Reproduction

Non-steroidal anti-inflammatory drugs (NSAIDs), which inhibit the prostaglandin synthase (PTGS) enzymes PTGS1 and PTGS2, may present a risk when consumed during early pregnancy. The impact of PTGS enzyme inhibition by NSAIDS on the three-dimensional organization and function of uterine compartments in context of implantation success is not well known. Here we show that pre-implantation treatment of mouse pregnancy with indomethacin, an NSAID that inhibits both PTGS1 and PTGS2, results in embryo crowding, delayed embryo implantation, defective implantation chamber formation that eventually results in significant pregnancy loss. These effects are dose dependent as lower doses of indomethacin do not show these effects on pregnancy. When embryo spacing defects were observed, a significant proportion of embryos that showed delayed growth or resorption were located in distinct decidual sites, suggesting that additional mechanisms beyond spatial distribution are at play. These findings highlight the potential risks associated with NSAID use during early pregnancy and underscore the need for further research into the underlying molecular mechanisms and the development of safer pain management strategies during pregnancy.

Uterine luminal fluid (ULF) composition plays a major role in cell-to-cell communication between the receptive endometrium and an invading blastocyst. ULF is made up of secretions from the uterine glands and uterine epithelial cells (UEC). However, the cellular mechanisms regulating these exocytotic secretions are not yet understood. This study investigated the role of extracellular vesicles (EVs) during early pregnancy using Transmission Electron Microscopy (TEM). TEM analysis at time of fertilisation (TOF) Day 1 and at time of receptivity (TOR) Day 5.5 revealed EVs present, with an abundance at TOR. Exocytosis signalling in UECs, by SNARE proteins syntaxin 2 (syn2) and SNAP23, was also examined. Immunofluorescence microscopy showed both syn2 and SNAP23 to be present in the apical area of UECs at TOR. Western blot and immunofluorescence quantification revealed a significant increase in syn2 and SNAP23 at TOR compared to TOF. SNAP23 colocalization with apical actin showed SNAP23 was in the luminal space contributing to ULF. Overall, this data shows EVs, syn2 and SNAP23 (potential receptivity marker) are present in ULF and may together create a favourable microenvironment for blastocyst implantation. Summary statementDuring uterine receptivity SNAREs participate in the secretion of ULF. EVs and SNAP23 are present in the uterine luminal space during uterine receptivity. SNAP23 has the potential to be used as a receptivity marker.

Study questionDoes the oestrogen receptor isoform, ER46, contribute to regulation of endometrial function?\n\nSummary answerER46 is expressed in endometrial tissues during the proliferative and secretory phases and is the predominant ER isoform in first trimester decidua. ER46 is abundantly expressed in uterine NK (uNK) cells and localised to the cell membrane. Activation of ER46 regulates the function of human uNK cells by increasing cell motility.\n\nWhat is known alreadyOestrogens acting via their cognate receptors are essential regulators of endometrial function and play key roles in establishment of pregnancy. ER46 is a 46kDa truncated isoform of full length ER (ER66, encoded by ESR1) that contains both ligand and DNA binding domains. Expression of ER46 in human endometrium has not been investigated previously. ER46 is located at the cell membrane of peripheral blood leukocytes and mediates rapid responses to oestrogens. UNK cells are a phenotypically distinct (CD56brightCD16-) population of tissue-resident immune cells that regulate vascular remodelling within the endometrium and decidua. We have shown that oestrogens stimulate rapid increases in uNK cell motility. Previous characterisation of uNK cells suggests they are ER66-negative but expression of ER46 has not been characterised. We hypothesise that uNK cells express ER46 and that rapid responses to oestrogens are mediated via this receptor.\n\nStudy design, size, durationThis laboratory-based study used primary human endometrial (n=24) and decidual tissue biopsies (n=30) as well as uNK cells which were freshly isolated from first trimester human decidua (n=18).\n\nParticipants/materials, setting, methodsPrimary human endometrial and first trimester decidual tissue biopsies were collected using methods approved by the local institutional ethics committee (LREC/05/51104/12 and LREC/10/51402/59). The expression of oestrogen receptors (ER66, ER46 and ER{beta}) was assessed by qPCR, western blot and immunohistochemistry. Uterine Natural Killer (uNK) cells were isolated from first trimester human decidua by magnetic bead sorting. Cell motility of uNK cells was measured by live cell imaging: cells were treated with oestradiol (E2)-BSA (10nM equivalent), the ER{beta}-selective agonist 2,3-bis (4-hydroxyphenyl)-propionitrile (DPN; 10nM) or vehicle control (DMSO).\n\nMain results and the role of chanceER46 was detected in proliferative and secretory phase tissues and was the predominant ER isoform in first trimester decidua samples. Immunohistochemistry revealed ER46 was co-localised with ER66 in cell nuclei during the proliferative phase but detected in both the cytoplasm and cell membrane of stromal cells in the secretory phase and in decidua. Triple immunofluorescence staining of decidua tissues identified expression of ER46 in the cell membrane of CD56-positive uNK cells which were otherwise ER66-negative. Profiling of isolated uNK cells confirmed expression ER46 and localised ER46 protein to the cell membrane. Functional analysis of isolated uNK cells using live cell imaging demonstrated that activation of ER46 with E2-BSA significantly increased uNK cell motility.\n\nLimitations, reasons for cautionExpression patterns in endometrial tissue was only determined using samples from proliferative and secretory phases. Assessment of first trimester decidua samples was from a range of gestational ages which may have precluded insights into gestation specific changes in these tissues. Our results are based on in vitro responses of primary human cells and we cannot be certain that similar mechanisms occur in situ.\n\nWider implications of the findingsE2 is an essential regulator of reproductive competence. This study provides the first evidence for expression of ER46 in human endometrium and decidua of early pregnancy. We describe a mechanism for regulating the function of human uNK cells via expression of ER46 and demonstrate that selective targeting with E2-BSA regulates uNK cell motility. These novel findings identify a role for ER46 in human endometrium and provide unique insight into the importance of membrane-initiated signalling in modulating the impact of E2 on uNK cell function in women.\n\nStudy funding/competing interest(s)These studies were supported by MRC Programme Grants G1100356/1 and MR/N024524/1 to PTKS. HODC was supported by MRC grant G1002033.

Ovarian stimulation (OS), utilized for the development of multiple ovarian follicles for IVF, induces supraphysiologic levels of E2 and an early rise in P4 that disrupt endometrial differentiation and decreases implantation rates or result in placental insufficiency and pregnancy complications. To improve pregnancy rates and reduce the risk of pregnancy complications associated with IVF, it is crucial to advance our molecular understanding of the molecular regulation of endometrial differentiation. Previous studies from our laboratory suggest G protein-coupled receptors (GPCRs) are important regulators of endometrial differentiation. To investigate this further, using a retrospective dataset, we identified all GPCRs expressed across the proliferative and secretory phase of the menstrual cycle and found that many members of the adhesion G protein-coupled receptor (ADGR) family are dynamically expressed. For each ADGR subfamily exhibiting differentially-expressed genes across the cycle, their expression was investigated by RT-PCR in the non-pregnant mouse uterus and decidua on E7.5 of pregnancy. For those genes expressed in the E7.5 decidua, their expression was further quantified by qPCR across early mouse pregnancy. The RT-PCR screen revealed expression of 13 ADGRs (4 of the 9 subfamilies) in E7.5 decidua and among these genes, many were differentially expressed between E0.5 and E5.5 or 6.5 and between E5.5 and E6.5. The dynamic expression of the ADGRs across the menstrual cycle and in early mouse pregnancy, suggests these ADGRs are E2- and/or P4-regulated genes. We therefore hypothesized that for these ADGR genes, mRNA expression would be disrupted in an OS cycle. This hypothesis was tested on endometrial biopsies collected in the secretory phase from prospective cohorts of women in natural and OS cycles. Consistent with the retrospective dataset, our data revealed that members of the ADGR gene family are expressed in the secretory phase of the natural menstrual cycle and for the first time, we show that their expression is altered by ovarian stimulation.

Endometrial glands are essential for fertility, consisting of ciliated and secretory cells that facilitate a suitable uterine environment for embryo implantation. This study sought to determine whether an endometrial gland specific transcriptome and splicing profile are altered in women with recurrent pregnancy loss. Our data provide a comprehensive catalogue of cilia and PAEP gene isoforms and relative exon usage in endometrial glands. We report a previously unannotated endometrial gland cilia transcript GALNT11 and its susceptibility to exon skipping. Key endometrial receptivity gene transcripts are also reported to change in endometrial glands of women with recurrent pregnancy loss. The endometrial gland cilia and PAEP targets identified in this study could be used to identify a perturbed endometrium, isolate causes of recurrent pregnancy loss and develop targeted therapies in personalised medicine.

The purpose of this study was to determine whether ovarian germline stem cells or any adult mitotic cell type contribute to the adult oocyte pool in vivo from early adult through to aging mice. Long-term thymidine analogue labelling assays using iododeoxyuridine (IdU) were performed to identify mitotic cells and their descendants by immunohistochemistry for IdU and germline markers Ddx4 and Oct4. C57BL/6 mice at 10 weeks, 5 months and 12 months old were given IdU for 21-30 days through their drinking water to label all dividing cells, followed by washout periods of 0-10 weeks. Over 60,000 ovarian somatic cells and oocytes were scored in 111 ovaries. No double labelling of IdU with germline stem cell markers was found in oocytes at any stage of maturity, in mice at any age. In contrast, IdU exposure during embryogenesis resulted in large numbers of labelled oocytes in postnatal mice, as expected. We were therefore unable to confirm mitotic activity in stem cells or any other progenitor replenishing the oocyte pool in C57BL/6 mice. There is no evidence that quiescent stem cells are activated when the oocyte pool is depleted by age.

Zona pellucida (ZP) plays a vital role in reproductive processes including oogenesis, fertilization and preimplantation development of embryo. The ZP of humans is composed of four glycoproteins (ZP1-ZP4), same as rats ZP. Our previous research reported a first case of human infertility due to ZP1 mutation, but the mechanism was unclear. Here we developed a genome editing in vivo rat model and a co-transfected in vitro cell model to investigate the pathogenic effect. In rat homozygous for the homologous mutation, ZP were absent in all of collected eggs. Further the growing and fully grown oocytes in the mutant ovaries completely lack a ZP but with detectable intracellular ZP1 protein. After mating with male rats, none of the mutant female rats got pregnant. Moreover, the co-transfected cell experiments and the ovarian experiments showed that the truncated ZP1 sequestered intracellularly ZP3 and ZP4 to impede their release outside, resulting in an intracellular accumulation of ZP1, ZP3 and ZP4, leading to absence of ZP in mutant oocytes. Our results clearly establish the causal role of ZP1 mutation on ZP defects and female infertility.\n\nSummary statementRat model mirrored completely the phenotypes observed in humans, infertility and abnormal eggs that lack a zona pellucida, through the negative effects of ZP1 mutation.

Sex development is an intricate and crucial process in all vertebrates that ensures the continued propagation of genetic diversity within a species, and ultimately their survival. Perturbations in this process can manifest as variations/differences of sex development (VSD/DSD). Primary gonadal somatic cells - the ovarian granulosa cells (GCs) in the case of women - represent the absolute model to investigate mechanism of disease in VSDs. Collection of these cells in humans is laborious and invasive, while classical animal models fail to recapitulate the human phenotype and function. Furthermore, in patients with the most severe forms of VSD gonadal cells are totally absent. It is therefore vital to develop an alternative cell-model. In view of this, we established an efficient method to reprogram donor-derived urinary progenitor cells (UPs) and differentiate iPSCs into granulosa-like cells (GLCs). The UPs presented a less invasive and high-quality cell source, improving the clinical applicability of the model along with utilising a non-integrative reprogramming method that eliminates alteration of the original genome. This novel GLC model closely resembles human GCs in morphology and marker gene expression of GC cell-fate and essential function. These results provide the prospect to generate patient-specific personalised GC models to investigate mechanism of disease in VSDs and could improve understanding of the intricacies in female gonadal development.

Recurrent implantation failure (RIF) is defined after three or more good quality embryo transfers following in vitro fertilisation without a successful pregnancy outcome. Many factors contribute to RIF however, the endometrial contribution remains unclear. Previous work by our group has identified a micro-RNA (miRNA) miR-185-5p as conserved across placental mammal irrespective of implantation strategies. We tested the hypothesis that miR-185-5p and the pathways it regulates, may be disrupted in the endometria of women with RIF. A human endometrial epithelial cells line (Ishikawa cells) was transfected with mimics or inhibitors for miR-185-5p for 24 (for implantation assay) or 48 hr (for proteomic analysis) along with non-targeting controls. There was a significant different in percentage attachment of BeWoW spheroids to cells transfected with miR-185-5p mimic compared to inhibitor (P<0.05). Transfection of epithelial cells with miR-185-5p altered expression of 1450 (mimic alone) and 509 (inhibitor alone) proteins respective of which, 146 were modified by both. Comparison of predicted targets of miR-185-5p, proteins modified by this study, and key endometrial genes from the literature determined genes and proteins associated with CNP family were further investigated in biopsies from individuals with (n=10) and without RIF (n=9) with CSNK1D expression significantly lower (p<0.05) in individuals with RIF. Collectively these data demonstrate that miR-185-5p modifies pathways that are important for successful implantation in humans.

Decidualisation is the hormone-dependent process of endometrial remodelling that is essential for fertility and reproductive health. It is characterised by dynamic changes in the endometrial stromal compartment including differentiation of fibroblasts, immune cell trafficking and vascular remodelling. Deficits in decidualisation are implicated in disorders of pregnancy such as implantation failure, intra-uterine growth restriction, and pre-eclampsia. Androgens are key regulators of decidualisation that promote optimal differentiation of stromal fibroblasts and activation of downstream signalling pathways required for endometrial remodelling. We have shown that androgen biosynthesis, via 5-reductase-dependent production of dihydrotestosterone, is required for optimal decidualisation of human stromal fibroblasts in vitro, but whether this is required for decidualisation in vivo has not been tested. In the current study we used steroid 5-reductase type 1 (SRD5A1) deficient mice (Srd5a1-/- mice) and a validated model of induced decidualisation to investigate the role of SRD5A1 and intracrine androgen signalling in endometrial decidualisation. We measured decidualisation response (weight/proportion), transcriptomic changes, and morphological and functional parameters of vascular development. These investigations revealed a striking effect of 5-reductase deficiency on the decidualisation response. Furthermore, vessel permeability and transcriptional regulation of angiogenesis signalling pathways, particularly those that involved vascular endothelial growth factor (VEGF), were disrupted in the absence of 5-reductase. In Srd5a1-/- mice, injection of dihydrotestosterone co-incident with decidualisation restored decidualisation responses, vessel permeability, and expression of angiogenesis genes to wild type levels. Androgen availability declines with age which may contribute to age-related risk of pregnancy disorders. These findings show that intracrine androgen signalling is required for optimal decidualisation in vivo and confirm a major role for androgens in the development of the vasculature during decidualisation through regulation of the VEGF pathway. These findings highlight new opportunities for improving age-related deficits in fertility and pregnancy health by targeting androgen-dependent signalling in the endometrium.

Meiosis is the hallmark of reproduction. Our understanding of early oocyte development was improved by studying the spatiotemporal dynamics and mechanisms governing meiosis in mice, however, our knowledge of the meiotic initiation process in humans remains limited. Here, we utilized three-dimensional (3D) analysis to determine spatiotemporal dynamics of meiotic initiation in fetal human ovaries. Similar to mice, we found that the first meiotic cells appear in clusters in the center of human fetal ovaries as early at 9 weeks and that the initiation of meiosis propagates as a radial wave. Between developing germ cells in fetal human ovaries, we detected a component of the intercellular bridge, TEX14 protein. 3D quantification of germ cells in ovaries collected at the end of first trimester revealed, for the first time, considerable variation in the number of meiotic cells between individuals and asynchronous mitotic-meiotic transition. In addition to illustrating the geography of meiotic initiation in ovaries from the first trimester, we extended our 3D analysis approach to second trimester and showed heterogeneous spatial distribution of meiotic and non-meiotic germ cells in human fetal ovaries. This study is an important step towards better understanding of 3D structure of developing ovary and early stages of meiosis in humans. Highlights- Organic solvent-based clearing methods and confocal microscopy can be implemented to visualize and quantify germ cells in the intact human fetal ovary. - Identification of a new, radial, pattern of meiotic initiation in the ovaries from the first trimester. - Immunolocalization of the intercellular bridge component TEX14 between developing germ cells in the fetal ovary - Maintenance of pre-meiotic germ cells in second trimester ovaries suggests less synchronous mitotic-meiotic stage transition.

Spermatozoa progressive motility is positively correlated with fertilization competence. Bulls ejaculates with progressive motility lower than 50% are routinely rejected through the process of straw preparation, designated for artificial insemination of dairy cows. We examined the quality and fertility competence of ejaculates with relative low progressive motility (55-60%, n = 5; control) with those of very low progressive motility i.e. below the lower threshold, (20-45%, n = 5; rejected). Analysis revealed a lower volume for the control vs. rejected samples. Dip-Quick staining revealed a higher proportion of spermatozoa with abnormal morphology in the rejected group, in particular those with detached heads. Activation of spermatozoa with calcium ionophore, resulted by a lower proportion of activated spermatozoa in the rejected group. In addition, a higher proportion of spermatozoa with DNA damage were recorded in the rejected vs. the control samples. Following in-vitro fertilization, the proportion of oocytes that developed to the 2- and 4-cell stage embryos did not differ between groups. However, the proportion of embryos that further developed to blastocysts, was higher in the control group. Transcript abundance of selected genes in the blastocysts and the apoptotic index did not differ between groups, suggesting that the forming blastocysts were of the same quality. It is suggested that in specific cases, for example genetically superior bulls, ejaculates with very low progressive motility can be used for in vitro production of embryo. Further in vivo examinations, i.e. artificial insemination or transferring of embryos derived from these inferior ejaculates, might clarified this point.

Defective endometrial stromal cell decidualization is a major cause of recurrent implantation failure (RIF), a condition with a prevalence of [~]15%. To treat RIF, a stronger understanding of the endometrial factors that regulate decidualization is required. Here we studied the role of the kisspeptin receptor (KISS1R) in regulating human endometrial stromal cell (HESC) decidualization. Our data revealed KISS1R inhibits HESC decidualization in vitro in a manner associated with a striking reduction in ESR1 protein levels. To determine whether KISSR inhibition of decidualization results from reduced ESR1 levels we expressed the dominant negative ESR1-46 isoform in decidualizing HESCs. We found that expression of ESR1-46 in decidualizing HESCs ablated the expression of ESR1-66 and ESR1-54 isomers, and blocked decidualization. Interestingly, when ESR1-64 was co-expressed with ESR1-46, ESR1-66 and ESR1-54 expression was restored and decidualization was rescued. Taken together, these results suggest that KISS1R inhibits HESC decidualization by downregulating ESR1 levels. Based on our findings, we suggest that by inhibiting HESC decidualization, KISS1R regulates the depth of embryo invasion of the stroma, a requirement for a successful pregnancy.

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.

Oestrogen and progesterone play essential roles in the release of mature oocytes, the priming and cycling of the uterine lining, and the maintenance of mammalian pregnancy. Progesterone is synthesized de novo at the embryo implantation site in the mouse, during decidualization of the endometrium. During early stages of pregnancy, the locally produced progesterone is thought to act as an immunosuppressant, preventing rejection of the fetal allograft at the fetal-maternal interface. However, both uterine natural killer cells and dendritic cells express glucocorticoid receptor rather than progesterone receptor. The importance of glucocorticoids in early pregnancy is inferred from the presence of steroid receptors and the 11{beta}-hydroxysteroid dehydrogenase enzymes, which modulate corticosterone action in the decidua, the trophoblast, the placenta, and the fetus. 11{beta}-hydroxylase is the last enzyme in the metabolism of cholesterol to corticosterone and, in a mouse model of 11{beta}-hydroxylase deficiency, complications of reproduction suggested its requirement for normal ovulation and uterine cell turnover. We present evidence that, in this model, folliculogenesis occurs normally but ovulation is inhibited, and abnormal uterine cell turnover ultimately leads to adenomyosis. Ovaries respond to a superovulation protocol by releasing oocytes and forming corpora lutea, and homozygous null blastocysts are capable of implantation, but the pregnancy is not maintained. We show that glucocorticoid is produced locally at the implantation site in control animals, revealing wide involvement of glucocorticoids in reproductive biology.

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.

BackgroundPremature ovarian insufficiency (POI) affects approximately 1% of women under 40 and is characterized by elevated levels of gonadotropins, reduced estradiol, impaired folliculogenesis, and infertility. Bone marrow-derived mesenchymal stem cell (BM-MSC)-based therapy has emerged as a promising regenerative strategy in preclinical POI models. This systematic review and meta-analysis evaluated BM-MSC-based interventions, including cell transplantation and secretome/extracellular vesicle administration, in animal models of POI. MethodsA systematic review and meta-analysis was conducted following PRISMA guidelines. PubMed, Web of Science, Scopus, ScienceDirect, and the Cochrane Library were searched from inception to February 19, 2025. Preclinical studies assessing BM-MSC-based interventions in animal models of POI were included. ResultsThirty-four studies comprising 1,357 animals were included. Compared with controls, BM-MSC-based therapy increased serum estradiol (standardized mean difference [SMD] 3.11; 95% confidence interval [CI] 2.38-3.84) and anti-Mullerian hormone (SMD 1.86; 95% CI 1.03-2.69), while reducing follicle-stimulating hormone (SMD -3.54; 95% CI -4.37 to -2.71) and luteinizing hormone (SMD -3.44; 95% CI -5.17 to -1.70). Follicular counts increased across developmental stages, with fewer atretic follicles. Reproductive outcomes improved, including normal estrous cycles (risk ratio [RR] 7.80; 95% CI 3.15-19.34), pregnancy occurrence (RR 3.72; 95% CI 2.14-6.44), and offspring number (SMD 1.57; 95% CI 1.04-2.09). ConclusionBM-MSC-based therapy consistently improved hormonal, follicular, and reproductive outcomes in preclinical POI models. More well-designed, standardized, and adequately controlled studies to confirm these findings are warranted. Systematic review registration: CRD42023449053

This study investigates the short and long-term effects of IFNT and PAG on the transcriptome of endometrium and blood leukocytes. Holstein heifers received intrauterine infusions of one of the following treatments: 20 mL of a 200 g/mL bovine serum albumin solution (BSA; vehicle) from day 14 to 16 of the estrous cycle (BSA), vehicle + 10 g/mL of IFNT from day 14 to 16 (IFNT3), vehicle + 10 g/mL of IFNT from day 14 to 19 (IFNT6), and vehicle + 10 g/mL of IFNT from day 14 to 16 followed by vehicle + 10 g/mL of IFNT + 5 g/mL of PAG from day 17 to 19 (IFNT+PAG). RNA-seq analysis was performed in endometrial biopsies and blood leukocytes collected after treatments. Acute IFNT signaling in the endometrium (IFNT3 vs BSA), induced differentially expressed genes (DEG) associated with interferon activation, immune response, inflammation, cell death, and inhibited vesicle transport and extracellular matrix remodeling. Prolonged IFNT signaling (IFNT6 vs IFNT3) altered gene expression related to cell invasion, retinoic acid signaling, and embryo implantation. In contrast, PAG induced numerous DEG in blood leukocytes but only 4 DEG in the endometrium. In blood leukocytes, PAG stimulated genes involved in development and TGFB signaling while inhibiting interferon signaling and cell migration. Overall, IFNT is a primary regulator of endometrial gene expression, while PAG predominantly affected the transcriptome of circulating immune cells during early pregnancy. Further research is essential to fully grasp the roles of identified DEG in both the endometrium and blood leukocytes. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=70 SRC="FIGDIR/small/591148v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@b51110org.highwire.dtl.DTLVardef@173433dorg.highwire.dtl.DTLVardef@4368e5org.highwire.dtl.DTLVardef@19bd118_HPS_FORMAT_FIGEXP M_FIG C_FIG Main findingsO_LIAcute IFNT signaling in the endometrium activates genes involved in inflammation while inhibiting vesicle transport in cells and ECM remodeling. C_LIO_LIProlonged IFNT signaling in the endometrium regulates genes involved in cell invasion and retinoic acid signaling. C_LIO_LIPAG alter gene expression in blood leukocytes more than in endometrium and may stimulate leukocyte differentiation while inhibiting leukocyte extravasation. C_LI

Proper action of the female sex steroids, 17{beta}-estradiol (E2) and progesterone (P4) on endometrium is essential for fertility. Beyond its role in regulating the cell cycle, cyclin A2 (CCNA2) also mediates E2 and P4 signaling in vitro, but a potential role in modulating steroid action for proper endometrial tissue development and function is unknown. To fill this gap in our knowledge, we examined human endometrial tissue from fertile and infertile women for CCNA2 expression and correlated this with pregnancy outcome. Functional assessment of CCNA2 was validated in vivo using a conditional Ccna2 uterine deficient mouse model while in vitro function was assessed using human cell culture models. We found that CCNA2 expression was significantly reduced in endometrial tissue, specifically the stromal cells, from women undergoing in vitro fertilization who failed to achieve pregnancy. Conditional deletion of Ccna2 from moue uterine tissue recapitulated the inability to achieve successful pregnancy which appears to be due to alterations in the process of decidualization, which was confirmed using in vitro models. From these studies, we conclude that CCNA2 expression during the proliferative/regenerative stage of the menstrual cycle acts as a safeguard allowing for proper steroid responsiveness, decidualization and pregnancy. When CCNA2 expression levels are insufficient there is impaired endometrial responsiveness, aberrant decidualization and loss of pregnancy. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=100 SRC="FIGDIR/small/545284v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@150b471org.highwire.dtl.DTLVardef@176a804org.highwire.dtl.DTLVardef@110a47dorg.highwire.dtl.DTLVardef@19f186c_HPS_FORMAT_FIGEXP M_FIG C_FIG

BackgroundPronuclear assessment appears to have the ability to distinguish good and bad embryos in the zygote stage, but paradoxical results were obtained in clinical studies. This situation might be caused by the robust qualitative detection of the development of dynamic pronuclei. Here, we aim to establish a quantitative pronuclear measurement method by applying expert experience deep learning from large annotated datasets. MethodsConvinced handle-annotated 2PN images (13419) were used for deep learning then corresponded errors were recorded through handle check for subsequent parameters adjusting. We used 790 embryos with 52479 PN images from 155 patients for analysis the area of pronuclei and the pre-implantation genetic test results. Establishment of the exponential fitting equation and the key coefficient {beta} 1was extracted from the model for quantitative analysis for pronuclear(PN) annotation and automatic recognition. FindingsBased on the female original PN coefficient {beta}1, the chromosome-normal rate in the blastocyst with biggest PN area is much higher than that of the blastocyst with smallest PN area (58.06% vs. 45.16%, OR=1.68 [1.07-2.64]; P=0.031). After adjusting coefficient {beta}1 by the first three frames which high variance of outlier PN areas was removed, coefficient {beta}1 at 12 hours and at 14 hours post-insemination, similar but stronger evidence was obtained. All these discrepancies resulted from the female propositus in the PGT-SR subgroup and smaller chromosomal errors. Conclusion(s)The results suggest that detailed analysis of the images of embryos could improve our understanding of developmental biology. FundingNone