Biology of Reproduction

The oviduct comprises 4 main regions: infundibulum (oocyte pick-up), ampulla (fertilization), isthmus (sperm capacitation and reservoir, preimplantation embryonic development), and uterotubal junction (UTJ; sperm and embryo transport). Mounting evidence in livestock and rodents suggest that gametes alter gene expression in secretory and ciliated epithelial cells of the oviduct. To elucidate whether adaptive interactions between the oviduct and gamete/embryo exist, we performed bulk RNA-sequencing on oviductal tissues collected from infundibulum+ampulla (IA) or isthmus+UTJ (IU) at various developmental stages (0.5, 1.5, 2.5-, and 3.5-days post coitus (dpc)) in mice. Samples were also collected during days 0.5, 1.5, 2.5, and 3.5 of pseudopregnancy (dpp). We found a strong region (IA vs. IU)-specific expression of large clusters of genes. The transition from 0.5 dpc to other pregnancy timepoints induces large sets of differentially expressed genes (DEGs) in pregnancy and pseudopregnancy in both IA and IU regions. Specifically, genes involved in pro-inflammatory responses were detected in both IU and IA regions. The presence of sperm at 0.5 dpc induces DEGs involved in pro-inflammatory responses in the IU region with an enrichment of biological processes for inflammatory cytokines, macrophage, and neutrophil recruitment. Additionally, DEGs are enriched in mitogen-activated protein kinase (MAPK) pathways along with genes in the Dusp family, Map3k8, Il1b, and Il1r2, among others. However, at 1.5 dpc we observed a strong shift to an anti-inflammatory condition in the IU region. These observations were absent in 0.5 and 1.5 dpp, suggesting that the DEGs observed for those inflammatory responses during pregnancy were likely induced by the presence of sperm. scRNA-seq analysis revealed that the inflammatory responsive genes were likely produced by secretory epithelial cells, compared to other cell types in the oviduct. In addition, multiple DEGs involved in pyruvate and glycolysis were enriched in the IU region, which could provide metabolic support for developing embryos. Lastly, we have also identified that there were cells that express immune markers in the oviduct, indicating that the oviduct is an immuno-dynamic tissue. In conclusion, our findings indicate that the oviduct is adaptive and responsive to the presence of sperm and embryos in a spatiotemporal manner. In this report, we intend to disseminate our findings on the transcriptional profiles during different stages of pregnancy. The complete study and validation at the protein level are currently underway and will be updated as soon as the data are available.

Structured AbstractO_ST_ABSPurposeC_ST_ABSTo determine if rescue in vitro maturation (IVM) of human oocytes can be improved by co-culture with ovarian support cells (OSCs) derived from human induced pluripotent stem cells (hiPSCs). MethodsFertility patients undergoing conventional ovarian stimulation for oocyte cryopreservation or IVF donated denuded immature germinal vesicle (GV) and metaphase I (MI) oocytes for research, which were allocated between either the control or intervention cultures. Fertility patients aged 25 to 45 years old donated immature oocytes under informed consent, with no additional inclusion criteria. The 24-28 hour OSC-IVM culture condition was composed of 100,000 OSCs in suspension culture with human chorionic gonadotropin (hCG), recombinant follicle stimulating hormone (rFSH), androstenedione and doxycycline supplementation. The Media-IVM control lacked OSCs and contained the same supplementation. Primary endpoints consisted of MII formation rate and morphological quality assessment. Additionally, metaphase spindle assembly location and oocyte transcriptomic profiles were assessed compared to in vivo matured MII oocyte controls. ResultsWe observed significant improvement in maturation outcome rates ([~]1.7X) for oocytes that underwent IVM with OSCs. Specifically, the OSC-IVM group yielded a maturation rate of 62% {+/-} 5.57% SEM versus 37% {+/-} 8.96% SEM in the Media-IVM (p=0.0138, unpaired t-test). Oocyte morphological quality between OSC-IVM and the Media-IVM control did not significantly differ. OSC-IVM resulted in MII oocytes with no instances of spindle absence and no significant difference in position compared to in vivo matured IVF-MII controls. OSC-IVM treated MII oocytes display a transcriptomic signature significantly more similar to IVF-MII controls than the Media-IVM control MII oocytes did. ConclusionThe novel OSC-IVM platform is an effective tool for rescue maturation of human oocytes obtained from conventional stimulation cycles, yielding oocytes with improved nuclear and cytoplasmic maturation. OSC-IVM shows broad utility for application in modern fertility treatment to improve the total number of available mature oocytes for fertility treatment.

The human ovary orchestrates sex hormone production and undergoes monthly structural changes to release mature oocytes. The outer lining of the ovary (cortex) has a key role in defining fertility in women as it harbors the ovarian reserve. It has been postulated that putative oogonial stem cells exist in the ovarian cortex and that these can be captured by DDX4 antibody isolation. We analysed on a single cell level the transcriptome and cell surface antigen profiles of over 24,000 cells from high quality ovarian cortex samples from 21 patients. Our single cell mapping reveals transcriptional profiles of six main cell types; oocytes, granulosa cells, immune cells, endothelial cells, perivascular cells, and stromal cells. Cells captured by DDX4 antibody are perivascular cells, not oogonial stem cells. Our data does not support the existence of germline stem cells in adult human ovaries thereby reinforcing the dogma of a limited ovarian reserve.

Ovarian tissue cryopreservation and subsequent autologous transplantation has allowed resumption of endocrine function as well as fertility in certain populations. However, graft function is short-lived due to ischemia and aberrant follicular activation post-transplantation. While many studies have focused on gene expression, we wanted to determine whether cryopreservation itself had a deleterious effect on regulatory elements that might influence transcriptional integrity and graft performance. In this study, we used Omni-ATAC to assess genome-wide chromatin accessibility in primary human follicles before and after cryopreservation. Omni-ATAC from fresh ovarian follicles identified active regulatory elements expected to be functional in oocytes and granulosa cells, and gene ontology was consistent with RNA translation/processing and DNA repair. While promoter accessibility was largely maintained in cryopreserved ovarian follicles, we observed a widespread increase in the number of accessible enhancers. Transcription factor motif analysis and gene ontology suggested that this dysregulation was focused around the epithelial-mesenchymal transition. Indeed, transcription factor binding was noted in major pathways involved in this transition: TGF-{beta} and Wnt signaling. Overall, our work provides the first genomic analysis of active regulatory elements in matched fresh and cryopreserved ovarian follicles as they undergo the process of ovarian tissue cryopreservation. Our data suggest that the process of cryopreservation activates an epithelial-mesenchymal transition state, which may lead to graft burn-out post-transplantation. Optimizing this technique in relation to this transition may therefore be an important step towards improving graft longevity and patient outcomes in fertility preservation. Summary sentenceCryopreservation of ovarian cortical tissue results in activation of differentiation and EMT pathways in follicles, which may explain graft burnout after autotransplantation.

Understanding the functions of maternal effect genes during oocyte growth is essential for elucidating the mechanisms of oogenesis and early embryonic development. However, conventional gene knockout and conditional knockout approaches require extensive breeding and are time-consuming. Here, we present a rapid in vitro gene functional analysis system that combines microinjection of mRNA, siRNA and plasmid DNA into mouse secondary follicles with a two-step oocyte growth culture system. Mouse secondary follicles were subjected to microinjection of mCherry mRNA and subsequently cultured for 15 days to produce fully grown oocytes. mCherry fluorescence persisted throughout the oocyte growth period but declined rapidly after fertilization. Despite minor cellular damage occasionally caused by microinjection, injected follicles developed normally and retained developmental competence. To evaluate the efficiency of gene suppression, we introduced siRNA targeting Dnmt3l, which is abundantly expressed during oocyte growth phase. Although Dnmt3l deficiency is known not to affect oocyte growth, we observed that oocyte growth was maintained normally despite a marked reduction in endogenous Dnmt3l mRNA levels in our knockdown model. These results demonstrate that this method enables efficient manipulation of gene expression specifically during oocyte growth while preserving developmental competence, providing a versatile platform for rapid functional screening of maternal effect genes in vitro.

The mammalian blastocyst undergoes two lineage segregations, i.e., formation of the trophectoderm and subsequently differentiation of the hypoblast (HB) from the inner cell mass, leaving the epiblast (EPI) the remaining pluripotent lineage. To clarify expression patterns of markers specific for these lineages in bovine embryos, we analyzed day 7, 9 and 12 blastocysts completely derived ex vivo by staining for OCT4, NANOG, SOX2 (EPI) and GATA6, SOX17 (HB) and identified genes specific for these developmental stages in a global transcriptomics approach. To study the role of OCT4, we generated OCT4-deficient (OCT4 KO) embryos via somatic cell nuclear transfer or in vitro fertilization. OCT4 KO embryos reached the expanded blastocyst stage by day 8 but lost of NANOG and SOX17 expression, while SOX2 and GATA6 were unaffected. Blastocysts transferred to recipient cows from day 6 to 9 expanded, but the OCT4 KO phenotype was not rescued by the uterine environment. Exposure of OCT4 KO embryos to exogenous FGF4 or chimeric complementation with OCT4 intact embryos did not restore NANOG or SOX17 in OCT4-deficient cells. Our data show, that OCT4 is required cell-autonomously for the maintenance of pluripotency of the EPI and differentiation of the HB in bovine embryos.

Paternal contributions to embryogenesis extend beyond DNA, yet the molecular cargo carried by sperm and its impact on development remain poorly defined. Aggresomes (AGG), cytoplasmic inclusions formed by misfolded proteins, are present in mammalian gametes, but their functional consequences are unclear. Here, we show that excessive AGG content in bovine sperm head compromises preimplantation embryo development. Using image-based flow cytometry, sperm from 32 sires were classified into low-, moderate-, and high-AGG groups. AGG levels were unrelated to sire age and did not affect in vitro capacitation or acrosome remodeling. However, embryos derived from high-AGG sires exhibited reduced blastocyst formation, delayed cleavage timing, and a higher incidence of developmental arrest at the 4-6 cell stage. Embryos from high-AGG sires also accumulated more AGG during development, showed elevated reactive oxygen species (ROS) levels, and displayed altered mitophagy dynamics. Supplementation with an ER stress inhibitor temporarily improved cleavage but did not enhance overall blastocyst formation, indicating a limited and stage-specific effect. In vivo, embryos from high-AGG sires showed lower transferable quality compared with those from low-AGG sires. These findings establish sperm head AGG content as a novel paternal determinant of embryo quality. By linking sperm-borne misfolded protein aggregates to disrupted developmental pathways in the resulting embryo, our study reveals a previously unrecognized mechanism of paternal influence on fertility and suggests new opportunities for molecular screening for male fertility. Significance StatementSperm contribute more to the embryo than DNA alone, yet the consequences of sperm-borne molecular cargo for early development remain largely unknown. We show that aggregates of misfolded proteins in the sperm head, a marker of disrupted protein quality control, impair preimplantation embryo development in cattle. Sires with elevated sperm aggregate content produce embryos that cleave later, arrest more frequently, and reach the blastocyst stage at lower rates, both in vitro and in vivo. These embryos carry greater aggregate loads, show heightened oxidative stress, and display dysregulated mitochondrial clearance. Our findings establish paternal proteostasis as a determinant of embryo quality and identify a class of sperm defects invisible to conventional semen analysis, opening new avenues for molecular fertility screening.

In vitro culture of cryopreserved ovarian follicles has the potential to extend fertility options for young women seeking to preserve ovarian tissue prior to undergoing cancer treatments. Successful implementation of this strategy has been elusive and likely requires a more complete understanding of the microenvironment of the developing ovarian follicles, including ovarian oxygen concentrations. The oxygen tension within the reproductive tract plays a crucial role in follicular development and oocyte maturation. While in vitro culture systems often use atmospheric oxygen (21%), the native environment in vivo is significantly lower, ranging from 1.5% to 8.7%. This study aimed to investigate the effects of reduced oxygen tensions (3% and 5%) on follicle survival, growth, antrum formation, and hormone production in cultured secondary follicles from rhesus macaques (macaca mulata). A total of 300 follicles were isolated from 7 animals and cultured under three oxygen conditions: 3%, 5%, and 21% O2. Follicle survival and antrum formation were assessed weekly by microscopy and Kaplan-Meier survival analysis, while growth dynamics and hormone levels (estradiol, progesterone, AMH, and inhibin B) were monitored throughout the culture period. Results demonstrated that follicles cultured at 3% and 5% oxygen exhibited significantly higher survival rates and antrum formation compared to those cultured at 21% O2. No significant differences in survival were observed between the 3% and 5% oxygen groups. Growth dynamics revealed distinct patterns, with both low oxygen groups promoting more robust and sustained follicle growth, while atmospheric oxygen led to rapid degeneration. Hormonal analysis showed that follicles in 21% O2 had elevated early hormone production but exhibited reduced long-term viability. In contrast, 3% and 5% oxygen delayed hormone production, reflecting a more stable and sustained follicular environment. These findings underscore the importance of low oxygen tensions in mimicking the physiological conditions of the reproductive tract, improving follicular development, and supporting optimal hormonal function in vitro. This study suggests that further reducing oxygen levels to 3% may offer additional advantages for long-term follicle viability and function in reproductive technologies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=71 SRC="FIGDIR/small/652505v1_ufig1.gif" ALT="Figure 1"> View larger version (17K): org.highwire.dtl.DTLVardef@11fcb62org.highwire.dtl.DTLVardef@61c0beorg.highwire.dtl.DTLVardef@1b5328forg.highwire.dtl.DTLVardef@10a7538_HPS_FORMAT_FIGEXP M_FIG C_FIG

In IVF clinics, preimplantation genetic testing (PGT) is a common practice that involves a biopsy and cryopreservation of embryos for genetic evaluation. When testing fails - or is required for already cryopreserved embryos - multiple freeze-thaw cycles occur. Though known to impact live birth rates, the exclusive influence of cryopreservation has not been elucidated. Here, we evaluate the effect of repeated cryopreservation on embryo health and implantation potential. Blastocyst-stage murine embryos were subjected to one, two or three freeze-thaw cycles with fresh embryos serving as a control. Outcomes assessed included post-thaw survival rate, allocation of cells to the inner cell mass (ICM) vs. trophectoderm cell lineages, implantation potential and offspring health. Post-thawing, embryos that were subjected to three freeze-thaw cycles had a significantly lower survival rates compared to embryos that had undergone one cycle (P<0.001). Additionally, the number of ICM cells was significantly reduced in embryos subjected to two or three freeze-thaw cycles compared to fresh or single-cycle embryos (P<0.001). No statistically significant differences were found for pregnancy rate, number of implantations, viable fetuses or resorption sites between treatment groups. We did however, find a non-significant yet interesting trend: three freeze-thaw cycles were associated with a 20% decrease in viable fetuses and a 20% increase in resorption sites compared to one freeze-thaw cycle group. These findings demonstrate that repeated cryopreservation adversely affects embryo health and may decrease implantation potential. Consequently, caution is advised regarding the repeated application of cryopreservation in IVF clinics, underscoring the need for further research to optimise cryopreservation protocols.

Somatic cell nuclear transfer (SCNT) remains inefficient, limiting its practical use in cattle reproduction and research. This study investigated two complementary strategies to enhance handmade cloning (HMC): (1) holding bovine oocytes overnight and delaying maturation to enable a second round of SCNT and (2) using bovine-induced pluripotent stem cells (biPSCs) as donor nuclei to enhance developmental competence. Bovine oocytes were subjected to either conventional in vitro maturation (CONV; 20 h) or delayed maturation using a holding medium for 20 h before CONV (HOLD). Matured oocytes were used for SCNT, parthenogenetic activation (PA), or in vitro fertilization (IVF) as controls. Handmade SCNT embryos were reconstructed using fibroblasts or biPSCs as donors, activated, and cultured for 7 days. Results showed no significant differences between CONV and HOLD groups in oocyte maturation, recovery after stripping, survival after zona removal, or cleavage and blastocyst development after SCNT. Fusion rates using fibroblasts were comparable between groups (42.6{+/-}6.0% vs. 50.3{+/-}9.8%), with biPSCs showing significantly higher fusion rates in CONV group (85.7{+/-}8.2% vs. 50.5{+/-}8.8%, P<0.05). Among fused embryos, biPSCs produced higher blastocyst rates (33.3{+/-}16.7%) compared with fibroblast donors (21.9{+/-}12.6%, P<0.05). Across all reconstructed embryos, cleavage and blastocyst development were also greater with biPSCs (odds ratios 3.4 and 2.7 respectively). These findings indicate that delaying maturation offers flexible timing for SCNT without compromising competence. Moreover, biPSCs enhance embryo developmental outcomes, supporting their use as superior donor cells for advancing cloning efficiency and applications in reproductive biotechnology.

Background and aimsAssisted reproductive technologies (ART), including in vitro fertilisation (IVF), account for over 10 million births worldwide. ART-conceived young offspring show altered cardiovascular phenotypes, including cardiac remodelling and raised blood pressure, but the mechanisms remain unclear. Mitochondrial disturbance during preimplantation development may link early ART exposure to later cardiac dysfunction. However, to our knowledge, no one has assessed mitochondrial function in adult offspring from IVF pregnancies. In this study, investigated the effects of IVF and embryo vitrification on blastocyst mitochondrial redox balance and metabolism, and determined whether these effects persisted into the adult heart. Methods and ResultsIGS-CD1 mouse blastocysts from naturally mated donors or IVF were transferred fresh or after vitrification-warming. IVF reduced blastocyst total, trophectoderm and inner cell mass cell number, while vitrification lowered the inner cell mass proportion and increased apoptosis. Both exposures depolarised mitochondrial membrane potential and depleted glutathione; reactive oxygen species rose with an interaction, being highest in vitrified IVF embryos. IVF reduced live birth rate and litter size. In the adult offspring, high-resolution respirometry of isolated mitochondria from left ventricle revealed reduced oxidative phosphorylation capacity with an increased H2O2 production, altered OXPHOS subunit abundance and reduced complex I, III and IV activities. ConclusionsIVF and vitrification impose distinct disturbance on preimplantation embryo redox states and bioenergetics, and this early disturbance is followed into adulthood with a reduced mitochondrial aerobic capacity and increased basal ROS production. These results have important implications for IVF practices and suggest that mitochondria may be permanently programmed by this procedure. Graphical Summary O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/744765v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@1cd1bd7org.highwire.dtl.DTLVardef@ded6b8org.highwire.dtl.DTLVardef@1e2ddf7org.highwire.dtl.DTLVardef@15abc84_HPS_FORMAT_FIGEXP M_FIG C_FIG IVF and vitrification impose distinct and partly independent effects on the preimplantation embryo that persist into the adult offspring heart. At the blastocyst stage, IVF reduced cell number and vitrification altered lineage allocation, while both exposures lowered mitochondrial membrane potential ({Delta}{Psi}m) and glutathione (GSH) and raised reactive oxygen species (ROS); vitrification additionally increased apoptosis. After embryo transfer, IVF reduced live birth rate and litter size, whereas vitrification altered postnatal growth trajectory. In adult offspring, ventricular mitochondria, vitrification reduced OXPHOS capacity and IVF reduced LEAK respiration, while both exposures increased H2O2/ O2 flux, reduced respiratory chain enzyme activities and altered OXPHOS subunit abundance.

Cannabis (marijuana) is the most widely used recreational drug in the USA accounting for about 62 million users in 2024. Among cannabis users, 26% are of prime reproductive age (18-25 years). Delta-9 tetrahydrocannabinol (THC) is the principal psychoactive component of cannabis and has been detected in human seminal fluids. Although abundant evidence indicates adverse effects of THC exposure on spermatogenesis in different species, acute effects of THC on postejaculatory sperm including fertilization potential and subsequent carryover effects on embryo development are largely unknown. The present study was designed to provide missing information on structural and mechanistic effects of THC exposure to postejaculatory sperm function by evaluating sperm indices often overlooked or masked during clinical evaluation. A bovine embryo continuum model was employed to determine effects of THC on sperm structure, kinematics, bioenergetics, and binding mechanisms. Effects of THC on the sperm genomic and epigenomic landscape were determined, complemented by paternal carry over effects on embryo development as a human translational model to elucidate paternal effects on future development, and to mirror sperm exposure during transport within the female reproductive tract. Cryopreserved bovine sperm from three bulls were independently exposed to physiologically relevant concentrations of THC (0 and 32nM, n = 2 individual replicates/bull) for 24 h under non-capacitating conditions at 25{degrees}C followed by quantification of sperm kinematics at 37{degrees}C. Samples of THC-exposed sperm and vehicle-control (0.1% DMSO) were collected in replicate following immediate addition of THC (0 h) and again at 24 h. DNA damage, acrosome integrity, bioenergetics, changes to DNA methylation and embryo development were quantified. Data were analyzed by logistic regression with a generalized linear mixed effect model. Computer-assisted sperm assessment revealed a reduction in progressive motility of THC-exposed sperm after 24 h while other parameters were not affected. Acrosome integrity as determined by flowcytometric analysis with FITC-PSA was severely compromised in THC-exposed sperm (P [&le;] 0.05), despite no detectable difference in capacitation status using merocyanine staining. Similarly, DNA integrity as determined by TUNEL assay was significantly impaired after 24 h of THC exposure (P [&le;] 0.05). Mechanistic effects of THC were explored through characterization of the transmembrane G-protein coupled cannabinoid 1 receptor (CB1). CB1 is expressed in the post-acrosomal region and its abundance decreased as compared to unexposed sperm. Alterations to the methylation landscape of sperm were then determined after 24 h of THC exposure through whole-genome Enzymatic Methyl Sequencing. PCA analysis indicated that sperm from different males formed distinct clusters, implying individual differences among bulls, while the effects of THC exposure produced tighter clusters. Paternal carryover effects on embryos derived by in vitro fertilization from THC exposed sperm had reduced 2-cell cleavage, 8-16 cell morula development, and reduced blastocyst development compared to unexposed sperm (46% vs. 33%). In conclusion, post-ejaculatory mammalian sperm exposure to THC compromises acrosome integrity, induces DNA damage, changes the sperm methylome, and reduces developmental potential. Collectively, these data implicate new considerations for recreational and clinical use of cannabis that impact cellular and molecular mechanisms important for sperm function with detrimental consequences for gamete interaction and embryo development.

The fat-tailed dunnart, Sminthopsis crassicaudata, is an emerging laboratory-based marsupial model for research on comparative biology, reproduction and conservation. In females, the reproductive cycle lasts 31 days and approximately 10 oocytes are ovulated per cycle. Assisted reproductive technologies (ART) play a crucial role in marsupial conservation, but developing protocols to harvest large numbers of oocytes remains a key challenge. Specifically, producing sufficient mature metaphase II (MII) oocytes in dunnarts continues to be difficult. Ovarian follicle stimulation is common practice to achieve superovulation in many species and typically requires treatment of prepubertal female animals to avoid the impacts of endogenous hormone cycling. Alternatively, adult females can be stimulated during the intermediate or follicular phase. In this study, we aimed to develop a high-yield induced-ovulation protocol to collect higher number of MII oocytes from adult, cycling, female dunnarts. We first synchronized the female dunnart reproductive cycles using luteinizing hormone-releasing hormone (LHRH). The reproductive cycles were monitored by examining cytology of vaginal lavage samples. After administering four LHRH injections given at three-day intervals, 88.9% (n=36) of the adult female dunnarts responded to the treatment, with their estrous cycles synchronized at the diestrous stage. We then induced ovarian follicle development through two PMSG injections over 6 days, followed by hCG administration to trigger ovulation. By combining estrous cycle synchronization with PMSG stimulation, we achieved 77.8% (n=36) ovulation success and obtained an average of 20.1{+/-}9.1 (n=28) MII oocytes per adult dunnart. These data demonstrated that estrous cycle synchronization followed by the PMSG-hCG treatment yields consistent, highly efficient induced-ovulation in adult dunnarts. This approach of combining estrus synchronization and follicle stimulation to produce sufficient MII oocytes for ART purpose could be applied to other valuable marsupial species to support conservation efforts. Summary SentenceSuperovulation and robust production of mature MII oocytes can be induced by 10-day estrus synchronization using LHRH followed by 6-day PMSG stimulation of follicle growth in adult fat-tailed dunnarts, a marsupial species. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=93 SRC="FIGDIR/small/671172v1_ufig1.gif" ALT="Figure 1"> View larger version (33K): org.highwire.dtl.DTLVardef@14c6685org.highwire.dtl.DTLVardef@decf75org.highwire.dtl.DTLVardef@64330org.highwire.dtl.DTLVardef@12538a4_HPS_FORMAT_FIGEXP M_FIG C_FIG

We created the c.1286C>G stop-gain mutation found in a family with primary ovarian insufficiency (POI) at age 30 years. The Eif4enif1 C57/Bl6 transgenic mouse model contained a floxed exon 10-19 cassette with a conditional knock-in cassette containing the c.1286C>G stop-gain mutation in exon 10. The hybrid offspring of CMV-Cre mice with Eif4enif1WT/flx mice were designated Eif4enif1WT/{Delta} for simplicity. A subset of female heterozygotes (Eif4enif1WT/{Delta}) had no litters. In those with litters, the final litter was earlier (5.4{+/-}2.6 vs. 10.5{+/-}0.7 months; p=0.02). Heterozygous breeding pair (Eif4enif1WT/{Delta} x Eif4enif1WT/{Delta}) litter size was 60% of WT litter size (3.9{+/-}2.0 vs. 6.5{+/-}3.0 pups/litter; p<0.001). The genotypes were 35% Eif4enif1WT/flx and 65% Eif4enif1WT/{Delta}, with no homozygotes. Homozygote embryos did not develop beyond the 4-8 cell stage. The number of follicles in ovaries from Eif4enif1WT/{Delta} mice was lower starting at the primordial (499{+/-}290 vs. 1445{+/-}381) and primary follicle stage (1069{+/-}346 vs. 1450{+/-}193) on day 10 (p<0.05). The preantral follicle number was lower starting on day 21 (213{+/-}86 vs. 522{+/-}227; p<0.01). Examination of ribosome protected mRNAs (RPR) demonstrated altered mRNA expression. The Eif4enif1 stop-gain mice replicate the POI phenotype in women. The unique mouse model provides a platform to study regulation of protein translation across oocyte and embryo development in mammals.

STUDY QUESTIONCan network modelling of single cell transcriptomic data identify cellular developmental trajectories of fallopian tube (FT) epithelium and reveal functional and pathological divergence from the endometrium? SUMMARY ANSWERA bidirectional secretory and ciliated differentiation trajectory was apparent from a novel OVGP1+ progenitor population of FT epithelial cells. A causal network model of whole transcriptome action in the FT and endometrium revealed specific functional divergence between secretory cells of these tissues. The network model reflected the latest ectopic pregnancy genome wide association study (GWAS), invoking MUC1 and other candidate genes in mature secretory cells for ectopic and eutopic implantation. WHAT IS KNOWN ALREADYThe fallopian tube forms the in vivo peri-conceptual environment, which has a significant impact on programming offspring health. The fallopian tube epithelium establishes this environment, however the epithelial cell types are poorly characterised in health and disease. STUDY DESIGN, SIZE, DURATIONPublicly available benign FT single cell RNA sequencing (scRNA-seq) samples from thirteen women across three studies were combined. Endometrial scRNA-seq samples from thirteen women from one study were used to demonstrate transcriptomic differences between the epithelia of the two tissues. Network models of transcriptomic action were constructed with hypergraphs. PARTICIPANTS/MATERIALS, SETTING, METHODSA meta-analysis of FT scRNA-seq samples was performed to identify epithelial populations. Differential gene expression assessed differences between fallopian tube and endometrial epithelial scRNA-seq data. Functional differences between secretory cells in the tissues were characterised using hypergraph models. To identify associations with ectopic pregnancy, expression quantitative trait loci (eQTLs) from a recent GWAS were mapped onto the network models. MAIN RESULTS AND THE ROLE OF CHANCEEpithelial cells (n=14,360) were clustered into 8 secretory and ciliated epithelial populations in the meta-analysis of 3 scRNA-seq datasets. A novel OVGP1+ epithelial progenitor cell was also identified, and its bi-directional differentiation to mature secretory or mature ciliated populations was mapped by RNA velocity analysis. This progenitor exhibited a high velocity magnitude (12.47) and low confidence (0.69), a combination strongly indicative of multipotent progenitor status. Comparing FT epithelial cells with endometrial epithelial cells revealed 5.3-fold fewer shared genes between FT and endometrial glandular secretory cells than between FT and endometrial ciliated cells, suggesting functional divergence of secretory cells along the reproductive tract. Hypergraphs were used to identify highly coordinated regions of the transcriptome robustly associated with functional gene networks. In the FT secretory cells, these networks were enriched for lipid (FDR<0.002) and immune (FDR<0.00007) related pathways. We mapped eQTLs from a GWAS meta-analysis of 7070 women with ectopic pregnancy over a range of significance (P = 1.68 x 10-21- 5.8 x 10-4) to the hypergraphs of FT and endometrium. Of the 22 genes present in the hypergraphs, 13 of these clustered as highly coordinated genes. This demonstrated the functional importance of MUC1 in the FT and endometrium, (GWAS Study P = 5.32x10-9) and identified additional genes (SLC7A2, CLDN1, GLS, PEX6, PLXNA4, NR2F1, CLGN, PGGHG, ANKRD36) implicated in ectopic pregnancy and eutopic pregnancy. LIMITATIONS, REASONS FOR CAUTIONThe sample size of reproductive age women was limited in previous studies, and though causal network modelling was used and previous mechanistic data supports candidate gene involvement, no in vitro or in vivo validation of candidate was performed. WIDER IMPLICATIONS OF THE FINDINGSThese findings consolidate the existing single cell transcriptomic datasets of the FT to provide a comprehensive understanding of epithelial populations and define functionally distinct secretory cells that contribute to the peri-conceptual environment of the FT. We further implicate the role of MUC1 and secretory cells in ectopic pregnancy and suggest future targets for investigating embryo implantation in the FT and endometrium.

Artificial insemination (AI) is commonplace in commercial pig breeding, and as such, ensuring sperm sample quality is of utmost importance to avoid reduced farrowing rates and litter sizes. Here, we have used high-throughput nuclear morphometric analysis to compare pig sperm samples categorised as meeting the AI standard (AIS) or not meeting the AI standard (N-AIS). We find that pig sperm nuclei are asymmetric, and that samples contain phenotypic shape abnormalities that show continuous variation. Samples classed as N-AIS have more abnormally shaped sperm than AIS samples, but conventional analysis misses many. The specific phenotypes identified suggest aspects of spermiogenesis that may be disrupted and indicate future avenues for improving pig sperm quality. This method reveals a significant difference in sperm head morphology between AIS and N-AIS pig sperm samples and has the potential to be further developed as a high-throughput tool for sperm head morphology assessment both in the pig breeding industry and in other species.

Nulliparous mares produce lighter and smaller foals compared to mares having previously foaled, with effects observed at least until 4 months of age. The need for a first gestation priming for the uterus to reach its full capacity has been proposed to explain this observation. Embryo developmental defects could be hypothesized but effects of maternal parity on the embryo have only been described once, in old mares, thus combining effects of parity and old age. The aim of this study was to determine effects of mare parity on embryo gene expression. Day-8 post ovulation blastocysts were collected from young (5/6 years old) nulliparous (YN, N=6) or multiparous (YM, N=4) non-nursing Saddlebred mares, inseminated with the semen of one stallion. Pure (TE_part) or inner-cell-mass-enriched (ICMandTE) trophoblast were obtained by embryo bisection for RNA sequencing (paired end, non-oriented, Illumina, NextSeq500). Deconvolution was performed on the ICMandTE dataset. Differential expression, with embryo sex and diameter as cofactors and gene set enrichment analysis (GO BP, KEGG, REACTOME databases) were performed using a false discovery rate <0.05 cutoff. Only a few genes were altered (ICM: n=18; TE: n=6) but several gene sets were perturbed (ICM: n=62; TE: n=50) by maternal parity. In YM, only pathways related to transcription, RNA processing and vesicle transport functions were enriched in the ICM whereas only pathways related to RNA localization were enriched in TE. In YN, while only gene sets related to ribosomes and extracellular matrix were enriched in the ICM, functions related to energy and lipid metabolism, lipid transport and interleukin-1 signaling were enriched in the TE. In conclusion, several genes and pathways are affected in embryos collected from nulliparous mares, with different effects on TE and ICM. Embryo development is altered in nulliparous mares, which could partially explain the term phenotype. Whether differences in gene expression result/induce poor embryo-maternal communication remains to be determined.

Despite numerous advances in in vitro fertilization (IVF) techniques since its first success in 1978, almost half of the patients treated remain childless. The multifactorial nature of IVF treatment means that success is dependent on variables, including the quality of oocytes. Therefore, new technologies are needed to objectively and quantitatively examine how each oocyte can be selected or optimized to achieve for the best possible outcomes for patients. Here, we report an optimized digital polymerase chain reaction (dPCR) for direct absolute quantification of nucleic acids within 3.5 h without the need for sample extraction or purification. Using individual oocytes, the developed method demonstrated absolute quantification with a linear dynamic range of 0.65 - 33 copies/{micro}L (r2=0.999), high accuracy and excellent reproducibility of <10% relative standard deviation. The method then identified the variable expression of Gapdh (0.72-16.95 copies/oocyte), Hprt1 (1.05-19.05 copies/oocyte) and ATPase 6, (5.55-32358.15 copies/oocyte) in ovaries even from the same mouse. Finally, dPCR was used to validate extracellular microRNAs from oocytes incubated with a toxic unsaturated very-long chained ceramide. This study therefore shows the feasibility of dPCR for the rapid and sensitive absolute quantification of DNA/RNA and extracellular miRNA for the study of oocytes.

Female age is the single greatest factor influencing reproductive performance. It is widely known that mitochondrial dysfunction plays a key role in reproductive senescence. Ovarian bioenergetics includes a sophisticated metabolic synergism between oocytes and human mural granulosa cells (GCs), which is crucial for oocyte maturation during follicular growth. These cells are believed to be potential biomarkers of oocyte quality. It has been proposed that alterations in their energy metabolism could lead to infertility. We investigated if there is an age-related effect on the energy metabolism of human mural granulosa cells. We performed an observational prospective cohort and experimental study including 127 women that underwent in vitro fertilization cycles allocated to two groups: a control group comprising oocyte donors aged less than 35 years and a group of infertile women aged over 38 years. The bioenergetics of cumulus cells and purified mural GCs were determined from oxidative phosphorylation parameters, aerobic glycolysis and adenine nucleotide levels. We have found that human mural GCs and cumulus cells present a high glycolytic profile and that the follicular fluid is critical to sustain their energy metabolism. GCs from older women present lower mitochondrial respiration and glycolysis than those from young donors which is not accompanied by a lower respiratory capacity. The diminished energy metabolism leads to a decrease in the total cellular energy charge. We conclude that, as women age, mural granulosa cells exhibit a reduction in their energy metabolism that is likely to influence female reproductive potential.

Periods of elevated ambient temperature challenge the bodys ability to maintain internal homeostasis, and heat stress poses particular risks during pregnancy. Epidemiological studies associate gestational heat exposure with higher rates of congenital anomalies such as hypospadias, yet the direct link between gestational heat exposure and reproductive anomalies remains unknown. In this study, we examined the effects of intermittent heat exposure on reproductive development in male mouse offspring. Pregnant dams either remained at constant temperature of 22{degrees}C (control) or were exposed to 38{degrees}C for 2 hours daily (experimental) from embryonic day (E)10 to E18, modeling intermittent heat exposure during mid-to-late gestation. Embryos were collected at E18 for analysis. While heat exposure did not affect pregnancy outcomes, including placental development, litter size, sex ratio, or fetal growth, male embryos exhibited significantly reduced anogenital distance and increased hypospadias scores, which are both markers of disrupted androgen signaling. Despite these phenotypic changes, expression of genes involved in androgen synthesis in the fetal testis, as well as gene expression in external genitalia, remained unchanged. Instead, transcriptomic analysis revealed significant alterations in testicular pathways related to RNA splicing and mRNA processing. Together, these findings reveal that maternal heat stress disrupts reproductive development of male offspring, with altered gene regulatory processes being a potential driver.