre were cultured with or without ET-1, and cumulus cells were subsequently denuded using hyaluronidase and cultured in α-MEM. A GeneChip® Human Transcriptome Array was applied to explore differences in the whole-genome transcriptome profiles of cumulus cells treated with or without ET-1. Real-time quantitative PCR and Western blotting were used respectively to examine Cx26 mRNA and protein levels in cumulus cells. Changes in cAMP levels in both oocytes and cumulus cells after ET-1 treatment were measured using an enzyme-linked immunosorbent assay. MAIN RESULTS AND THE ROLE OF CHANCE: Cumulus cells from MII-stage oocytes exhibited upregulated ET-1 expression, compared to those from GV-stage oocytes. The addition of ET-1 to the culture medium enhanced the GVBD rate of cumulus cell-enclosed human oocytes. Whole-genome transcriptome microarray analyses revealed significantly downregulated Cx26 expression in cumulus cells after ET-1 treatment, and this action was blocked by an ETRB antagonist. The involvement of Cx26 was further supported by the finding that ET-1 treatment led to decreased cAMP levels in oocytes but increased cAMP levels in cumulus cells. LARGE SCALE DATA: Microarray data are published in the GEO database (GSE97684). LIMITATIONS, REASONS FOR CAUTION: The heterogeneity of human COCs collected from patients undergoing IVF might affect the maturation results in vitro. Although we focused on the effects of ET-1 on human oocyte maturation in the present study, mammalian oocyte maturation is a complicated process involving many endocrine and paracrine factors. WIDER IMPLICATIONS OF THE FINDINGS: Our present study suggests that in vitro, human GV-stage oocyte maturation could be enhanced by adding ET-1 to the culture medium. In the present study, we explored the molecular mechanisms by which ET-1 initiates the resumption of meiosis and demonstrated that ET-1 promotes oocyte maturation by downregulating the expression of the gap junction protein Cx26 in cumulus cells. These results expand our understanding of the molecular mechanisms underlying mammalian oocyte maturation and provide a basis for better in-vitro maturation strategies. STUDY FUNDING AND COMPETING INTERESTS: This work was supported by grants from the China Natural Science Foundation (Grant Nos. 81170567 and 81370761). The authors declare that they have no conflicts of interest associated with this manuscript.

Intra-individual purifying selection on mitochondrial DNA variants during human oogenesis.

STUDY QUESTION: Does selection for mtDNA mutations occur in human oocytes? SUMMARY ANSWER: We provide statistical evidence in favor of the existence of purifying selection for mtDNA mutations in human oocytes acting between the expulsion of the first and second polar bodies (PBs). WHAT IS KNOWN ALREADY: Several lines of evidence in Metazoa, including humans, indicate that variation within the germline of mitochondrial genomes is under purifying selection. The presence of this internal selection filter in the germline has important consequences for the evolutionary trajectory of mtDNA. However, the nature and localization of this internal filter are still unclear while several hypotheses are proposed in the literature. STUDY DESIGN, SIZE, DURATION: In this study, 60 mitochondrial genomes were sequenced from 17 sets of oocytes, first and second PBs, and peripheral blood taken from nine women between 38 and 43 years of age. PARTICIPANTS/MATERIALS, SETTING, METHODS: Whole genome amplification was performed only on the single cell samples and Sanger sequencing was performed on amplicons. The comparison of variant profiles between first and second PB sequences showed no difference in substitution rates but displayed instead a sharp difference in pathogenicity scores of protein-coding sequences using three different metrics (MutPred, Polyphen and SNPs&GO). MAIN RESULTS AND THE ROLE OF CHANCE: Unlike the first, second PBs showed no significant differences in pathogenic scores with blood and oocyte sequences. This suggests that a filtering mechanism for disadvantageous variants operates during oocyte development between the expulsion of the first and second PB. LARGE SCALE DATA: N/A. LIMITATIONS, REASONS FOR CAUTION: The sample size is small and further studies are needed before this approach can be used in clinical practice. Studies on a model organism would allow the sample size to be increased. WIDER IMPLICATIONS OF THE FINDINGS: This work opens the way to the study of the correlation between mtDNA mutations, mitochondrial capacity and viability of oocytes. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by a SISMER grant. Laboratory facilities and skills were freely provided by SISMER, and by the Alma Mater Studiorum, University of Bologna. The authors have no conflict of interest to disclose.

Ultrastructural Changes and Methylation of Human Oocytes Vitrified at the Germinal Vesicle Stage and Matured in vitro after Thawing.

AIM: The study aimed to assess the effect of in vitro vitrification and maturation on the nuclear configuration, cytoplasmic maturation and global DNA methylation pattern of human germinal vesicle (GV) stage oocytes. METHODS: Human oocytes from infertile women were randomly assigned to one of 3 groups: (i) metaphase II (MII) oocytes matured in vivo (vivo-MII, n = 56) as controls; (ii) MII oocytes matured in vitro (vitro-MII, n = 106); and (iii) MII oocytes that were vitrified at the GV stage, warmed and matured in vitro (cryo-MII, n = 122). All MII oocytes were fixed and immunofluorescence staining for spindle, chromosome, mitochondrion and cortical granules (CGs) were performed; examination was done using immunofluorescence laser scanning confocal microscope. The expression of 5-methycytosine in these MII oocytes was also assessed. RESULTS: No significant difference was observed between vitro-MII and cryo-MII groups with respect to oocyte maturation rate (72.4 vs. 78.3%). No significant differences were observed in the distribution of mitochondria, migration of CG and global DNA methylation pattern among the 3 study groups. However, the abnormal configuration of spindle and chromosome was significantly higher in cryo-MII group (78.9 and 84.2%) as compared to that in the vitro-MII (45.0 and 50.0%, p < 0.05) and vivo-MII groups (27.3 and 36.4%, p < 0.05). CONCLUSION: GV oocytes appeared to resist vitrification and retain their potential for maturation to MII stage oocytes after thawing. However, GV oocytes vitrification combined with in vitro maturation could affect the organization of spindle and chromosome.

Expression and localization of tubulin cofactors TBCD and TBCE in human gametes.

The tubulin cofactors TBCD and TBCE play an essential role in regulation of the microtubule dynamics in a wide variety of somatic cells, but little information is known about the expression of these cofactors in human sperm and oocytes. In this study, we focused on the investigation of the presence of, and the differential distribution of, the tubulin cofactors TBCD and TBCE in human sperm and during human oocyte maturation. We performed expression assays for TBCD and TBCE by reverse transcription-polymerase chain reaction (RT-PCR), western blot and immunofluorescence and verified the presence of both cofactors in human gametes. TBCD and TBCE were located mainly in the middle region and in the tail of the sperm while in the oocyte the localization was cytosolic. The mRNA of both tubulin cofactors were present in the human oocytes but not in sperm cells. This finding gives a first insight into where TBCD and TBCE could carry out their function in the continuous changes that the cytoskeleton experiences during gametogenesis and also prior to fertilization.

A spontaneous increase in intracellular Ca2+ in metaphase II human oocytes in vitro can be prevented by drugs targeting ATP-sensitive K+ channels.

STUDY QUESTION: Could drugs targeting ATP-sensitive K(+) (K(ATP)) channels prevent any spontaneous increase in intracellular Ca(2+) that may occur in human metaphase II (MII) oocytes under in vitro conditions? SUMMARY ANSWER: Pinacidil, a K(ATP) channel opener, and glibenclamide, a K(ATP) channel blocker, prevent a spontaneous increase in intracellular Ca(2+) in human MII oocytes. WHAT IS KNOWN ALREADY: The quality of the oocyte and maintenance of this quality during in vitro processing in the assisted reproductive technology (ART) laboratory is of critical importance to successful embryo development and a healthy live birth. Maintenance of Ca(2+) homeostasis is crucial for cell wellbeing and increased intracellular Ca(2+) levels is a well-established indicator of cell stress. STUDY DESIGN, SIZE, DURATION: Supernumerary human oocytes (n = 102) collected during IVF/ICSI treatment that failed to fertilize were used from October 2013 to July 2015. All experiments were performed on mature (MII) oocytes. Dynamics of intracellular Ca(2+) levels were monitored in oocytes in the following experimental groups: (i) Control, (ii) Dimethyl sulfoxide (DMSO; used to dissolve pinacidil, glibenclamide and 2,4-Dinitrophenol (DNP)), (iii) Pinacidil, (iv) Glibenclamide, (v) DNP: an inhibitor of oxidative phosphorylation, (vi) Pinacidil and DNP and (vii) Glibenclamide and DNP. PARTICIPANTS/MATERIALS/SETTINGS/METHODS: Oocytes were collected under sedation as part of routine treatment at an assisted conception unit from healthy women (mean ± SD) age 34.1 ± 0.6 years, n = 41. Those surplus to clinical use were donated for research. Oocytes were loaded with Fluo-3 Ca(2+)-sensitive dye, and monitored by laser confocal microscopy for 2 h at 10 min intervals. Time between oocyte collection and start of Ca(2+) monitoring was 80.4 ± 2.1 h. MAIN RESULTS AND THE ROLE OF CHANCE: Intracellular levels of Ca(2+) increased under in vitro conditions with no deliberate challenge, as shown by Fluo-3 fluorescence increasing from 61.0 ± 11.8 AU (AU = arbitrary units; n = 23) to 91.8 ± 14.0 AU (n = 19; P < 0.001) after 2 h of monitoring. Pinacidil (100 µM) inhibited this increase in Ca(2+) (85.3 ± 12.3 AU at the beginning of the experiment, 81.7 ± 11.0 AU at the end of the experiment; n = 13; P = 0.616). Glibenclamide (100 µM) also inhibited the increase in Ca(2+) (74.7 ± 10.6 AU at the beginning and 71.8 ± 10.9 AU at the end of the experiment; n = 13; P = 0.851. DNP (100 mM) induced an increase in intracellular Ca(2+) that was inhibited by glibenclamide (100 µM; n = 9) but not by pinacidil (100 µM; n = 5). LIMITATIONS, REASONS FOR CAUTION: Owing to clinical and ethical considerations, it was not possible to monitor Ca(2+) in MII oocytes immediately after retrieval. MII oocytes were available for our experimentation only after unsuccessful IVF or ICSI, which was, on average, 80.4 ± 2.1 h (n = 102 oocytes) after the moment of retrieval. As the MII oocytes used here were those that were not successfully fertilized, it is possible that they may have been abnormal with impaired Ca(2+) homeostasis and, furthermore, the altered Ca(2+) homeostasis might have been associated solely with the protracted incubation. WIDER IMPLICATIONS OF THE FINDINGS: These results show that maintenance of oocytes under in vitro conditions is associated with intracellular increase in Ca(2+), which can be counteracted by drugs targeting K(ATP) channels. As Ca(2+) homeostasis is crucial for contributing to a successful outcome of ART, these results suggest that K(ATP) channel openers and blockers should be tested as drugs for improving success rates of ART. STUDY FUNDING/COMPETING INTERESTS: University of Dundee, MRC (MR/K013343/1, MR/012492/1), NHS Tayside. Funding NHS fellowship (Dr Sarah Martins da Silva), NHS Scotland. The authors declare no conflicts of interest.