Until recently, no repeatable protocol for equine in vitro fertilization (IVF) had been established, despite decades of trying. Recent studies have demonstrated reproducible IVF outcomes under specific experimental conditions, bringing equine IVF into a new perspective. Prolonged incubation of stallion spermatozoa in a TALP based medium supplemented with penicillamine, hypotaurine and epinephrine (PHE) appears to be important factors of protocols that support sperm capacitation and markedly improved IVF outcomes. The sperm capacitation procedure has now been applied successfully using both fresh and frozen-thawed stallion semen, with the latter requiring a shorter pre-incubation period. Here, we review the molecular, structural, and metabolic processes that drive capacitation in stallion spermatozoa, with comparisons to better-characterized mechanisms in other mammals, focusing on changes in sperm membrane composition, energy metabolism, cytoplasmic content, and on the influences of seminal plasma and the female reproductive tract. Because the newly developed IVF protocol appears to be shorter with frozen-thawed semen, special attention is given to capacitation-like changes triggered by cryopreservation, and their consequences for sperm function and fertilizing capacity. Importantly, although successful fertilization can be achieved under these conditions, this does not constitute direct evidence that the molecular mechanisms governing stallion sperm capacitation have been fully elucidated. Rather, these protocols provide a robust experimental platform for investigating these mechanisms in the horse.

The Japanese eel (Anguilla japonica) is a commercially important species whose captive reproduction remains challenging. Bone morphogenetic protein 15 (BMP15) is an oocyte-derived factor that plays a key role in regulating folliculogenesis; however, its function in Japanese eel is unclear. In this study, the full-length Japanese eel bmp15 cDNA was cloned and bioactive recombinant BMP15 protein was produced. Weekly co-administration of recombinant BMP15 with FSH and LHRHa for 10 weeks significantly promoted ovarian development, as indicated by increased gonadosomatic index (GSI), larger oocyte area, and elevated serum vitellogenin (VTG) levels compared to hormone treatment alone. Integrated multi-omics analyses suggested that BMP15 exerts its biological functions through a coordinated regulatory network involving anabolic metabolism, glycerophospholipid biosynthesis, nutrient transport, extracellular matrix remodeling, and suppression of steroidogenesis. These findings are consistent with a mechanistic model in which BMP15 simultaneously promotes growth, maintains follicular homeostasis, and optimizes oocyte quality by controlling maturation timing. Our findings identify BMP15 as a potential key regulator of ovarian development in Japanese eel and highlight its potential as a novel homologous factor for improving artificial maturation strategies in eel aquaculture.

Intrauterine infusion of platelet-rich plasma shows clinical promise as a regenerative therapy for patients with repeated implantation failure, yet the precise cellular and molecular mechanisms governing its in vivo effects on the human endometrium remain poorly understood. To elucidate these mechanisms at high resolution, we performed single-cell RNA sequencing on paired endometrial biopsies collected from patients during the proliferative phase, both before and shortly after intrauterine platelet-rich plasma infusion. Our analysis reveals that platelet-rich plasma acts as a potent modulator of the endometrial microenvironment by inducing extensive transcriptional reprogramming within resident cell populations. Alongside the direct stimulatory effects of PRP on various cell lineages, stromal cells emerged as key mediators contributing to this regenerative response, suppressing developmental plasticity pathways while concomitantly upregulating functions crucial for tissue regeneration, including cytoskeletal remodeling, pro-angiogenic signaling, and immune modulation. These reprogrammed stromal cells are predicted to serve as functional hubs, potentially driving enhanced paracrine networks that support robust angiogenesis through endothelial cell expansion and promote a growth-favorable epithelial phenotype via secreted growth factors. Notably, platelet-rich plasma infusion also induced a marked expansion and activation of specific tissue-resident natural killer cell subsets, indicating a recalibration of the local immune landscape. This study highlights that platelet-rich plasma orchestrates a multifaceted regenerative program where direct cellular stimulation converges with reprogrammed stromal-driven paracrine signaling to facilitate vascularization, epithelial growth, and immunological recalibration in the endometrium.

Placenta insufficiency, arising from impaired trophoblast (Tr) function, contributes to early embryo loss and fetal growth restriction. Sirtuin (SIRT) 1 and SIRT6 are redox modulators in early embryonic development, yet their roles in porcine Tr remain unclear. This study examined the expression and roles of SIRT1/6 in porcine Tr during early pregnancy. Conceptus and Tr tissues from days 12 to 30 pregnant gilts were analyzed for SIRT1/6 expression. Then, day-15 Tr cells were treated with SIRT1/6 modulators to assess gene expression, cell viability, cell-cycle proteins, cell adhesion, cytokine synthesis, and reactive oxygen species (ROS) generation under control and hydrogen peroxide (H2O2)-induced oxidative stress. SIRT1 mRNA expression increased with placenta development (days 15-30 of gestation), while its protein levels remained unchanged. SIRT6 protein abundance was greater during implantation (day-15 conceptuses) and at placentation onset (day-20 Tr) compared with day-30 Tr. SIRT1 activation stimulated mRNA expression of mitochondrial biogenesis regulators (NFE2L2, TFAM) and respiratory chain components (MT-ND1-2, MT-ATP6/8). The SIRT6 activator induced NRF1, TFAM, and all 13 mitochondrial respiratory subunit genes (MT-ND1-6, MT-CYB, MT-CO1-3, MT-ATP6/8). Both activators stimulated cell proliferation, possibly by shortening mitosis as evidenced by reduced phospho-H3-S10 levels. IL6 synthesis was upregulated, while IFNγ production was decreased in these cells. SIRT6 activation counteracted H2O2-induced oxidative stress, reducing ROS and supporting cell viability, while amplifying stress-induced mitochondrial pathways. In conclusion, SIRT1/6 may support porcine Tr development and function. SIRT6 appears to modulate the response of peri-implantation conceptuses to oxidative stress, suggesting its potential as a target for improving prenatal survival.

Antenatal corticosteroid (ACS) therapy, typically using dexamethasone (Dex), is a cornerstone of improving preterm infant survival by accelerating lung maturation. However, its systemic effects on other developing organs remain poorly understood. Here, we reveal a previously unrecognized impact of prenatal Dex exposure on fetal testis development, with implications for male reproductive health. Analysis of fetal testes from a Dex-based ACS mouse model revealed that Dex suppressed androgen synthesis by reducing Leydig cell number and downregulating steroidogenic pathways. Dex also activated immune programs, induced CD163+ M2c macrophages and IL-10 signaling, and promoted endothelial expansion. In response to aberrant pro-angiogenic signals, vascular architecture was disrupted: capillary density and pericyte gene expression declined, while blood vessel diameter increased. Transcriptomic analyses revealed downregulation of androgen response and cholesterol metabolism pathways, alongside upregulation of immune and coagulation signatures. Analyses of fetal ovaries revealed a sexually dimorphic and organ-specific effect of ACS treatment, in which ovarian gene expression during this window of development remained unaffected. Our findings uncover an immune-vascular mechanism linking prenatal Dex to impaired steroidogenesis, providing new insight into potentially broad effects of glucocorticoid exposure.

Direct experimental analysis of the mammalian oviduct is constrained by limited tissue access and the short lifespan of ex vivo preparations. Extracellular matrix-embedded three-dimensional epithelial organoids provide longer-term in vitro models. However, their inward-facing apical surface and the absence of supporting stromal cells limit physiological studies of the oviduct, including ciliary activity and maternal-embryonic interactions. Here, we provide a step-wise protocol detailing the generation of mouse and human oviductal assembloids in which epithelial cells form an outward-facing (apical-out) layer around a stromal core. Epithelial and stromal cells from adult mouse oviducts or human Fallopian tubes are isolated, expanded separately, and subsequently aggregated in a rotational culture system. The protocol also outlines morphological and immunostaining criteria for confirming cellular organization, whole-mount detection of external cilia, measurement of ciliary beat frequency, and co-culture of mouse assembloids with preimplantation embryos. Mouse and human assembloids retained epithelial and stromal identity and displayed cilia at the accessible outer surface. In a proof-of-concept experiment, embryos co-cultured with the assembloids developed to blastocysts at a rate similar to that of in vivo-derived blastocysts. This reductionist system provides a straightforward and tractable model to investigate oviduct physiology and embryo-maternal communication while allowing direct manipulation and observation of the epithelial interface.

In pigs, OCT4 expression is maintained in the trophectoderm after the first lineage differentiation, whereas it is rapidly downregulated in mice. Nevertheless, the functional role and downstream mechanisms of OCT4 in the TE in pigs remain entirely unexplored. By TE-specific depletion, we demonstrate that OCT4 is crucial for TE proliferation and expansion in pigs. Mechanistically, OCT4 directly binds to and activates the proto-oncogene JUN, which in turn represses the anti-proliferative gene BTG2 by binding to its promoter. Disruption of this OCT4-JUN-BTG2 axis results in BTG2 accumulation and cell cycle arrest. Notably, knockdown of BTG2 significantly rescues the proliferation defect induced by TE-specific OCT4 depletion. This establishes a model in which OCT4 drives placental morphogenesis, offering key insights into placental evolution with direct implications for livestock reproduction.

Pelvic organ prolapse (POP) is a leading cause for sow mortality in the United States. Recent work has evaluated biological factors associated with POP, and differences have been observed within the vaginal microbiota of sows at high risk using 16S rRNA gene amplicon sequencing. Additional functional studies are needed to better understand the relationship of dysbiosis of the vaginal microbiome with POP risk in sows. The current study's objective was to provide information about the functional potential of the sow vaginal microbiome, and to identify candidate genes and organisms that may be associated with POP. For this, metagenome shotgun sequencing was conducted on DNA extracted from 16 vaginal swab samples from late gestation sows. Of the 16 samples, 8 were from sows at high risk and 8 from sows at low risk for POP. Of the 8 samples from sows at high risk for POP four subsequently experienced POP. Subsequent contigs were annotated to generate a gene catalog of the vaginal microbiome of sows. The contigs were binned into metagenome assembled genomes (MAGs), resulting in 10 high-quality MAGs identified as Mannheimia varigena, Corynebacterium maris, Turicibacter bilis, Staphylococcus hyicus, Streptococcus dysgalactiae, Anaerococcus prevotii, Actinobacillus rossii, Prevotellaceae, Methanobrevibacter, and Veillonella caviae. MAGs classified as Streptococcus dysgalactiae and Staphylococcus hyicus contained potential virulence factors that are linked to the weakening of the connective tissue of the reproductive tract. This work provides initial insights into the functional potential of the vaginal microbial communities in late gestation sows in relation to POP and reproductive health.

Cesarean scar pregnancy (CSP) has posed a serious threat to women's reproductive health. The view on its mechanism remained controversial. Here, we performed a single-cell sequencing on villi and decidua from three normal and three cesarean scar pregnancies in patients with a history of cesarean delivery. Enrichment analysis was performed to elucidate the functional roles of various cell types. Pseudotime analysis was utilized to infer the differentiation of trajectory. SCENIC provided insights into transcription regulation. CellChat was employed to explore intercellular interactions. A single-cell atlas of the maternal-fetal interface microenvironment in patients with a history of cesarean delivery was conducted to elucidate aberrant activities associated with CSP. Significant alterations were observed in trophoblast differentiation and function, including changes in angiogenesis, migration, and interactions with the extracellular matrix (ECM). A marked reduction in decidual stroma cells was observed, accompanied by abnormal ECM remodeling. Neutrophils (Neu) emerged as the principal immune cells, with elevated numbers and activation. Inflammatory pathways such as NF-κB in Neu increased. Cellular interactions between stroma cells and trophoblasts were mainly COLLAGEN, FN1, and LAMININ, and between immune cells and trophoblasts were VISFATIN and CXCL. This comprehensive single-cell analysis has elucidated the cellular heterogeneity at the maternal-fetal interface in patients with a history of cesarean delivery and shed light on distinct cellular abnormalities associated with CSP.

Early human pregnancy (11-15 weeks) is a critical period when placental dysfunction begins but remains clinically undetectable, preventing early intervention for pregnancy complications. This highlights a pressing need for circulating biomarkers that can signal the onset of a complication. Circulating extracellular vesicles (EVs) carry bioactive cargo that reflect originating cell activity in real-time, positioning them as early biomarkers of pregnancy complications. While pregnancy studies have identified plasma EV cargo alterations across complications, studying small EVs alone-the field standard-or bulk EVs together obscures pathological changes that may manifest preferentially in one population. To address this, we performed the first multi-omic comparison of small and large plasma EVs from healthy pregnancies at 11-13 weeks (n=10) to establish subtype-specific cargo differences and provide a reference for normal pregnancy EV biology. We quantified mitochondrial DNA (mtDNA), performed transcriptomics (mRNAs, miRNAs) and proteomics to define small and large EV signatures. Large EVs were preferentially enriched for mitochondrial cargo including DNA, transcripts, and proteins as well as hypoxia response, endocrine signaling, and VEGF signaling related transcripts and proteins. In contrast, small EVs were enriched for canonical EV markers and basement membrane proteins. Additionally, we found that EV miRNA composition was similar across EV subtypes with only 7 miRNAs differentially abundant between small and large EVs. These distinct small and large EV cargo signatures underscore the importance of profiling both populations for liquid biopsy potential. Together these data provide a baseline for future studies linking EV signature alterations to placental dysfunction and adverse pregnancy outcomes.

Ovulation links folliculogenesis to fertilization and pregnancy, yet vertebrates have evolved contrasting ovulatory strategies that reflect ecological and social pressures. Spontaneous ovulators rely on internally generated cyclic signals, whereas induced ovulators couple follicular rupture to external stimuli such as copulation or social cues. Despite these differences, both strategies converge on a conserved hypothalamic-pituitary-ovarian cascade in which a GnRH-driven preovulatory LH surge coordinates oocyte maturation, follicle rupture, and corpus luteum formation. Arcuate Kisspeptin/Neurokinin B/Dynorphin (KNDy) neurons function as the GnRH pulse generator, while anteroventral periventricular nucleus/Preoptic Area (AVPV/POA) neurons are the GnRH surge generator by integrating estradiol-mediated positive feedback to gate the timing and amplitude of this surge. This review aims to contrast spontaneous and induced ovulation across vertebrates, emphasizing how both strategies converge on a common final pathway. The review then examines how diverse sensory and environmental cues are wired into this core axis in taxa such as camelids, rabbits, cats, honeycomb groupers, most birds, and zebrafish. Finally, ecological and social contexts are considered, highlighting how ovulation mode shapes paternity monopolization, mating systems, and the adaptive balance between male reproductive success and female control over fertilization.

Obesity and metabolic syndrome are major contributors to male infertility. These conditions disrupt the hypothalamic-pituitary-gonadal (HPG) axis and compromise testicular steroidogenesis and spermatogenesis through mechanisms that include altered aromatase activity, insulin resistance, chronic inflammation, and oxidative stress. Irisin, an exercise-induced myokine, has emerged as a metabolic signal with potential effects on male reproductive function. This review summarizes irisin biology, proposed mechanisms of action, and putative functions of irisin in male reproduction, with emphasis on obesity-associated fertility impairment. Studies in high-fat diet-induced obese rodent models indicate that exogenous irisin can improve metabolic homeostasis, restore testosterone production, increase expression of key steroidogenic enzymes, and mitigate obesity-associated reductions in spermatogenesis and sperm quality. These effects are commonly attributed to anti-inflammatory, antioxidant, and cytoprotective actions, as well as modulation of cellular energy metabolism through AMP-activated protein kinase and mitogen-activated protein kinase signalling. Irisin has also been reported to influence central neuroendocrine pathways involved in gonadotropin regulation and to improve sexual behaviour in rodent models of metabolic- and antidepressant-induced sexual dysfunction. However, the physiological relevance of irisin in men's reproductive physiology remains incompletely defined. Clinical evidence is largely associative, and data on irisin receptor expression and downstream signalling in the male reproductive tract are limited. Key gaps also include the regulation of irisin secretion and potential tissue-specific actions across stages of male reproductive development. Translational studies are needed to clarify irisin's actions within the HPG axis and gonads, and to evaluate its potential to mitigate obesity-related male reproductive dysfunction.

The transient receptor potential cation channel subfamily V member 1 (Trpv1) is a polymodal sensor of noxious stimuli, including heat and pain. However, its contribution to mammalian reproductive physiology remains incompletely understood. Here, we evaluated reproductive phenotypes and transcriptomic responses in a Trpv1-knockout mouse model under normal and heat-stress conditions. Vaginal-smear profiling showed altered estrous cyclicity in Trpv1-deficient females. Bulk RNA-seq and Smart-seq2 analyses identified tissue- and condition-dependent gene-expression changes associated with Trpv1 deficiency, including extracellular-matrix-related transcriptional signatures in heat-stressed oocytes. In males, Trpv1 deficiency was associated with altered sperm-motility measurements under heat stress, changes in steroidogenesis-related transcripts, and testicular stress-response signatures. These findings identify candidate molecular pathways associated with Trpv1 deficiency and thermal challenge. Further studies, including mating, fertilization, embryo-development, protein-level, and cellular functional assays, are required to determine the consequences for gamete competence and fertility.

Polycystic ovary syndrome (PCOS) is a common and complex endocrine and metabolic disorder whose etiology has not yet been fully elucidated. Emerging research has revealed an association between PCOS pathogenesis and post-translational modifications of proteins. However, the effect of neddylation on PCOS pathogenesis has not been investigated. In this study, key proteins of the neddylation pathway (Nedd8, neddylation-activating enzyme (NAE), and Cullin-1/2/3) were significantly upregulated in the granulosa cells of patients with PCOS; this was accompanied by enhanced apoptosis (increased Bax and Caspase-3, decreased Bcl-2) and elevated expression of the androgen synthesis-related proteins, namely: androgen receptor (AR) and Cytochrome P450 Family 17 Subfamily A Member 1(CYP17A1). In the in vitro DHT-induced PCOS KGN cell model, key neddylation pathway proteins (Nedd8, NAE, and Cullin-1) were upregulated. Overexpression of Nedd8 and inhibition of neddylation by MLN4924 promoted apoptosis. However, inhibition of neddylation by MLN4924 reversed AR/CYP17A1 protein expression. In a rat model of PCOS, MLN4924 treatment significantly suppressed the expression of Nedd8, NAE, and Cullin-1/2/3 in the ovarian tissue, restored estrous cycles, alleviated ovarian morphological abnormalities (characterized by increased corpora lutea and reduced cystic follicles), and decreased serum testosterone and LH levels. MLN4924 significantly promoted ovarian granulosa cell apoptosis, but reduced the expression of androgen synthesis-related proteins. Together, these findings indicate the unrecognized role of neddylation in PCOS, while highlighting that MLN4924 represents a potential therapeutic approach for PCOS treatment.

Centromeres ensure faithful chromosome segregation, yet the satellite arrays typically underlying these loci exhibit remarkable variation in sequence, organization, and size across individuals and species. Recent advances in long-read genome assemblies and experimental model systems have begun to reveal that this diversity is not simply tolerated but can directly influence centromere function during female meiosis. Asymmetry between centromeric satellite arrays of homologous chromosomes can lead to asymmetries in kinetochore proteins and spindle interactions, biased chromosome orientation on the spindle, and ultimately non-Mendelian inheritance by centromere drive. Emerging evidence further suggests that centromeric satellite asymmetry can increase meiotic chromosome segregation errors and aneuploidy, particularly in the context of small arrays. In this review, we discuss recent progress linking centromeric satellite variation with meiotic chromosome behavior, centromere drive, and segregation fidelity.