Biology of Reproduction

Conventional semen cryopreservation involves equilibration at 4{degrees}C and optimum freezing rates. We hypothesized that a cholesterol-based semen extender obviates the need for equilibration, minimizing total processing time for semen cryopreservation. Experiments were conducted to determine the effects of semen extender (egg yolk- or cholesterol-based) and freezing method (routine or fast) on post-thaw sperm characteristics and fertility of beef and bison semen. In Experiment 1, beef semen diluted in tris-egg yolk-glycerol (TEYG) or cholesterol-cyclodextrin tris-glycerol (CCTG) extender underwent routine or fast freezing method. Cholesterol from animal and plant origins were compared. The routine method included 90-min equilibration at 4{degrees}C and routine freezing (RE-RF, total time 97 min) whereas the fast method included no equilibration and fast freezing (NE-FF, total time 14 min). Post-thaw sperm quality was assessed by CASA, and in vitro fertilization. Post-thaw sperm motility was not affected by the origin of cholesterol (animal or plant), but was lowest in the TEYG NE-FF group (24% vs 43-51%, P < 0.05). In vitro cleavage and blastocyst development rates did not differ between RE-RF and NE-FF groups. In Experiment 2, bison semen was diluted in TEYG or plant-CCTG extender and frozen as in Experiment 1. Post-thaw sperm motility was lowest in the TEYG NE-FF group (10% vs 39-51%, P < 0.05). In Experiment 3, beef semen diluted in TEYG or plant-CCTG extender underwent either a routine (RE-RF) or modified freezing (NE-RF, total time 25 min) method. Post-thaw sperm characteristics did not differ between extenders but were greater using routine freezing (RE-RF) compared to the modified method of freezing (NE-RF). Pregnancy rates were similar between extenders (TEYG vs plant-CCTG) using the modified freezing method without equilibration and insemination at 72 h after progesterone device removal. In conclusion, beef and bison semen diluted in cholesterol-based extender may be cryopreserved without equilibration.

Plac1 is an X-linked gene essential for placental and embryonic development. A knockout (KO) mouse model was used to identify Plac1-regulated gene expression at E16.5 and E18.5 using gene expression microarray. Genes exhibiting at least 1.5-fold change in expression and FDR < .05 were considered significant. At E16.5, 717 genes were downregulated and 798 were upregulated in male KO placentas versus wild type (WT), whereas at E18.5, 1122 genes were downregulated and 1149 were upregulated. GO, KEGG, and IPA analyses revealed downregulated genes were enriched for Rho GTPase-mediated and actin-cytoskeleton based processes that transmit extracellular cues through canonical signaling pathways, including Integrin, GPCR, Wnt, Notch, VEGF, BMP and TGF-beta, documented to impact trophoblast development, vasculogenesis, vascular tone, branching morphogenesis, and immunomodulation. Furthermore, a preeclampsia-associated transcriptomic signature was induced that strengthened over time. By contrast, upregulated genes reflected immune activation and adaptations to oxidative stress resulting from impaired placental function. These findings indicate that Plac1 supports signaling required to maintain placental structure and regulatory function. Its absence disrupts essential regulatory processes and triggers cellular stress and immune activation, contributing to fetal growth restriction, increased risk for embryopathy and preeclampsia, consistent with the Developmental Origins of Health and Disease (DOHaD) framework.

Vitamin D signaling has recognized roles in female reproductive physiology, but its effects at the chromatin level in endometrial stromal cells are still unclear. Here, we investigated how the active form of vitamin D, 1,25-dihydroxyvitamin D3, or calcitriol, influences the accessible chromatin landscape of human endometrial stromal cells. Assay for transposase-accessible chromatin using sequencing (ATAC-seq) was performed on T-HESCs treated with either a vehicle or 1,25(OH)2D3. Ligand treatment increased overall chromatin accessibility, shown by higher ATAC-seq signal intensity, while causing only minor changes in the total number of called peaks. Peak annotation revealed that accessible regions were spread across both promoter-proximal and distal genomic areas. Integrating this data with CUT&RUN and RNA sequencing showed that most vitamin D-responsive cistromic modifications and transcripts were linked to nearby open chromatin, though fewer were associated with regions that were significantly differentially accessible. These results suggest that 1,25(OH)2D3-dependent transcription mainly occurs within a permissive, pre-accessible chromatin environment. This study offers new evidence that active vitamin D influences the epigenomic landscape of human endometrial stromal cells, establishing the chromatin-based molecular response to a chemically-defined VDR ligand, 1,25(OH)2D3, relevant to stromal differentiation and preparation for decidualization. HighlightsO_LIFirst evidence suggesting the direct impact of active vitamin D, 1,25-dihydroxyvitamin D3, 1,25(OH)2D3, enhanced the signal intensity of chromatin accessibility in human endometrial stromal cells C_LIO_LIMost accessible chromatin regions were shared between vehicle and ligand-treated human endometrial stromal cells C_LIO_LI1,25(OH)2D3-responsive transcription occurs largely within pre-accessible chromatin in human endometrial stromal cells C_LIO_LIAssay for transposase-accessible chromatin sequencing (ATAC-seq) defines a chromatin-level pharmacologic response to a chemically defined VDR ligand in human endometrial stromal cells C_LI

Study questionDoes the human placenta utilize the creatine phosphagen system for energy homeostasis during development? Summary answerComponents of the creatine (Cr)-creatine kinase (CK)-phosphocreatine (PCr) system are dynamically expressed by the trophoblast and mesenchymal compartments throughout gestation wherein creatine kinase is required for cellular ATP metabolism, cell cycle, and proliferation of trophoblast cells. What is known alreadyThe Cr-CK-PCr system maintains ATP homeostasis in tissues with high energy demand and is required for proliferation, migration, and invasion of tumor cells. The term human placenta can synthesize and transport creatine locally. Early placental development involves trophoblast proliferation, an event requiring ATP, but the role of the creatine phosphagen system during early placental development remains unknown. Study design, size, durationWe performed immunohistochemistry (IHC) and immunofluorescence (IF) for different components (biosynthesis, transport, utilization) of the Cr-Ck-PCr system in human placentae (n=3/group) across gestation including first trimester, second trimester, and term. Using primary human trophoblast stem cells (hTSCs) and trophoblast organoids (TO), we determined the role of the creatine phosphagen system in trophoblast growth by functional inhibition of creatine kinase. Participants/materials, setting, methodsIHC/IF were performed in human placentae across gestation for proteins involved in biosynthesis (AGAT and GAMT), transport (SLC6A8, SLC22A15, and SLC6A13) and utilization (CKB and CKMT1) of creatine to determine the presence of the creatine phosphagen system locally in the placenta. For delineating the functional importance of this system in placental development, cyclocreatine (cCr), a creatine analogue, was used for functional inhibition of CK. Primary hTSCs were culture in medium containing 0 (control), 1, 10, 20 mM cCr for 48 hours followed by analysis of cell growth (cell count), cell cycle (EdU incorporation assay), apoptosis (Annexin V/PI flow cytometry), energy metabolism (Sea horse mito-stress and glycolytic stress tests), and gene expression (qPCR). Primary TO were also treated with 20mM cCr for 6 days in vitro to determine the role of Cr-CK-PCr system in placental development. Main results and the role of chanceAGAT localized to the fetal villous mesenchyme, while GAMT was broadly expressed in the trophoblast and fetal mesenchyme compartments across gestation. CKB localized primarily to fetal mesenchyme with strongest expression at term. CKMT1 was broadly expressed in all trophoblast subtypes. SLC6A8 was abundant in early syncytiotrophoblast but absent at term, where its expression shifted to fetal blood vessels. SLC22A15 was expressed in the endothelial cells of fetal capillaries across gestation. In primary hTSCs, cyclocreatine (20mM) treatment reduced proliferation (P<0.001), decreased expression of trophoblast epithelial marker EGFR (P<0.05), induced G0/G1 and G2/M arrests (P<0.0001), enhanced early and late apoptosis (P<0.0001), and downregulated GPX8 expression (P<0.05). Seahorse analysis revealed marked reductions (P<0.01) in mitochondrial (basal, maximal, and ATP-linked) and glycolytic (rate, capacity, and reserve) function compared to controls. In primary human TO, cyclocreatine treatment reduced the growth of organoids (P<0.05) as well the expression of EGFR (P<0.05). Large scale dataN/A Limitations, reasons for cautionFurther experiments assessing apoptosis, cellular stress and redox imbalance may provide more mechanistic role of the creatine phosphagen system in trophoblast metabolism and function. Since the functional role of the Cr-CK-PCr system was investigated in vitro, findings of this study should be taken with caution for implications of in vivo placental development. Nevertheless, reproducible results of reduced growth of trophoblast cells using both 2D and 3D cultures is highly suggestive of the importance of the creatine phosphagen system in early placental development. Wider implications of the findingsThis study provides foundational knowledge that the placenta contains the creatine phosphagen system, known for ATP homeostasis, and that this system ensures proper cell division, survival and placental development. Dysregulation of components of Cr-CK-PCr system in placenta has been observed in pregnancy disorders such as preeclampsia and fetal growth restriction warranting continued investigation into mechanisms and potential remediation using creatine supplementation. Stem cells share similar metabolic features so findings of this study can be implicated in other stem cells models as well. Study funding/competing interest(s)This work was supported by CIRM EDUC4-12804 Interdisciplinary Stem Cell Training Grant and a Lalor Foundation Postdoctoral Fellowship awarded to NS, and by the California Institute for Regenerative Medicine (DISC0-13757) and the National Institute of Child Health and Human Development (R01-HD096260) award to FS. The authors have no competing interest to declare.

IntroductionHealthy and diseased placentae alike often display some degree of pathology. However, quantitative techniques to characterize common pathologies and their relationship to local maternal hemodynamics in healthy primate placentae are currently limited. MethodsPlacentae from seven rhesus macaques were imaged by MRI at three time points across mid-to late-gestation, to quantify placental blood volume, flow, and perfusion from maternal spiral arteries across pregnancy. Near term, we collected placental cotyledons, digitized hematoxylin/eosin-stained slides, then segmented and annotated sub-tissues and major pathologies (intervillous gaps, fibrin deposition, villous agglutination, inflammatory agglutination, and stromal mineralization) within each cotyledon. Individual pathologies were assessed in relation to each other and MRI perfusion metrics, in a cotyledon-specific manner. Parallel analyses were performed to investigate both basic (Spearman correlation) and animal variance-negated (dimensionality-reduction) relationships. ResultsCotyledons with increased stromal mineralization demonstrated low blood perfusion across pregnancy, alongside significant compensatory changes. Mineralization was further associated with decreased fetal weight, across all sub-tissues. Dimensionality reduction revealed maternal vascular malperfusion-associated pathologies as the largest contributor to dataset variance. Additionally, pathologies commonly associated with healthy placental function demonstrated low cotyledon blood flow and volume at all timepoints, with no evidence of compensatory changes across gestation. ConclusionsComprehensive digital annotation revealed several relationships connecting pathology and maternal blood perfusion in the healthy primate pregnancy, at the smallest functional unit of the placenta. This methodological framework embeds pathologist-refined morphological expertise into a quantitative, spatially resolved format that can ground, rather than be replaced by, unsupervised computational approaches to placental analysis.

In the terminal segment of the seminiferous tubules, SOX17 expression in the rete testis (RT) epithelium plays a crucial role in the formation of the Sertoli valve (SV), as revealed by phenotypic analyses of RT-specific Sox17 conditional knockout (cKO) mouse testes. In these RT-specific Sox17 cKO testes, SV disruption leads to the backflow of RT fluid into the seminiferous tubules, resulting in defective spermiogenesis and male infertility. Although valve deformation in the Sox17 cKO testes is likely caused indirectly by impaired downstream actions of Sox17 in the RT, the mechanisms by which SOX17 in RT influences SV formation in the seminiferous tubules remain unclear. To address this, we generated a novel AMH-Sox17 transgenic (Tg) mouse line carrying a human AMH promoter-driven Sox17 cDNA cassette. We analyzed the phenotypes of the Sertoli valve and spermatogenesis in AMH-Sox17 Tg mice, as well as in RT-specific Sox17 cKO; AMH-Sox17 Tg double mutant mice. Ectopic SOX17 (SOX17+) expression in Sertoli cells resulted in excessive Sertoli valve structures with acetylated tubulin bundles in the terminal segment of the AMH-Sox17 Tg testes, along with enhanced WNT4/RSPO1 signaling, suggesting the enhanced valve formation of ectopic SOX17+ Sertoli cells by themselves. Moreover, the AMH-Sox17 Tg could partially rescue the SV deformation and infertility in RT-specific Sox17 cKO mice, leading to proper SV formation, normal spermiogenesis and a partial recovery of male fertility in AMH-Sox17 Tg; RT-specific Sox17 cKO double mutant mice. These findings genetically demonstrate that ectopic SOX17+ Sertoli cells can compensate for SOX17 paracrine signaling in the RT, underscoring a key shared downstream pathway between RT and SV. Summary statementThe paracrine actions downstream of ectopic SOX17 expression in the Sertoli cells not only promote the valve formation, but also partially rescue the defective spermiogenesis of the rete testis-specific Sox17-null mice.

Current evaluation of male fertility is largely based on indirect sperm parameters such as viability, concentration, morphology, and motility; however, each of these parameters, alone or combined, has been shown to have limited predictive value for successful fertilization. To address this problem, we introduce hSPICER (human SPerm-Induced CEll-cell fusion Requiring JUNO), an assay that evaluates sperm function based on their ability to induce fusion of somatic cells expressing human JUNO (hJUNO), the egg-specific sperm receptor. Similarly to our previous discovery in mice, we found that human sperm can fuse with somatic cells expressing hJUNO on their surface (pseudo-eggs) and promote content mixing between cells in culture, as measured using a split GFP system. The assay is sensitive, specific, and species-dependent, requiring hJUNO for optimal signal. We generated a stable cell line expressing hJUNO, enhancing reproducibility and sensitivity. We also show that hSPICER is compatible with cryopreserved sperm and consistent over different days. Importantly, hSPICER values correlate with fertilization outcomes of patients during fertility treatments, indicating its potential as a functional diagnostic tool. Beyond diagnostic uses, hSPICER establishes a platform to explore sperm fusion mechanisms and to screen for therapeutic compounds and interventions to treat low fertility, enhance fertilization, and develop non-hormonal contraceptives for males and females, as well as quality assessment of semen samples in fertility clinics and sperm banks.

In eutherian mammals, blastocyst implantation is often associated with a quasi-inflammatory reaction in the endometrium, which is resolved with the establishment of the definitive placenta. This is understandable in the case of invasive placentation, since implantation entails a nidatory injury to the maternal tissue due to the invading blastocyst. Quasi-inflammatory processes have also been documented in pregnant pigs, even though the blastocyst only attaches to, rather than invades into, the endometrium of the uterus. In this study, we asked what processes in early porcine pregnancy lead to the resolution of attachment-associated inflammation. In generic wound healing the transition from a pro- to an anti-inflammatory state is caused by a corresponding transition from M1 to M2 polarized macrophages following efferocytosis by macrophages of apoptotic neutrophils. In order to determine whether this scenario applies to the pregnancy-related resolution of inflammation in the porcine uterus, we produced a series of bulk transcriptome samples spanning days (D) 13 to 25 of gestation. This time span corresponds to the transition from pre- to post-attachment stages of pregnancy. We found slower changes in the transcriptome between D20 and D25 than prior to D20, suggesting a turning point in pregnancy-related reprogramming. The turning point at D20 corresponds to the time of firm attachment of trophectoderm to uterine luminal epithelium and the cessation of IFNG signaling from the blastocyst. This transition coincides with increased expression of RNAs of genes implicated in resolution of inflammation and M2 polarization such as ARG1, MRC1/CD206, CD86, TGFb1 and IL10, as well as a significant increase in expression of HGPD, the enzyme that metabolizes prostaglandins. While immunoreactivity for ARG1 was found in putative macrophages in the sub-epithelial stratum compactum, other markers of M2 polarized macrophages were localized to non-immune cells: MRC1 was found on fibroblast-like stromal cells, CD86 on trophoblast cells, and IL10 in luminal and glandular epithelia. These results suggest that intrauterine immune regulation is decoupled from that of the rest of the body by engaging non-immune cell types as anti-inflammatory mediators during the peri-attachment period of pregnancy.

Pluripotent stem cells derived from livestock species represent valuable systems for studying early mammalian development and for establishing renewable, well-defined cell sources; however, direct comparative characterization of distinct pluripotent stem cell platforms in sheep remains limited. In this study, we established and evaluated two ovine pluripotent stem cell types: reprogrammed induced pluripotent stem cells (siPSCs) and embryonic disc-derived stem cells (sEDSCs). Both siPSCs and sEDSCs exhibited core features of pluripotency, including compact colony morphology, alkaline phosphatase activity, expression of key pluripotency-associated markers, and maintenance of a normal ovine karyotype. Flow cytometry and quantitative RT-PCR analyses revealed broadly overlapping yet distinguishable pluripotency marker expression profiles between the two cell types. Functional pluripotency was confirmed by embryoid body formation and in vitro differentiation into derivatives of all three germ layers. To further assess lineage-specific differentiation competence and compare functional outputs relevant to mesodermal differentiation, both pluripotent stem cell types were directed towards the adipogenic lineage. While siPSCs and sEDSCs were each capable of adipogenic differentiation, differences in differentiation efficiency and marker expression were observed. Together, these findings demonstrate that ovine siPSCs and sEDSCs share core pluripotency characteristics while retaining distinct molecular and functional properties, providing a robust comparative framework for studies of ovine pluripotency, lineage specification, and stem cell biology.

Background The use of Assisted Reproductive Technology (ART) is increasing worldwide. These treatments involve ovarian stimulation to enable multiple follicle recruitment, hence inducing supraphysiological estrogen levels. While most long-term follow-up of women undergoing ART has concerned cancer incidence, the long-term safety regarding cardiovascular and metabolic diseases remains under-explored. This study was performed to assess the risk of acute myocardial infarction, cerebral ischemic conditions, intracranial hemorrhage, type 2 diabetes mellitus, heart failure, aortic aneurysm or dissection, and chronic kidney disease in women that conceived with ART, and to investigate the role of the underlying infertility and its risk factors. Methods and Findings Swedish national registers allowed us to follow a nationwide cohort of 380,756 women from their first birth between 1992 and 2002 until the end of 2023. The safety of ART was evaluated by comparing women with infertility who conceived with and without ART, while adjusting for baseline differences in age, body mass index, country of origin, socioeconomic factors, pre-existing comorbidity, smoking and year. The role of infertility was additionally explored by comparing all women with and without infertility adjusting for age, as well as the aforementioned baseline characteristics. Cumulative risks were plotted using inverse-probability weighted Kaplan-Meier curves. To facilitate the comparison of groups we also estimated risk differences and ratios at 10-, 20-, and 30-years of follow-up. Use of ART was not associated with cardiovascular disease except for an excess risk of cerebral ischemic conditions, with a 30 year risk ratio of 1.43 (1.09; 1.89). With the exception of cerebral ischemic conditions, intracranial hemorrhage, aortic dissection, and chronic kidney disease, women with a history of infertility exhibited consistently higher risk of all outcomes, adjustment for differences in baseline characteristics explained some but not all of these elevated risks. Conclusions With the exception of ischemic cerebral conditions, the findings provide reassurance regarding the long-term cardiometabolic safety of ART use, while adding to the growing literature suggesting that infertility can act as a marker of womens cardiovascular and metabolic disease.

Early pregnancy requires a tightly regulated pro-inflammatory environment shared between the primitive placenta and decidua. While immune balance supports successful implantation and placental invasion, disruptions in immune signaling during this period can impair implantation and lead to embryo loss. In this study, we investigated the molecular mechanisms underlying immune imbalance during implantation using a trophoblast stem cell (TSC) model. TSCs were cultured in either stem cell or syncytiotrophoblast (STB) differentiation medium and treated with either lipopolysaccharides (LPS) or interferon beta (IFNB). RT-qPCR and Western blotting revealed that LPS failed to induce a pro-inflammatory cytokine response in TSCs or STBs. In contrast, IFNB triggered a strong antiviral response in both TSCs and STBs. RNA-sequencing of IFNB-treated TSC and STB 3D spheroids revealed subtle differences between the TSCs and STB responses to interferons. Both TSC and STB IFNB-treated spheroids mount an interferon-mediated antiviral response; however, STB spheroid genes associated with the type I interferon response, viral RNA/DNA sensing, and antigen processing were upregulated. We also compared the interferon response between the CT27 (female) and CT29 (male) TSCs and STBs. While STBs showed minimal differences, the CT29 TSCs exhibited a markedly stronger interferon response than the CT27 TSCs. Collectively, these findings suggest that the primitive placenta is selectively responsive to interferon signaling rather than direct pathogen-associated stimuli. This implies that maternal immune activation, rather than microbial invasion, likely drives that placental immune response and embryo success at this stage. Understanding these dynamics underscores the importance of the maternal immune balance in early pregnancy success.

Background: Literature on the role of thermal discomfort (heat- and cold-stress) on in-vitro fertilization (IVF) outcomes are scarce and inconclusive. This multi-center research examines association between heat stress and IVF treatment outcomes in Andhra Pradesh, which is prone to year around chronic heat stress. Methods: IVF data were abstracted from clinical chart review of all patients from three IVF from centers 2019 to 2023, which included time-stamped data on each IVF procedure, demographics and pre-existing comorbidities. Weather data were acquired from the National Climatic Data Center (NCDC). IVF outcomes were modelled with respect to time-lagged exposure to ambient temperature stratified by hyper- and hypo-thermic conditions using Poisson and logistic regressions depending on the scale of IVF outcomes adjusting for confounders. Results: Heat stress peaked in June, which corresponded with elevated number of spontaneous abortions/miscarriage (SAM). Under hypo- and hyper-thermic conditions a unit increase ambient temperature was associated with an 11% higher and an 8% lower number of oocytes retrieved, respectively. Adjusting for confounders, a 10 degree F increase in two-day lag heat stress was associated with a 30% higher odds of SAM (odds ratio ~ 1.03; 95% CI = 1.001 to 1.068; p-value < 0.043), and odds of PTB were 3 times higher when three day-lagged heat index (HI) was greater than 35 degree C (odds ratio 1.13 to 7.99; p < 0.05). Conclusion. Our findings warrant strategies to engage IVF patients in mitigating their exposure to thermal discomfort before and during the treatment.

During early development of the placenta, a subset of murine trophectoderm stem cells (TSCs) undergo endoreplication, an unusual form of cell division cycle that decouples DNA synthesis from cytokinesis, resulting in physiological polyploidy. Oscillations in CDK2 activity are essential for the orderly progression of the cell cycle to ensure replicated DNA is accurately partitioned into two daughter cells. However, it remains underexplored how the dynamics of CDK2 activity regulate endoreplication in the context of TSCs differentiation. To address this question, we leveraged the variability in cell fate decisions in an established in vitro system of TSCs differentiation that relies on removal of a growth factor, FGF4, to induce endoreplication. Using quantitative single-cell live confocal microscopy of a precise CDK2 biosensor, DHB-Venus, we identified at least three different outcomes upon FG4 removal: self-renewal, endoreplication, and migration. Our quantitative analyses showed high levels of Cdk2 activity in self-renewing cells whereas intermediate DHB-Venus turnover is linked to increased nuclear and cell size, indicating a shift to endoreplication. Importantly, we also characterize a third class of differentiating TSCs with migratory characteristics that correlate with low levels Cdk2 activity without a change in nuclear size. In sum, our results demonstrated a correlation between different fate outcomes and specific thresholds of CDK2 activity. Our findings show that TSCs can distinguish between different outcomes through modulating the central kinase of the cell cycle, CDK2, positioning it as a key regulator of early trophoblast differentiation. Summary StatementThis study investigates the oscillatory behavior of CDK2 activity during murine trophectoderm differentiation and its potential role in guiding cell fate decisions.

Background Delineating the cellular origins of extracellular vesicles (EVs) enables the detection of clinically relevant changes in dynamic and complex tissues, such as the endometrium, which are not characterizable through single biomarker assays. Transcriptome deconvolution into cellular composition using deep learning methods provides a means to explore this complexity. However, such computational methods have not been previously applied to EV bulk transcriptomes, and their efficacy in profiling EV population changes and concordance to tissue throughout the menstrual cycle remains unknown. Methods This observational cross-sectional study utilized a deconvolutional generative deep learning algorithm, BulkTrajBlend, trained on a comprehensive human endometrial single-cell RNA sequencing (scRNA-seq) atlas. The model was applied to deconvolve paired bulk transcriptomes from endometrial tissue and uterine fluid EVs (UF-EVs) across the proliferative (P, n=4), early-secretory (ES, n=5), mid-secretory (MS, n=5), and late-secretory (LS, n=5) phases from healthy, fertile women. To validate generalizability, independent UF-EV datasets (ES, n=12; MS, n=12) obtained via different laboratory protocols were included. Deconvolved pseudo-single-cell (pSC) profiles from UF-EV data were subsequently integrated with Visium spatial transcriptomics slides of human endometrium (P, n=2; MS, n=4; ES, n=2). Results We developed a foundation model-based approach utilizing self-supervised learning to determine the cellular origin of EVs from their transcriptomic profiles. By mapping the generated pSC profiles to spatial transcriptomic data, we evaluated spatial origins of EVs. The statistical analysis demonstrated that UF-EV transcriptome deconvolution reflects the dynamic changes in the cellular composition of endometrial tissue across the menstrual cycle phases. The ability to distinguish accurately between proliferative and decidualizing menstrual cycle phases (ROC-AUC = 0.98) using cellular profile of deconvoluted UF-EVs transcriptome enables non-invasive profiling of endometrial tissue. Conclusions Our findings indicate the feasibility of determining endometrial tissue cellular composition using UF-EV transcriptomics. This methodology enables refined, non-invasive endometrial testing, avoiding invasive biopsy procedures. Based on deconvolution results, we are able to correlate UF-EV content to tissue, and distinguish between menstrual cycle phases. These results build toward a multifactorial screening method for abnormalities within the endometrium.

Pregnancy leads to many adaptations in the maternal body, most of which are reversible. However, reproductive experience can also result in permanent effects. Here, we investigated how pregnancy influences the somatotrophic system and the lasting effects of reproductive experience on the maternal organism. Reproductive experience induced a pronounced increase in lean body mass and longitudinal growth in both wild-type and growth hormone (GH)-deficient mice compared with age-matched virgins. Body growth was primarily observed during the first pregnancy, whereas a second gestation was mostly associated with increased adiposity. Data from a cohort of women with isolated GH deficiency (IGHD) caused by a loss-of-function mutation in the GHRHR gene revealed that nulliparous women were 7 cm shorter than those with one or more pregnancies. Increased GH secretion was observed in pregnant wild-type mice but not in pregnant GHRHR-deficient mice. Pregnancy-induced body growth is preserved despite disruption of GH-, ghrelin-, and estrogen-related signaling pathways. In conclusion, reproductive experience induces permanent changes in the maternal organism, promoting body growth in models that allow this response. Pregnancy-induced body growth appears to be independent of GH action. These findings underscore the need for further studies to investigate the long-lasting consequences of reproductive experience in females.

BackgroundPreeclampsia (PE) is a hypertensive disorder of pregnancy characterized by systemic inflammation, oxidative stress, and endothelial dysfunction. Although maternal vascular dysfunction is well established in PE, the mechanisms underlying fetal vascular injury remain poorly understood. We investigated whether inflammatory signaling activates NADPH oxidase 5 (NOX5) and contributes to oxidative stress and dysfunction in human umbilical arteries from pregnancies complicated by PE. MethodsUmbilical arteries and serum samples were obtained from normotensive pregnant women (NP) and women with PE. Vascular reactivity, nitric oxide (NO) bioavailability, reactive oxygen species (ROS) generation, cytokine levels, and NOX isoform expression were evaluated in human umbilical arteries and EA.hy926 endothelial cells. Pharmacological inhibition of NOX5, TNF- neutralization, Ca{superscript 2} channel blockade, and siRNA-mediated NOX5 silencing were used to investigate mechanisms. ResultsPE umbilical arteries exhibited increased vasoconstrictor responses, oxidative stress, and NOX5 expression, accompanied by impairment of NO bioavailability. NOX5 inhibition reversed vascular hyperreactivity in PE vessels. Exposure of normotensive umbilical arteries to PE serum reproduced the PE vascular phenotype, characterized by enhanced ROS generation, reduced NO levels, and hypercontractility. In endothelial cells, PE serum induced TNF--dependent Ca{superscript 2} influx, oxidative stress, and reduced NO production. Both pharmacological and genetic inhibition of NOX5 prevented these alterations. ConclusionsPE promotes fetal vascular dysfunction through activation of a TNF-/Ca2+/NOX5 signaling pathway that amplifies oxidative stress and impairs NO bioavailability. These findings identify NOX5 as a previously unrecognized mediator of umbilical artery dysfunction in PE and suggest the TNF-/Ca2+/NOX5 axis as a potential therapeutic target in hypertensive pregnancies.

Polycystic ovary syndrome (PCOS) is linked to adverse pregnancy outcomes and increased cardiometabolic risk in offspring, yet the placental mechanisms underlying these risks remain poorly understood. Metformin is prescribed during PCOS pregnancies despite limited mechanistic justification. Using multi-modal molecular analyses of placentas from healthy controls and women with PCOS randomized to placebo or metformin (PregMet trial), restricted to uncomplicated pregnancies, we characterized direct PCOS associated placental alterations independent of confounding complications. PCOS placentas showed transcriptional downregulation across multiple cell types and shifts in cell type proportions. Specifically, syncytiotrophoblasts exhibited reduced expression activity of growth hormone receptor signaling and glycosaminoglycan biosynthesis. Endothelial cells displayed diminished receptor tyrosine kinase pathway activity, including VEGFC, despite increased cell proportion and hypervascularity. Intercellular communication networks were globally suppressed, including reductions in PDGF signaling from Hofbauer cells to fibroblasts. Notably, metformin did not reverse most PCOS-associated molecular alterations and induced transcriptional changes correlated to birth weight and childhood BMI. These findings indicate that PCOS-associated placental features are driven by cell type specific dysregulation of growth factor, angiogenic signaling pathways that are largely unresponsive to metformin. This underscores the need to develop mechanism based, placenta targeted therapeutic alternatives for future pregnancy management.

This study aims to elucidate the association of circadian rhythm disruption with male testosterone levels and reproductive health using integrated epidemiological and experimental evidence. In the UK Biobank (n = 38,562), rest-activity rhythm amplitude was associated with lower serum testosterone levels (-0.21 nmol/L comparing the lowest vs. highest quartiles) and increased risks of orchitis and hydrocele (hazard ratios: 1.23 and 1.14, respectively). These findings were replicated in an occupational study of shift workers in China (n = 118), where shift work was independently associated with decreased testosterone levels ({beta} = -0.301, P = 0.015). In mouse models, circadian disruption induced testicular and epididymal atrophy, spermatogenic disorders, and suppressed circulating testosterone levels, accompanied by downregulation of key steroidogenic proteins. Together, these findings provide converging evidence that circadian rhythm disruption impairs testosterone synthesis, potentially through dysregulation of steroidogenesis, highlighting circadian rhythm as a modifiable environmental determinant of male reproductive health.

Development and use of assisted reproductive technologies (ARTs) in non-domestic species provides novel tools for species conservation. As a first step towards in vitro embryo production, we developed an OPU technique for two antelope species, scimitar horned oryx (Oryx dammah) and roan antelope (Hippotragus equinus) utilizing a custom-made needle guide and existing OPU equipment utilized by livestock and human practitioners. Females were anesthetized and placed in sternal recumbency for transvaginal OPUs. Prior to OPUs (36 - 45 hours), SHO and roan were either hormonally stimulated with follicle stimulating hormone (FSH, 140 or 250IU) as a single injection or not. A total of 32 and 26 OPUs were completed in SHO (n=10) and roan (n=7), respectively, representing one to four OPUs per animal at monthly intervals. A total of 141 oocytes were recovered from 215 follicles in SHO and 31 oocytes from 58 follicles in roan. FSH dose (250IU) increased (P<0.05) the number of follicles aspirated and the number of oocytes recovered in SHO. No effects of FSH were observed in roan (P>0.05). Good quality oocytes were recovered from all females and procedures were conducted in four consecutive months with no evidence of scar tissue buildup or reduced capacity to recover quality oocytes. These ARTs can be used to develop in vitro embryo production tools for population management and the preservation of female genetics; bolstering genetic diversity and guarding against extinction.

In mammals, a small population of spermatogonial stem cells (SSCs) is established shortly after birth. These cells self-renew and produce sperm for the entirety of a males reproductive lifespan, passing the genome on to the next generation. Thus, establishment of a population of SSCs with high genomic integrity is essential. SSCs are derived from a much larger precursor population of male germ cells (MGCs) that differentiate during fetal life. During the last third of gestation, MGCs undergo a prolonged period of G0 cell cycle arrest during which they sustain high levels of transcription and acquire epigenetic programming for SSC fate. Although these differentiation steps can cause cellular and genomic damage, it has been unclear whether selection for germ cell quality occurs during G0 arrest since no classic markers of cell death have been detected. In this study, we utilize a mouse model to characterize a population of MGCs that begin to accumulate markers if cell death, such as AnnexinV (AnV) and propidium iodide (PI), at E16.5. The AnV- and PI-positive MGC population is characterized by low expression of the RNA-binding protein, Dead End 1 (DND1), and exhibit dsDNA breaks and mitochondrial dysfunction. Interestingly, we do not see evidence of an active cell death cascade until the time of birth, where we see phosphorylation of MLKL, a hallmark of a necroptotic cell death mechanism. Based on these findings, we propose that variable cellular health is an important basis for selection of the SSC precursors. Significance StatementSpermatogonial stem cells (SSCs) are essential for reproductive fitness, yet how their precursors are selected during development is not known. Utilizing a mouse model, this study describes high levels of cellular damage within a subset of male germ cells (MGCs) during G0 arrest. The damaged MGC population was marked by low expression of the RNA-binding protein, DND1, and was strongly associated with mitochondrial dysfunction and dsDNA breaks. We observed signs of non-apoptotic cell death by embryonic day (E)16.5 and the appearance of necroptotic markers in MGCs at the time of birth. This study uncovers previously unknown heterogeneity in the MGC pool and points to MGC health as an important source of selection during G0 arrest.