Molecular Human Reproduction

Study questionDoes application of human stem cell-derived ovarian support cells (OSCs) for in vitro maturation (IVM) have a safe reproductive toxicity profile? Summary answerThe use of OSC-IVM co-culture improves blastocyst formation in a mouse model and results in healthy live births with no evidence of reprotoxicity. What is known alreadyAbbreviated stimulation to obtain immature oocytes combined with a successful IVM offers a promising alternative to traditional in vitro fertilization, reducing hormonal doses and making IVF shorter and safer. Recently, we developed an OSC platform derived from human induced pluripotent stem cells (hiPSCs) that replicate dynamic ovarian function in vitro, enhancing human oocyte maturation and yielding an improved blastocyst formation rate compared to commercial IVM options. However, the reproductive toxicity profile, commonly assessed via murine multigenerational models, for OSC-IVM remains unknown. Study design, size, durationA total of 70 B6/CBA 6-8-week-old stimulated female mice were used in this study to collect immature mouse oocytes (n=2,025) at the germinal vesicle (GV) stage. Half of these oocytes were retrieved denuded (denuded oocytes condition, n= 930), while the remaining oocytes were kept with the cumulus cells (COCs condition, n= 1,095) to simulate the two possible dispositions of oocytes during clinical practice. Oocytes from each condition, denuded oocytes and COCs, were randomly assigned to either commercially available traditional IVM media (MediCult-IVMTM, Origio) group (control group) or the same traditional IVM media supplemented with human OSCs (FertiloTM, Gameto Inc.) to form the OSC-IVM group (test group). Participants/materials, setting, methodsOocytes from each condition, denuded oocytes and COCs, were subjected to in vitro culture for 18-20 hours. After IVM, metaphase II (M2) oocytes were inseminated by intracytoplasmic sperm injection (ICSI) and cultured to assess blastocyst formation in vitro. Embryos that reached the blastocyst stage on day five were vitrified using Kitazatos protocol in preparation for embryo transfers. A group of M2 oocytes and blastocyst embryos were employed for quality analyses by immunofluorescence. Vitrified blastocysts were warmed and transferred to pseudopregnant females (4-5 embryos per uterine horn), evaluating the F1 offspring. Pup characteristics were tracked, including weight, length, sex ratio, and physiology. Weekly monitoring assessed mouse behavior and development. At reproductive age, select F1 mice were outbred to wildtype mice to produce the F2 generation, analyzing live births, sex ratio, morphology, and behavior across groups. Moreover, hormonal and organ histological analyses were performed in F1 mice to further explore the overall health of the progeny. Main results and the role of chanceIn contrast to findings in humans, in mice OSC-IVM generally led to a decreased maturation rate compared to Traditional-IVM (68.6% {+/-} 14.1% versus 80.9% {+/-} 5.9%, p=0.0101). Subsequent embryo culture yielded significantly different fertilization rates between the four groups (p=0.0055). Specifically, OSC-IVM with COCs significantly differed from Traditional-IVM with denuded oocytes (89.5 {+/-} 10.5 versus 96.5 {+/-} 4.8, p=0.0098). There were no differences in the cleavage rates (p=0.7547). However, there was a significant distinction in the blastocyst formation (p=0.0068), wherein OSC-IVM with COCs showed a greater formation rate compared to Traditional-IVM for both denuded oocytes and COCs (56.1% {+/-} 19.2% versus 41.5% {+/-} 15.9% and 38.0% {+/-} 16.2%; p=0.0408, and p=0.0063). Spindle morphology analysis demonstrated normal spindle morphology in denuded oocytes and COCs under both Traditional-IVM and OSC-IVM. Moreover, embryo analysis showed no significant difference in inner cell mass count (p=0.1550). Following embryo transfers, analysis of live births showed no significant distinctions between groups regarding delivery, sex ratio, pup length, developmental and behavioral abnormalities, hormonal values or histopathological anomalies in the F1 generation. Evaluation of the F2 generation also showed no significant differences in live births, sex ratio, or developmental/behavioral abnormalities between groups, further validating the absence of long-term implications and transgenerational effects derived from OSC-IVM culture. Limitations, reasons for cautionAlthough this study was conducted in compliance with European Medicines Agency (EMA) ICH E6 (R2) Good clinical practice scientific guidelines to demonstrate the OSC safety, human clinical studies evaluating in vivo and live birth outcomes are necessary to corroborate the findings of this study. Wider implications of the findingsThis study provides evidence of the safety of using the OSC-IVM system, as evidenced by the lack of adverse effects on in vitro embryo development post OSC-IVM and on the health and fertility of offspring across successive generations in vivo. Trial registration numberN/A

Elucidating the underlining mechanisms underpinning successful fertilisation is imperative in optimising IVF treatments, and may lead to a specific diagnosis and therefore potential treatment for some infertile couples. One of the critical steps involves paternal chromatin reprogramming, in which compacted sperm chromatin packed by protamines is removed by oocyte factors and new histones, including histone H3.3, are incorporated. This step is critical for the formation of the male pronucleus, without which the zygote contains only 1 pronucleus (1PN), in contrast to normally fertilised zygotes with two-pronuclei (2PN). 1PN zygotes are a frequently observed phenomenon in IVF treatments, therefore aberrant mechanism of action controlling paternal chromatin repackaging may be an important cause of abnormal fertilisation. Hira is the main H3.3 chaperone that governs this protamine-to-histone exchange. In this study, we investigated the maternal functions of two other molecules of the Hira complex, Cabin1 and Ubn1 in the mouse. Loss-of-function Cabin1 and Ubn1 mouse models were developed: their zygotes displayed abnormal 1PN zygote phenotypes, similar to the phenotype of Hira mutants. We then studied human 1PN zygotes, and found that the Hira complex was absent in 1PN zygotes which were lacking the male pronucleus. This result confirms that the role of the Hira complex in male pronucleus formation has coherence from mice to humans. Furthermore, rescue experiments showed that the abnormal 1PN phenotype derived from Hira mutants could be resolved by overexpression of Hira in the mouse oocytes. In summary, we have provided evidence of the role of Hira complex in regulating male pronucleus formation in both mice and humans, that both Cabin1 and Ubn1 components of the Hira complex are equally essential for male pronucleus formation, and that this can be rescued. We present a proof-of-concept experiment that could potentially lead to a personalised IVF therapy for oocyte defects.

Numerous reports show that the epididymis plays a key role in the acquisition of sperm fertilizing ability but less information exists on its contribution to embryo development. Evidence from our laboratory showed that mammalian CRISP (Cysteine-Rich Secretory Proteins), known to be expressed in the epididymis, to regulate calcium (Ca2+) channels and to participate in fertilization, may also be relevant for embryo development. More specifically, we found that males with simultaneous mutations in Crisp1 and Crisp3 genes exhibited normal in vivo fertilization but impaired embryo development. In the present work, aimed to investigate the mechanisms underlying this reproductive phenotype, we observed that embryo development failure was not due to delayed fertilization as no differences in sperm transport within the female tract nor in in vivo fertilization were found shortly after mating. The observation that impaired embryo development was also found in eggs fertilized by epididymal sperm either after uterine insemination or in vitro fertilization, revealed that the defects were already present at epididymal level. Of note, eggs fertilized in vitro by mutant sperm exhibited impaired meiotic resumption not due to defects in Ca2+oscillations during egg activation, prompting us to examine potential sperm DNA defects. Interestingly, DNA fragmentation was found in cauda but not caput epididymal mutant sperm revealing that DNA integrity defects appear during epididymal maturation. Moreover, exposure of control sperm to mutant epididymal fluid significantly increased DNA fragmentation, indicating the relevance of the luminal environment for sperm DNA integrity. The finding that incubation of sperm with control epididymal fluid in the presence of Ca2+ also increased DNA fragmentation together with the higher intracellular Ca2+ levels detected in mutant sperm supports a dysregulation of Ca2+ homeostasis as the main responsible for DNA fragmentation and subsequent early development failure of mutant males. Together, our results support the contribution of the epididymis beyond fertilization, identifying CRISP1 and CRISP3 as novel male factors relevant for DNA integrity and early embryo development. Given the existence of human functional homologues of CRISP and the incidence of DNA fragmentation in infertile men, we believe these findings not only provide relevant information on the impact of epididymal factors on embryonic development but will also contribute to a better understanding, diagnosis and treatment of human infertility.

BackgroundMitochondrial dysfunction is a leading contributor to the decline in oocyte quality associated with maternal aging. Prior investigations of mitochondrial function in the ovarian follicle have largely treated the mitochondrial pool as a homogeneous population, reporting aggregate values that may obscure biologically meaningful differences between distinct mitochondrial subpopulations. The present study addresses this limitation by characterizing mitochondrial subpopulation dynamics in oocytes and cumulus granulosa cells at single-organelle resolution using fluorescence-activated mitochondria sorting (FAMS). ResultsAnalysis of the aggregate mitochondrial population in mouse oocytes revealed no significant age-associated differences in mitochondrial DNA copy number or membrane potential, a result that would previously have been interpreted as evidence of minimal age-related mitochondrial change. Subpopulation analysis revealed this conclusion to be incomplete: aged oocytes showed significantly elevated mitochondrial DNA copy number specifically within the high membrane potential and small mitochondrial subpopulations, with no significant differences in the low membrane potential or large subpopulations. NMN supplementation normalized mitochondrial DNA copy number in the high membrane potential and small subpopulations toward young levels while producing an opposing effect in large mitochondria, demonstrating subpopulation-specific rather than uniform rejuvenation. In cumulus cells, significant age-associated changes were detectable at the aggregate level, including a reduction in mitochondrial DNA copy number and an elevation in membrane potential, and subpopulation analysis further resolved these findings. The age-associated reduction in cumulus cell mitochondrial DNA copy number was driven predominantly by the high membrane potential subpopulation. NMN supplementation exerted opposing effects on small and large cumulus cell mitochondrial subpopulations, increasing mitochondrial DNA copy number above both young and aged levels in small mitochondria while further reducing it below aged levels in large mitochondria. ConclusionsViewing the mitochondrial pool as a heterogeneous mixture of functionally distinct subpopulations rather than a uniform population reveals age-associated alterations in oocytes and cumulus cells that are undetectable by aggregate analysis. NMN supplementation exerts subpopulation-specific effects in both cell types, identifying specific mitochondrial subtypes as more precise targets for future mechanistic investigation of age-associated infertility than the mitochondrial pool considered in aggregate.

Non-obstructive azoospermia (NOA) is the absence of sperm in the ejaculate due to spermatogenic failure. Fifty percent of NOA cases are unexplained but may arise from unidentified genetic mutations. Variants in TEX11 have been identified in men with NOA; and Tex11 knockout in mice causes NOA. Here we attempt to validate three TEX11 variants discovered in NOA patients by knocking them into the orthologous region of the mouse genome using CRISPR/Cas9 gene editing. Compared to wild type (144.2 {+/-} 9.87 mg; 1.7 {+/-} 0.5 million sperm/cauda epididymis 4.8 {+/-} 1.3 pups/breeding), Tex11D mice (frameshift mutation) had reduced testis weight (28.33 {+/-} 1.16 mg); no sperm in the epididymis; and were infertile with a maturation arrest testicular phenotype. We did not observe any spermatogenesis or fertility defects Tex11A mice (missense mutation). Tex11L mice had reduced testis weight (87.5 {+/-} 14.79 mg) and epididymal sperm counts (0.33{+/-}0.13 million/cauda epididymis) but an incompletely penetrant infertility phenotype (5.4 {+/-} 1.13 pups/breeding) with one third of mice being infertile. Infertile Tex11L mice also had a distinct epididymal phenotype with reduced sperm density in the caput and no sperm in the cauda, which was filled with amorphous material.

Recurrent pregnancy loss (RPL) affects 1-2% of women trying to conceive, yet in many cases the causes remain unclear. Endometrial function is central to the establishment and maintenance of pregnancy, and endometrial dysfunction may underlie RPL. A greater understanding of the endometrial cell populations and their interactions in women with and without RPL may identify markers of endometrial receptivity and the likelihood of pregnancy success. Single cell RNA sequencing was performed on RPL (n = 3) and control (n = 4) endometrial biopsies collected at days 21-24 of the menstrual cycle, the window of implantation. 10,022 cells were clustered and nine major individual cell types were characterised. Further analysis identified six distinct endometrial stromal cell (EnSCs) and three natural killer (NK) cell sub-populations. In RPL, there were changes in the abundance of specific endometrial stromal and NK cell subpopulations with associated differences in cellular communication between the cell types related to the Wnt pathway and angiogenesis. This is consistent with NK cell signalling orchestrating the difference in abundance of stromal cells and regulating processes needed for successful implantation. These changes in RPL endometrium provide further evidence for an endometrial cause of RPL and identify specific mechanisms for future study.

Study questionWhich proteins underpin oocyte developmental competence, as modelled by oocytes of variable competence matured in vivo, or matured in vitro under different conditions (capacitation in vitro maturation (CAPA) or standard in vitro maturation (IVM))? Summary answerSignificant differences in the global proteome were observed in both oocytes and their corresponding cumulus cells depending on the mode of oocyte maturation, with key variations in eukaryotic translation, autophagy and endocytosis pathways within oocytes, and changes in reactive oxygen species detoxification and serine biosynthesis in cumulus cells. What is known alreadyWithin the ovarian follicle, mammalian oocytes must acquire the necessary molecular machinery to support successful fertilisation and embryonic development. Close contact with the surrounding cumulus cells ensures coordinated nuclear and cytoplasmic maturation of the oocyte, along with the accumulation of proteins stored within the oocyte in cytoplasmic lattices and endo-lysosomal vesicular assemblies. Study design, size, durationThis basic science study utilised a mouse model to assess proteomic changes across three oocyte competence models. Key proteins identified in mouse oocytes were also assessed in discarded immature human germinal vesicle (GV) oocytes and MII oocytes following rescue-IVM. Three oocyte maturation methods were tested: i) in vivo maturation, (ii) CAPA and (iii) standard IVM. In vivo maturation served as a positive control group, whereby metaphase II (MII) mature oocytes were collected from mice stimulated with pregnant mare serum gonadotropin (PMSG) and triggered with human chorionic gonadotropin (hCG), simulating full ovarian stimulation. For the in vitro maturation groups, immature cumulus oocyte complexes (COCs) were collected from mildly stimulated (23 hr PMSG) mice. For the standard IVM group, immature COCs were matured in media containing amphiregulin and epiregulin for 18 hours. For the CAPA group, COCs were held for 24 hours in pre-IVM conditions in the presence of c-type natriuretic peptide (CNP), oestradiol, insulin and follicle stimulating hormone (FSH), and then matured via IVM in media containing FSH, amphiregulin and epiregulin. Four biological replicates were performed for mouse proteomics experiments, three biological replicates performed for mouse immunocytochemistry experiments and six replicates were performed for embryology experiments. Participants/materials, settings, methodsFour to six-week-old C57BL/6JAusb mice were used for all mouse experiments. Embryology outcomes were used to confirm the variation in oocyte developmental competence between the three maturation groups. For the in vivo, CAPA and IVM groups, mature MII COCs were collected and separated into oocytes and cumulus cells. Oocytes and cumulus cells were subjected to mass spectrometry and bioinformatic analysis was performed using Proteome Discoverer and Ingenuity Pathway Analysis, with data validated by immunofluorescence. To assess conservation of proteins in human oocytes, 49 oocytes were collected from 36 patients following assisted reproduction technology (ART) cycles and subject to immunofluorescence. Rescue-IVM was also performed with half of the human oocyte cohort to obtain MII oocytes. Main results and the role of chanceProteomic profiling identified around 1600 proteins in mouse oocytes and 3100 in mouse cumulus cells across all three treatment groups (at least 2 peptides per protein). Differential expression analysis and pattern analysis collectively revealed a signature of proteins that were consistently differentially expressed between in vivo and in vitro oocyte maturation systems (log2FC of {+/-} 1 and a p-value [≤] 0.05). These subsets of proteins were mapped to biological processes including eukaryotic translation, autophagy and endocytosis pathways within oocytes. Orthogonal validation of clathrin, ribosomal protein L24 and eukaryotic initiation factor 2A supported the proteomic findings and expression was conserved in human oocytes. Changes in reactive oxygen species detoxification and serine biosynthesis were observed in mouse cumulus cells, with fluorescence intensity changes in ferredoxin-1 and phosphoglycerate dehydrogenase supporting the dysregulation of cumulus cell processes during in vitro maturation. Large scale dataThe mass spectrometry data are available via ProteomeXchange with identifier PXD073269. Limitations, reasons for cautionThe foundational mechanisms of oocyte developmental competence remain elusive, particularly in humans where MII oocytes are heterogenous in quality within the same stimulation cycle and patient. In this study, C57Bl6/J mice were used as the model species, allowing precise control over differing models of oocyte quality and capacity to analyse large numbers of oocytes. However, care is required when interpreting the significance of these findings in mice to mechanisms regulating human oocyte quality. Nonetheless, the in vivo stimulation and both IVM protocols used in this study are clinically relevant and developmentally matched. This study has also not addressed oocyte developmental competence in gonadotropin-free IVM oocytes, which is now a clinical reality. Wider implications of the findingsThis study confirms that mouse oocytes, matured in vitro in two clinically relevant systems, show reduced developmental competence when compared to in vivo matured oocytes. Through examination of the global proteome in oocytes, molecular pathways including eukaryotic translation, autophagy and endocytosis were dysregulated in in vitro oocytes. Recent findings have revealed the critical role of these pathways to developmental competence in the context of in vivo development. In cumulus cells, changes in reactive oxygen species detoxification and serine biosynthesis were observed, adding to the extensive knowledge around metabolic activity in cumulus cells as a critical facet of oocyte quality. Combined, this data suggests that the necessary processes of protein storage and degradation in oocytes and metabolism in cumulus cells constitute important components of oocyte quality. These processes appear suboptimal in current IVM systems, providing a future research direction to optimise IVM protocols with consideration to these protein pathways. Study funding/competing interestsThis study was funded by a National Health and Medical Research Council Investigator Fellowship (APP1023210) awarded to R.B.G. and by a gift from Open Philanthropy. The following competing interests are declared: R.B.G.is a consultant to Dioseve Inc.. L.E.W is a co-founder, shareholder, director and advisor of Jumpstart Fertility Inc.. L.E.W. is also an advisor and shareholder in EdenRoc Sciences, the parent company of Metro Biotech NSW and Metro Biotech, and in Life Biosciences LLC and its daughter companies. His UNSW Industry Scientia position is partly funded by Proto Axiom. All other authors have no competing interests to disclose.

Spermatogonial differentiation is controlled by distinct spatial and temporal activities of metabolizing enzymes and signaling molecule formation. In vitro models are reductionist ways to examine the interactions between Sertoli cells and spermatogonial stem cells, which may be therapeutic targets for infertility that cause non-obstructive azoospermia, due to either Sertoli-cell only syndrome, hypospermatogenesis or maturation arrest. Here, we find that nanomolar doses of isotretinoin are sufficient to drive Stra8 expression in vitro, an interaction that is both dose-dependent and inhibitable. We compare complex in vitro models (CIVMs) seeded from cells isolated post-natal day 5 (PND 5) and post-natal day 10 (PND 10) Sprague Dawley rat testis. The CIVMs maintain metabolic capacity to produce bioactive retinoids form retinol. We also investigate the impact of common media supplementations on spermatogonial phenotype and find that they can impact the expression of Stra8 and Plzf as markers of early differentiating and undifferentiated spermatogonia, respectively. These results highlight the power of in vitro models to investigate the dynamics of the spermatogonial niche.

In vitro fertilization (IVF) is a non-coital method of conception used to treat human infertility. Although IVF is viewed as largely safe, it is associated with adverse outcomes in the fetus, placenta, and adult offspring life. Because studies focusing on the effect of IVF on the male reproductive system are limited, we used a mouse model to assess the morphological and molecular effects of IVF on male offspring. We evaluated three developmental stages: 18.5-day fetuses and 12- and 39-week-old adults. Regardless of age, we observed changes in testicular-to-body weight ratios, serum testosterone levels, testicular morphology, gene expression, and DNA methylation. Also, sperm showed changes in morphology and DNA methylation. To assess multigenerational phenotypes, we mated IVF and naturally conceived males with wild-type females. Offspring from IVF males exhibited decreased fetal weight-to-placental weight ratios and changes in placenta morphology regardless of sex. At 12-weeks-of-age, offspring showed higher body weights and differences in glucose, triglycerides, insulin, total cholesterol, HDL and LDL/VLDL levels. Both sexes showed changes in gene expression in liver, testes and ovaries, and decreased global DNA methylation. Collectively, our findings demonstrate that male IVF offspring exhibit abnormal testicular and sperm morphology and molecular alterations and transmit defects multigenerationally.

ObjectiveTo understand the immediate impact that testicular heat stress has on isolated populations of precursor male germ cells including Spermatocytes and Spermatids. DesignMice were given testicular heat stress and pre-cursor male germ cells were immediately isolated. RNA sequencing was performed and validated using qPCR. SubjectsThis work was carried out in adult male CD1 mice. ResultsUsing next-generation RNA sequencing 134 differentially expressed transcripts were found to be differentially expressed upon exposure to testicular hyperthermia, 93% of which were upregulated. In addition, testicular hyperthermia induced 395 differential splicing events and altered the usage of 61 polyadenylation sites. To explain these observations and understand why transcript abundance appears to favour upregulation following testicular hyperthermia, we assessed global piRNA levels and found an overall, rapid reduction. Concomitantly, we observed an increase in transposable element RNA (LINE1) and protein (ORF1p) abundance. Furthermore, increased LINE1 expression appeared to be correlated with DNA damage in the male germline. At 24 hours post heat stress, piRNA levels recovered close to control levels, coincident with a significant reduction in LINE1 transcript expression within spermatocytes. ConclusionTesticular hyperthermia needs to be considered in the context of all reproductive outcomes. Affected spermatozoa are likely to be genetically compromised, leading to adverse outcomes such as infertility or loss of embryo following fertilization.

Cystic fibrosis transmembrane conductance regulator (CFTR) is a prominent chloride channel that governs mucous secretion in multiple organs, including the reproductive tract. According to earlier reports, defective CFTR results in infertility due to congenital bilateral absence of the vas deferens (CBAVD). However, obstruction in the vas deferens is not the only reason CFTR deficiency causes male infertility. The mechanism underlying the loss of mature sperm owing to CFTR deficiency remains elusive. This study aimed to assess the role of CFTR in spermatogenesis, for which 6- and 8-week-old male mice with Cftr+/+, Cftr+/-, and Cftr-/- genotypes were chosen. Furthermore, we assessed the correlation between CFTR deficiency and delayed development of the reproductive system, anomalous apoptosis activation in spermatogenesis, and ionic alterations of the testis lumen. The results demonstrated that the growth of Cftr-/- mice were delayed, with underweight reproductive organs and mild hypospermatogenesis. CFTR depletion destabilizes spermatogenesis by producing abnormal sperm and triggers activation of the Bax/Bcl-2 ratio in Cftr-/- and Cftr+/-mice, causing caspase-mediated irreversible intrinsic apoptosis. Stage-specific apoptosis in germ cells targeted the sexually mature mice, and the testis microenvironment affirmed that ion concentrations influence sperm capacitation. The blood pH determines apoptosis induction, as CFTR is a bicarbonate transporter. In conclusion, Cftr-/- mice were infertile because CFTR deficiency generated an ionic imbalance in the testis lumen, leading to Bax expression and Bcl-2 blockage, which triggered caspases or further activation of voltage-dependent anion-selective channel 1 (VDAC1). Cumulatively, cytochrome C was released due to altered mitochondrial membrane potential. Eventually, anomalous up-regulated apoptosis activation affected spermatogenesis, thus rendering the Cftr-/- male mice infertile. The results supplied new insights into CFTR modulation in reproduction: an imbalanced testicular microenvironment due to CFTR deficiency affects spermatogenesis and fertility in mice through the overactivation of spermatocyte caspase signalling, thus driving us to focus on updated treatments for CFTR deficiency-caused infertility.

The human luteinising hormone chorionic gonadotropin receptor (LHCGR) is a G-protein coupled receptor activated by both human chorionic gonadotropin (hCG) and luteinizing hormone (LH), two structurally related gonadotropins with essential roles in ovulation and maintenance of the corpus luteum. LHCGR expression predominates in ovarian tissues where it elicits functional responses through cyclic adenosine mononucleotide (cAMP), Ca2+ and extracellular signal-regulated kinase (ERK) signalling. LHCGR has also been localized to the human endometrium, with purported roles in decidualization and implantation. However, these observations are contentious. In this investigation, transcripts encoding LHCGR were undetectable in bulk RNA sequencing datasets from whole cycling endometrial tissue and cultured human endometrial stromal cells (EnSC). However, analysis of single-cell RNA sequencing data revealed cell-to-cell transcriptional heterogeneity and identified a small subpopulation of stromal cells with discernible LHCGR transcripts. In HEK-293 cells expressing recombinant LHCGR, both hCG and LH elicited robust cAMP, Ca2+ and ERK signals that were absent in wild type HEK-293 cells. However, none of these responses were recapitulated in primary EnSC cultures. In addition, proliferation, viability and decidual transformation of EnSC were refractory to both hCG and LH, irrespective of treatment to induce differentiation. Although we challenge the assertion that LHCGR is expressed at a functionally active level in the human endometrium, the discovery of a discrete subpopulation of EnSC that express LHCGR transcripts may plausibly account for the conflicting evidence in the literature.

Study questionCan single-nuclei and bulk RNA sequencing technologies be used to elucidate novel mechanisms of ovarian insufficiency in Turner Syndrome (TS)? Summary answerUsing single-nucleus and bulk RNA sequencing approaches, we identified novel potential pathogenic mechanisms underlying ovarian insufficiency in TS including and beyond X chromosome haploinsufficiency. What is known alreadyTurner syndrome (TS) is the most common genetic cause of Primary Ovarian Insufficiency (POI) in humans. Morphological analyses of human fetal 45,X ovaries have demonstrated fewer germ cells and marked apoptosis established by 15-20 weeks post conception (wpc); however, we do not understand why POI develops mechanistically in the first instance. Study design, size, durationSingle-nucleus RNA sequencing (snRNA-seq): two 46,XX and two 45,X (TS) human fetal ovaries at 12-13 wpc. Bulk RNA sequencing: 19 human fetal ovary, 20 fetal testis, and 8 fetal control tissue (n=47 total samples; Carnegie Stage 22-16wpc). Participants/materials, setting, methodsTo identify novel potential mechanisms of ovarian insufficiency in TS and to characterise X chromosome gene expression in the 45,X ovary, we performed snRNA-seq of peri-meiotic 46,XX (n=2) and 45,X (n=2) fetal ovaries at 12-13 weeks post conception (wpc); and 2) a bulk RNA sequencing time-series analysis of fetal ovary, testis, and control samples across four developmental timepoints. Main results and the role of chanceGerm and somatic cell subpopulations were mostly shared across 46,XX and 45,X ovaries, aside from a 46XX-specific/45,X-depleted cluster of oogonia ("synaptic oogonia") containing genes with functions relating to sex chromosome synapsis; histone modification; intracellular protein regulation and chaperone systems. snRNA-seq enabled accurate cell counting localised to individual cell clusters; the 45,X ovary has fewer germ cells than the 46,XX ovary in every germ cell subpopulation, confirmed by histopathological analysis. The normal sequence of X-chromosome inactivation and reactivation is disrupted in 45,X ovaries; XIST was not expressed in 45,X somatic cells but was present in germ cell clusters, albeit with lower expression than in corresponding 46,XX clusters. The 45,X ovary has a globally abnormal transcriptome, with low expression of genes with proteostasis functions (RSP4X); cell cycle progression (BUB1B); and OXPHOS mitochondrial energy production (COX6C, ATP11C). Genes with higher expression in 45,X cell populations were enriched for apoptotic functions (e.g., NR4A1). Limitations, reasons for cautionLimitations include the relatively small sample size of the snRNA-seq analysis and the focus on a fixed meiotic timepoint which may overlook a dynamic process over time. Wider implications of the findingsWe characterise the human fetal peri-meiotic 45,X ovary at single-cell resolution and offer insights into novel pathogenic mechanisms underlying ovarian insufficiency in TS. Although asynapsis due to X chromosome haploinsufficiency likely plays a significant role, these data suggest meiotic failure and ovarian insufficiency may be a combinatorial process characterised by periods of vulnerability throughout early 45,X germ cell development Study funding/competing interest(s)This research was funded in whole, or in part, by the Wellcome Trust Grants 216362/Z/19/Z to SMcG-B and 209328/Z/17/Z to JCA. Human fetal material was provided by the Joint MRC/Wellcome Trust (Grant MR/R006237/1) Human Developmental Biology Resource (http://www.hdbr.org). Research at UCL Great Ormond Street Institute of Child Health is supported by the National Institute for Health Research, Great Ormond Street Hospital Biomedical Research Centre (grant IS-BRC-1215-20012).

BackgroundThe mammalian mature spermatozoon has a unique chromatin structure in which the vast majority of histones are replaced by protamines during spermatogenesis and a small fraction of nucleosomes are retained at specific locations of the genome. The chromatin structure of sperm remains unresolved in most livestock species, including the pig. However, its resolution could provide further light into the identification of the genomic regions related to sperm biology and embryo development and it could also help identifying molecular markers for sperm quality and fertility traits. Here, for the first time in swine, we performed Micrococcal Nuclease coupled with high throughput sequencing on pig sperm and characterized the mono-nucleosomal (MN) and sub-nucleosomal (SN) chromatin fractions. ResultsWe identified 25,293 and 4,239 peaks in the mono-nucleosomal and sub-nucleosomal fractions, covering 0.3% and 0.02% of the porcine genome, respectively. A cross-species comparison of nucleosome-associated DNAs in sperm revealed positional conservation of the nucleosome retention between human and pig. Gene ontology analysis of the genes mapping nearby the mono-nucleosomal peaks and identification of putative transcription factor binding motifs within the mono-nucleosomal peaks showed enrichment for sperm function and embryo development related processes. We found motif enrichment for the transcription factor Znf263, which in humans was suggested to be a key regulator of the genes with paternal preferential expression during early embryo development. Moreover, we found enriched co-occupancy between the RNAs present in pig sperm and the RNA related to sperm quality, and the mono-nucleosomal peaks. We also found preferential co-location between GWAS hits for semen quality in swine and the mono-nucleosomal sites identified in this study. ConclusionsThese results suggest a clear relationship between nucleosome positioning in sperm and sperm and embryo development.

Oocytes from many invertebrate and vertebrate species exhibit unique endoplasmic reticulum specializations (cortical ER clusters) thought be essential for egg activation. In examination of cortical ER clusters, we observed they were tethered to previously unreported fenestrae within the cortical actin layer. Further, studies demonstrated sperm preferentially bind to plasma membrane overlying the fenestrae, establishing close proximity to underlying ER clusters. Moreover, following sperm-oocyte fusion, cortical ER clusters undergo a previously unrecognized global maturational change in volume, shape, and calreticulin content that persists through sperm incorporation, before dispersing at the pronuclear stage. These changes did not occur in oocytes from females mated with Izumo1 -/- males demonstrating that gamete fusion plays an important role in ER cluster maturation. In addition to these global changes seen at sites distant to the sperm, highly localized ER modifications were noted at the sperm binding site as cortical ER clusters surround the sperm head during incorporation, then form a diffuse cloud surrounding the decondensing sperm nucleus. This study provides the first evidence that cortical ER clusters interact with the fertilizing sperm, indirectly through a previous unknown lattice work of actin fenestrae, then directly during sperm incorporation. These observations raise the possibility that oocyte ER cluster-sperm interactions provide a competitive advantage to the oocyte, which may not occur during assisted reproductive technologies such as intracytoplasmic sperm injection. Summary StatementSperm-oocyte interactions stimulate global changes in cortical endoplasmic reticulum cluster structure as well as localized responses at the sperm binding site.

Heat shock proteins play essential roles as molecular chaperones, enacting protein folding and preventing of protein aggregation. In a previous study, a predicted damaging homozygous non-synonymous genetic variant was detected in the heat shock protein gene HSPA4L. Here, we used RNA interference in Drosophila melanogaster to explore the role of the heat shock protein A member 4 family (HSPA4) family in male fertility. Expression of the fly orthologue of the mammalian HSPA4 and HSPA4L genes, Hsc70Cb, was ablated in the male germline using two RNAi lines and the Nanos-Gal4 driver. Strong knockdown of Hsc70Cb in male germ cells resulted in male sterility, characterised by the absence of germ cells in testes and the over-proliferation of the testis soma. A less robust knockdown of Hsc70Cb in the male germline resulted in a sperm individualisation defect and a failure of sperm release into the seminal vesicle (analogous to the epididymis). When human HSPA4 or HSPA4L cDNA was introduced into infertile Hsc70Cb mutant flies, a partial rescue was observed, whereby in the robust Hsc70Cb knockdown setting germ cells progressed to the spermatocyte stage before undergoing cell death. Collectively, the absence of sperm in the Hsc70Cb (line 1) is consistent with the infertile man harbouring a homozygous HSPA4L genetic variant, supporting the hypothesis that HSPA4L is required for male fertility in humans and flies and highlighting the utility of the fly as a model of human spermatogenesis. In briefHsc70Cb is essential for spermatogonia survival and sperm individualisation in Drosophila. This study highlights the conserved roles of the HSPA4 family across animals and the utility of flies as a model organism for male fertility research.

Study questionCould follicular fluid-derived extracellular vesicles (ffEVs) benefit human oocyte rescue in vitro maturation (rIVM)? Summary answerSupplementation of rIVM culture with ffEVs isolated from mature follicles enhanced oocyte maturation rates by >20%, inducing changes in oocyte protein profile and organelle distribution. What is already knownIVM involves the culture of immature germinal vesicle (GV) oocytes under set laboratory conditions to allow for their transition to mature metaphase II (MII) stage, which is confirmed by the extrusion of the first polar body. Efficient IVM could circumvent aggressive controlled ovarian stimulation (COS), reduce the cost and broaden the repertoire of infertility treatments. Animal studies suggest that extracellular vesicles (EVs), membranous nanosized vesicles containing different molecular content (e.g. nucleic acids, proteins) and present in the ovarian follicular fluid could enhance oocyte maturation. The uptake of ffEVs by bovine, equine and feline oocytes, but not human, has been demonstrated. Study design, size, durationWomen undergoing transvaginal oocyte retrieval after COS (n=83) were recruited to donate follicular fluid (n=54 single follicles) and/or immature GV oocytes (n=95). We aimed to: a) define differences in the protein cargo of ffEVs derived from human follicles containing mature (MII-ffEVs, n=10) versus immature (GV-ffEVs, n=5; metaphase I MI-ffEVs, n=5) oocytes, b) demonstrate the capacity of human GV oocytes to uptake MII-ffEVs and c) determine the effect of MII-ffEVs supplementation on oocyte maturation. Participants/materials, setting, methodsffEVs were isolated by ultracentrifugation. The protein content of ffEVs was analysed by mass spectrometry. The uptake of fluorescently-labelled MII-ffEVs by GV oocytes (n=15) was assessed by confocal microscopy. GVs were cultured for rIVM in a timelapse incubator with MII-ffEVs (n=45 GVs) or without (n=40 GVs) and extrusion of polar body denoted maturation. The impact of MII-ffEVs supplementation on IVM-matured oocytes was assessed through single-cell proteomics and intracellular organelles appearance on transmission electron microscopy (TEM). Main results and the role of chanceWe identified 1340 proteins in ffEVs, with proteins such as F12, IGKV1-39, FREM2, and C1QC being significantly enriched in MII-ffEVs. GV oocytes internalised MII-ffEVs, and their supplementation for 48 hours increased the oocyte maturation rate compared to control by 22.8{+/-}9.4% (77.8% vs 55% maturation rate respectively; p-value=0.0372). Proteomic analysis of ffEV-supplemented mature oocytes (n=6) revealed 56 differentially abundant proteins (DAPs) compared to not supplemented mature oocytes (n=5). Among them, 37 DAPs were in higher abundance in ffEVs- supplemented mature oocytes including Hyaluronan Synthase 1 (HAS1) that is associated with oocyte maturation (6.55 fold increase). Electron microscopy showed differences in oocyte organelle distribution and appearance, particularly that of endoplasmic reticulum (RE) and RE-mitochondria complexes. Functional enrichment analysis of differentially abundant proteins during ffEV-oocyte interaction revealed regulation of endoplasmic reticulum, steroid biosynthesis, and keratin organisation pathways. Large scale dataN/A Limitations, reasons for cautionThis study utilised immature oocytes from COS cycles, therefore the results should be interpreted within the context of rIVM potential. Winder implications of the findingsThese results provide new insights into the role of ffEVs in enhancing oocyte maturation, offering potential improvements for clinical rIVM protocols and inspire the development of global IVM supplements based on ffEVs or associated specific cargo. Study funding/competing interest(s)This work was funded by an EMDO research fellowship and a FAN research grant (Fonds zur Forderung des akademischen Nachwuchses) from the University of Zurich. What does it mean for the patientsInfertility rates are rising, with 17% of couples worldwide needing help to get pregnant, often through treatments like in vitro fertilisation (IVF). IVF usually involves using hormones to stimulate the ovaries to produce multiple eggs, which can be tough on a womans health, both physically and emotionally, and can be very expensive. In vitro oocyte maturation (IVM) is a gentler alternative, where eggs are matured outside the body, reducing risks and costs. However, IVM isnt as effective as IVF yet, mainly because the current methods are not perfect. Our research is exploring a new approach to improve IVM by adding extracellular vesicles from follicular fluid to the egg culture. This could help the eggs mature better, leading to higher success rates and giving more options to couples struggling with infertility.

BACKGROUNDDuring spermiogenesis, histones are exchanged by protamines (PRMs) in spermatids, which results in DNA hypercondensation and protection. Rodents and primates express two PRMs (PRM1 and PRM2) in a species-specific ratio. Maintaining this ratio is necessary for functional chromatin reorganization and alteration is associated with sub- or infertility in mice and humans. Prm1 and Prm2 deficient mice are infertile, while Prm1+/- males are subfertile showing a severely altered PRM ratio. Prm2+/- males are fertile and display a protamine ratio comparable to WT. OBJECTIVESHere, we addressed the question whether loss of one allele of Prm1 and one allele of Prm2 affects fertility. MATERIAL AND METHODSDouble heterozygous (dHET) mice lacking one allele of Prm1 and one allele of Prm2 were generated and analyzed RESULTSdHET males were infertile with sperm showing retention of histones and TNPs, high levels of PRM2 precursor and decreased levels of mature PRM2. In mature sperm the PRM ratio and the total PRM content was not altered. However, CMA3 staining revealed incomplete protamination and sperm nuclei appeared more rounded and slightly bigger, suggesting impaired DNA-hypercondensation. In dHET sperm, DNA degradation was apparent, but to a lower level compared to sperm from Prm1 and Prm2 deficient males. Increased 8-OHdG levels suggested oxidative stress in the epididymis of dHET mice. However, a fraction of dHET sperm were capable of fertilization, with embryonic development up to 8-cell stage. DISCUSSION AND CONCLUSIONThese results suggest, that male factor infertility might not be reliably detected by measuring PRM1/PRM2 ratio but rather by determining the level of protamination by e.g. CMA3 analysis and pre-PRM2 retention.

Study questionWhat specific proteomic changes and molecular markers associated with in vitro aging occur in human oocytes? Summary answerSingle-cell proteomic analysis revealed the protein dynamics during the in vitro aging of oocytes. Subsequent functional analysis highlighted key biological processes affected in aged oocytes and identified several genes as candidate key factors for oocyte and early embryo quality. What is known alreadyOvulated oocytes undergo a time-dependent degradation process if fertilization does not occur within a specific window. This aging process leads to morphological, molecular, and epigenetic changes that can compromise oocyte quality and subsequent embryo development. In previous studies, transcriptome sequencing in humans and mice has revealed serial changes in aging oocytes including oxidative stress, mitochondrial dysfunction, DNA damage, and alterations in cell cycle regulation. These changes can lead to apoptosis, chromosomal abnormalities, and epigenetic modifications, all of which negatively affect oocyte quality. However, research regarding protein level changes and regulatory mechanisms in human oocytes during in vitro aging remains unclear. Study design, size, durationUtilizing "All-in-one" single-cell proteomics, we investigated the protein expression levels in human oocytes at the germinal vesicle (GV) stage and metaphase II (M II) stage following 24 hours of in-vitro aging. We analyzed four groups of oocytes: fresh GV (n=8), in vitro-aged GV (n=9), fresh M II (n=11), and in vitro-aged M II (n=11). This approach allowed for a detailed comparison of proteomic changes associated with in vitro aging in oocytes at different developmental stages. Participants/materials, setting, methodsOocytes at GV and MII stage were collected for "All-in-one" single-cell quantitative proteomic respectively. Differentially expressed genes were analyzed using the R package DESeq2 or DEP (filtered with q-value[≤]0.05 and Foldchange[≥]1.5). EGSEA package was used to perform pathway analysis. Main results and the role of chanceThe proteomic analysis of fresh and in vitro-aged human GV and MII oocytes identified 3,268 proteins. In GV oocytes, compared to fresh samples, 73 proteins were upregulated and 90 were downregulated. Gene Ontology (GO) enrichment analyses revealed these DEPs were involved in key biological processes such as lysosome organization, spindle assembly and oxidative stress response. Gene Set Enrichment Analysis (GSEA) revealed a significant down regulation of genes associated with protein translation initiation and methylation pathways, while those in the apoptosis mitochondrial changes were upregulated. In MII oocytes, 63 proteins were upregulated and 69 were downregulated. GO analysis highlighted their involvement in cytoplasmic translation, ribosome biogenesis and oocyte development. GSEA showed that aged MII oocytes exhibited a downregulation of gene sets in protein translation but an upregulation in membrane fusion pathways compared to fresh oocytes. Furthermore, we identified proteins with consistent expression trends across both GV and MII stages, including MRFAP1 and MT2A, as candidate biomarkers for human oocyte aging in vitro. Limitations, reasons for cautionSingle cell proteomic techniques may not fully capture the dynamic range of proteins present within oocytes, leading to potential underrepresentation of low-abundance proteins that could play crucial roles. Furthermore, the heterogeneity within oocytes introduces variability that can obscure the identification of consistent proteomic signatures associated with oocyte quality or aging. Wider implications of the findingsThese findings not only provide an understanding of the protein changes underlying the in vitro aging of human oocytes but also offer potential biomarkers and intervention targets for future research and clinical applications in assisted reproductive technologies. Study funding/Competing interest(s)This project received funding from the National Natural Science Foundation of China (NO.82471693, 22574175); the Natural Science Foundation of Hunan Province (NO. 2024JJ4100), the Hunan Provincial Grant for Innovative Province Construction (2019SK4012), the Hundred Youth Talents Program of Hunan Province (to S.Z.), the Major Scientific Program of CITIC Group (No. 2023ZXKYB34100) and Scientific Research Foundation of Reproductive and Genetic Hospital of CITIC-XIANGYA (YNXM-202313, YNXM-202319, YNXM-202211). The authors have no conflicts of interest to declare.

BackgroundEndometriosis is a common reproductive disease with a heterogeneous presentation. Classification attempts have thus far not offered insight into its cause or its symptoms. Endometriosis may result from the migration of shed endometrium to the peritoneal cavity. However, there are cases reported in girls without uteruses and men. While a non-retrograde menstruation origin of ectopic tissue is certain in these cases, we propose using DNA methylation age (DNAm age) to distinguish between retrograde and non-retrograde etiologies.\n\nMethodsUsing publicly available DNA methylation data and Horvaths pan-tissue epigenetic clock, we compared DNAm age and epigenetic age acceleration (EAA) of ectopic lesions to eutopic endometrium of diseased and control endometrium. We examined EAA in cancer metastasis and teratomas to control for migration and developmental origin.\n\nResultsDisease status does not change DNAm age of eutopic endometrium, but the effect of ectopic status was profound: -16.88 years (p = 4.82 x 10-7). There were no differences between EAA of primary/metastatic tumor pairs, suggesting that the observed effect is not due to migration. Immature or mature teratoma compartments decreased DNAm age by 9.44 and 7.40 years respectively, suggesting that developmental state correlates with DNAm age.\n\nConclusionsEctopic endometriotic tissue exhibits decelerated DNAm age, similar to that observed in teratomas composed of multipotent tissue. The migration process does not change DNAm age and eutopic endometrium is concordant with chronological age regardless of disease status. We conclude that DNAm age of ectopic lesions can classify endometriosis into distinct subtypes that may be clinically relevant.