Biology of Reproduction

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.

Background: The placenta has a unique transcriptomic profile, including microRNAs that are secreted into maternal circulation throughout pregnancy. MicroRNAs are small, non-coding RNA that post-transcriptionally regulate gene expression. Spontaneous preterm birth (sPTB) is associated with substantial differences in both placental pathophysiology and placental gene expression compared to term birth. We aimed to generate microRNA signatures of sPTB and map them to target genes using a microRNA-mRNA network. Methods: This study was conducted within the Conditions Affecting Neurocognitive Development and Learning in Early childhood (CANDLE) study. Placental samples were collected at delivery, and RNA was isolated for mRNA and microRNA sequencing. To investigate sPTB, this study excluded placental samples of participants with iatrogenic indications for PTB or induced labor. We examined differences in microRNA expression in participants who delivered before 37 weeks (N=35) compared to term participants (N=404) in a series of covariate-adjusted linear regression models. We used paired placental microRNA and mRNA expression data from this cohort to validate associations between computationally predicted microRNA-mRNA pairs and establish a microRNA-mRNA network. Results: Expression of 7 microRNAs were increased in sPTB (FDR<0.05) and were inversely correlated with sPTB-associated genes involved in immune signaling. Expression of 12 microRNAs were decreased in sPTB, including 4 members of the maternally expressed chromosome 14 microRNA cluster (miR-376a-3p, miR-376c-3p, miR-377-3p, and miR-381-3p). These microRNAs were predicted to negatively regulate oxidative phosphorylation genes that were increased in sPTB. The associations between miR-376c-3p and miR-377-3p and oxidative phosphorylation were confirmed in microRNA knockdown experiments. Conclusions: This study highlights potential biological mechanisms by which placental microRNA dysfunction might contribute to sPTB and highlights putative sPTB biomarkers that may be detectable in maternal circulation.

The distribution of fat-soluble compounds (including antioxidants) in follicular fluid (FF) remains sparsely documented in relation to in vitro fertilization (IVF) outcomes and existing studies have reported diverging associations. This study aimed to describe plasma and FF concentrations of fat-soluble micronutrients in women undergoing IVF and to analyze their adjusted associations with ovarian function, embryo development and pregnancy outcomes. In 2021-2022, plasma and FF samples were collected from 82 women (first IVF cycle) at oocyte puncture, along with lifestyle data covering the three preceding months. Eleven compounds (two tocopherols, three xanthophylls, five carotenes and retinol) were quantified. All compounds were detected in both compartments (lowest in FF) except phytoene, undetectable in FF. Plasma and FF -tocopherol concentrations were positively associated with plasma estradiol levels before oocyte puncture (both p<0.01) while FF -carotene and lycopene were inversely associated with plasma progesterone concentrations (p=0.01 and 0.02, respectively). Plasma phytofluene and phytoene were positively associated with mature oocyte rate (p=0.03 and p=0.01, respectively), while FF retinol was negatively associated (p=0.03). Carotenes, tocopherols and retinol were inversely associated with later IVF outcomes: fertilization rate (p<0.001 for plasma g-tocopherol, 0.02 for FF retinol), top-quality embryo (p=0.02 for plasma phytofluene), biochemical pregnancy at day 7 post-embryo transfer (p=0.05 for plasma -tocopherol, 0.02 for plasma -carotene), clinical pregnancy (p=0.03 for plasma -tocopherol, 0.01 for plasma phytoene) and live birth (p=0.04 for plasma -tocopherol, 0.02 for plasma phytoene). Plasma and FF g-tocopherol were positively associated with embryo fragmentation (both p<0.05). Finally, among xanthophylls, only plasma {beta}-cryptoxanthin was positively associated with plasma progesterone concentrations (p=0.02). Our findings of heterogeneous associations between tocopherols, carotenes, retinol and IVF outcomes across the stages of IVF suggest a beneficial effect limited to early outcomes and support a complex and context-dependent role of these compounds in female reproduction. This manuscript has been submitted to PlosOne on August 19, 2026.

The major events of female germline establishment, from primordial germ cell (PGC) specification to assembly of primordial follicles, occur during embryonic/fetal development. This study presents a single-cell RNA-sequencing atlas of the bovine fetal ovary at four gestational timepoints: estimated day 50, and timed pregnancies at days 70, 90, and 120, capturing the progression of PGCs through commitment, meiotic entry, and early oocyte growth. Fourteen transcriptionally distinct cell populations were identified, including stromal, epithelial, endothelial, immune, somatic support cell, and germ cell lineages. Sub-clustering of the germ cell population resolved six developmental stages (PGCs, transitioning oogonia, proliferative oogonia, committed oogonia, meiotic prophase I oogonia, and oocytes), while that of the somatic support cell compartment revealed five granulosa cell subtypes (steroidogenic, pre-granulosa 1, pre-granulosa 2, pre-granulosa 3, and epithelial cells). Trajectory analysis reconstructed the developmental path of PGCs to oocytes, with sequential activation of meiotic and oocyte-specific gene programs. Representation of all six germ cell stages at day 120 pointed to asynchronous oogenesis in the fetal ovary, which was validated and shown to be region-specific by protein immunolocalization. Intercellular signaling networks between germ cells and the somatic niche were mapped, revealing strong interactions through BMP, KIT, IGF, IGFBP, WNT, and MDK pathways with temporal specificity across gestational ages. The bovine germ cell and pre-granulosa cell subtypes demonstrate significant transcriptional parallels with similarly-staged cells from human fetal ovaries, establishing the cow as a reliable model for human germ cell and ovarian development. Collectively, these data provide a developmental roadmap for bovine oogenesis at the single-cell resolution that advances fundamental understanding of gametogenesis and informs strategies for advanced assisted reproduction.

The cryopreservation of testicular tissue is crucial for maintaining male fertility; However, its efficacy is often compromised by oxidative stress and mitochondrial dysfunction. Trehalose, a natural cryoprotectant, demonstrates significant potential, yet its specific mechanisms, particularly in mitochondrial regulation, remain insufficiently characterized. This study aimed to investigate the cryoprotective effects of trehalose on testicular tissue from 18-21-day-old piglets, with a focus on mitochondrial metabolism. Samples were cryopreserved via a slow-freezing protocol in a modified standard solution containing 200 mmol/L trehalose. The protective effect was evaluated by measuring testosterone synthesis, blood testis barrier (BTB) and spermatogenesis. Additionally, protective outcomes were assessed by measuring cell viability, tissue morphology, reactive oxygen species (ROS) levels, apoptosis rates, and testosterone secretion following freeze-thaw cycles. Transcriptomic sequencing and bioinformatics analyses were conducted to elucidate the underlying molecular mechanism. Cryopreservation led to reduced testosterone synthesis and secretion, decreased levels of BTB-binding proteins, and impaired spermatogenesis. Results indicated that 200 mmol/L trehalose significantly improved cell survival, decreased apoptosis and ROS levels, and enhanced testosterone secretion. 200 mmol/L trehalose partially increased the expression of StAR and CYP11A1 genes associated with testosterone synthesis while it protected the tight junction proteins Claudin-11, ZO-1 and the gap junction protein Cx43. Consequently, it exerted a reproductive protective effect by increasing the expression of key spermatogenic regulators DDX25, HMGB2, acrosomal protein DYP19L2, and sperm tail proteins AKAP4 and CFAP44. Transcriptomic profiling demonstrated that trehalose predominantly restored the transcriptional expression of genes involved in the mitochondrial electron transport chain and oxidative phosphorylation pathways, including ND2, COX2, ATP8, ATP6, ND5, ND6 and CYTB. These findings indicate that trehalose primarily protects piglet testicular tissue during cryopreservation by enhancing mitochondrial function, thereby providing a molecular basis for optimizing cryopreservation protocols.

IntroductionPreeclampsia is characterized by a pro-inflammatory, anti-migratory and anti-angiogenic placental phenotype. Impaired spiral artery remodeling stemming from trophoblast dysfunction is a key pathogenic mechanism. Little is known about the processes that govern trophoblast function normally and in preeclampsia. In preeclampsia, placental Let-7b-5p is reduced. The objectives of this study were to determine the normal function of Let-7b-5p in human trophoblast cells, to examine whether the ssRNA sensors, Toll-like receptor (TLR) 7 and/or TLR8 are mediators of trophoblast Let-7b-5p function, and whether disruption of this pathway promotes a preeclampsia-like phenotype in the trophoblast. MethodsThe human first trimester trophoblast cell line, Sw.71, was transfected with a Let-7b- 5p mimic, a Let-7b-5p inhibitor, or scramble control. Cells were treated with or without the TLR7 inhibitor IRS661 or the TLR8 inhibitor CUCPT9a. Trophoblast migration was measured using a two-chamber assay and interactions with human endometrial endothelial cells (HEECs) was measured using a 3D matrigel model. Trophoblast anti-angiogenic sFlt-1 release was measured by ELISA and sFLT1 mRNA measured by RT-qPCR. ResultsTransfection of trophoblast cells with a Let-7b-5p mimic elevated migration through activation of TLR7 and TLR8, while in a TLR7-dependent manner, the Let-7b-5p mimic negatively regulated sFlt-1 production. Furthermore, inhibition of trophoblast Let-7b-5p reduced migration, elevated FLT1 mRNA expression and sFlt-1 release, and reduced trophoblast-endometrial endothelial cell interactions. ConclusionsThis study highlights a role for TLR7/TLR8-activating Let-7b-5p in promoting normal trophoblast function and endothelial interactions and that disruption in this miR-driven signaling pathway may be relevant to processes driving a pre-eclamptic placental phenotype. HighlightsTrophoblast migration is positively driven by Let-7b-5p activating TLR7 and TLR8 Let-7b-5p, via TLR7, negatively regulates trophoblast anti-angiogenic sFlt-1 production. Inhibition of trophoblast Let-7b-5p reduces trophoblast migration and normal interactions with endometrial endothelial cells, while sFlt-1 production is elevated. TLR7/TLR8-activating Let-7b-5p promotes normal trophoblast function and endothelial interactions and disruption in this miR-driven signaling pathway may promote a preeclamptic placental phenotype.

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. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=148 SRC="FIGDIR/small/743297v1_ufig1.gif" ALT="Figure 1"> View larger version (50K): org.highwire.dtl.DTLVardef@1917a7borg.highwire.dtl.DTLVardef@41d7org.highwire.dtl.DTLVardef@e2bf98org.highwire.dtl.DTLVardef@90c9f3_HPS_FORMAT_FIGEXP M_FIG C_FIG SummaryThe protocol for generating mouse and human oviductal assembloids by combining epithelial and stromal cells for studying oviductal function in an in vitro setting.

Healthy ovarian follicle development and ovulation require coordinated communication between oocytes and surrounding somatic cells. Although oocyte-secreted factors (OSFs), such as GDF-9 and BMP-15, are established regulators of this communication, the non-OSF maternal factors that support OSF delivery and signaling during late-stage follicle development remain poorly understood. Here, using an oocyte-specific Dhx36 knockout mouse model, we identify the G-quadruplex (G4) helicase DHX36 as a non-OSF maternal factor required for antral follicle development and hormone-induced ovulation. Dhx36 deficiency caused severe defects in granulosa cell proliferation and cumulus expansion, accompanied by impaired activation of SMAD2/3 and SMAD1/5/8, while ERK1/2 activation remained intact. Although the expression of major OSFs was largely unchanged, Dhx36-deficient oocytes exhibited disrupted microvilli and transzonal projections (TZPs), resulting in defective OSF delivery and impaired oocyte-cumulus communication. Proteomic, lipidomic, and ultrastructural analyses further revealed dysregulated phospholipid metabolism, membrane organization, autophagy, and organelle homeostasis, including abnormal lysosomal, mitochondrial, and endoplasmic reticulum structures. Integrative transcriptomic and proteomic analyses identified concordant downregulation of genes involved in these processes, whose promoters were enriched in potential G4 motifs. Consistently, Dhx36 deficiency was associated with reduced RNA polymerase II activity, while pharmacological G4 stabilization impaired transcription of selected genes. Together, these findings establish DHX36 as a maternal regulator that links oocyte intrinsic homeostasis to intercellular communication, suggesting that DHX36-dependent maintenance of membrane and organelle integrity is essential for OSF delivery, cumulus cell function, antral follicle development, and ovulation.

Recurrent urinary tract infection represents a substantial unmet public health in women. Local administration of estradiol has been shown to reduce recurrence, however in vitro models of the female urinary tract remain limited and the mechanisms underlying the effects of estradiol are incompletely understood. Here, we describe a novel iPSC organoid differentiation protocol and its application to establish a multilayered transwell barrier culture model. Estradiol treatment resulted in reduced expression of innate antimicrobial peptides and cytokines, together with increased expression of demannosylation pathways. Treatment of transwell cultures with a combination of female sex hormones reduced endogenous CXCL8 signaling, independently of a 24-hour uropathogenic Escherichia coli (UPEC) challenge. To our knowledge, this is the first iPSC organoid-derived model of the urinary tract, which provides a platform for investigating interactions between the urothelium, urobiome and hormonal environment.

Mammalian preimplantation development requires precisely coordinated lineage decisions to establish the trophectoderm (TE), inner cell mass (ICM), epiblast (EPI), and primitive endoderm (PrE). Glucose metabolism and epigenetic regulation are increasingly recognised as key determinants of lineage specification during preimplantation development. However, how glucose-dependent metabolic cues interface with epigenetic mechanisms to regulate embryonic cell fate remains poorly understood. Here, we investigated the role of glucose in regulating protein methylation by protein arginine methyltransferases (PRMT) in bovine preimplantation development. PRMT1 and its associated histone mark H4R3me2a were detected throughout bovine oocyte maturation and embryo development. Pharmacological inhibition of Type I PRMTs using two structurally distinct inhibitors, GSK3368715 and MS023, markedly reduced global protein asymmetric dimethylarginine (ADMA) and H4R3me2a levels. PRMT inhibition impaired blastocyst cell proliferation, reduced total cell number, and disrupted both first and second lineage decisions, as demonstrated by decreased CDX2- and SOX2-positive TE and ICM cells and reduced NANOG- and GATA6-positive EPI and PrE cell allocation. Mechanistically, Type I PRMT inhibition downregulated key components of the Hippo-associated TE programme, including YAP, TEAD4, and TFAP2C. Consistent effects were observed in mouse embryos, where MS023 treatment reduced ADMA, CDX2, YAP, and TFAP2C expression and impaired TE and ICM allocation. Collectively, our findings identify Type I PRMT-mediated ADMA as an essential epigenetic regulator of early mammalian lineage specification and support a conserved ADMA-Hippo regulatory axis linking arginine methylation to embryonic cell fate decisions. In briefType I protein arginine methyltransferase (PRMT)-mediated asymmetric dimethylarginine (ADMA) is required for proper lineage specification during mammalian preimplantation development. ADMA depletion disrupts Hippo signalling, cell proliferation, and trophectoderm and inner cell mass allocation in bovine and mouse embryos.

The blood-gonadal barrier protects the germline from parental exposures. A phenomenon known as intergenerational inheritance suggests that, exceptionally, this barrier can be surpassed with consequences for the subsequent generation. Specific diet regimes and early traumatic experiences have been among the chronic stressors shown to be able to lead to intergenerational inheritance in mammals. Less is known about how acute stress can affect the germline. Cardiac damage leads to several alterations in peripheral organs and, overall, affects blood flow, metabolism, and the immune response. Whether cardiac damage can also affect the reproductive system is not known and might offer new insights into the potential inheritance of cardiovascular disease. Here, we used zebrafish and mouse models to explore the intergenerational role of cardiac damage and repair. In the first week after a cardiac cryolesion, male zebrafish gonads and gametes activated responses associated with inflammation. In sperm, chromatin accessibility was found altered in response to cardiac cryolesion. Offspring of cryoinjured zebrafish males revealed changes in cardiac function and cardiac gene expression. Induction of systemic sterile inflammation in the paternal generation mimicked cardiac injury effects in the following generation, while anti-inflammatory treatments in the injured paternal generation partially recovered F1 cardiac features. Similar features were found in mouse testis after a neonatal injury, and in the hearts of their offspring, suggesting a conserved role of sterile inflammation as a vector for intergenerational transmission of cardiac injury.

Background: Early access to pregnancy assessment units now detects many tubal ectopic pregnancies (TEP) at a stage when they could resolve spontaneously, creating a management dilemma. Methods: We performed a hypothesis-generating exploratory analysis in a retrospective study to assess whether serum progesterone (P4) levels in women with TEP are associated with management outcome. Results: Ninety-one cases of TEP managed in a single centre over three years were analysed. Receiver operating characteristic (ROC) curve analysis was used to explore serum levels of progesterone (P4), first human chorionic gonadotropin (hCG) and peak hCG (alone and in combination) in relation with successful completion of expectant management. Decision-tree analysis using first hCG and P4 was additionally performed to explore clinical sequential risk stratification. 23% (n=21) successfully completed expectant management. P4 concentrations in the expectant management group (median 3 nmol/L, IQR 2.00 to 8.50) were significantly lower than in those requiring surgical or medical management (median 17 nmol/L, IQR 5.75 to 29.25; p=0.0002). Area under the ROC curve (AUC) values for P4, log10 first hCG, log10 peak hCG and P4 with log10 first hCG were 0.766, 0.814, 0.811 and 0.835, respectively, for predicting successful expectant management. However, hCG was not significantly outperformed. Nonetheless, Youden optimised thresholds for hCG and P4 are reported, alongside decision-tree analysis that identified sequential first hCG and P4 thresholds associated with successful expectant management. Conclusion: Lower P4 levels are associated with successful expectant management of TEP but they do not outperform hCG either alone or as an adjunctive marker.

Chronic inflammation disrupts hormonal balance in the Hypothalamic-Pituitary-Gonadal (HPG) axis, contributing to reproductive disorders. While immune cells in the hypothalamus and ovaries have been extensively studied, their impact on the pituitary remains largely unexplored. Our research identifies pituitary macrophages (PitMacs) as the dominant pituitary immune cell population with a role in regulating reproductive gonadotropin secretion both in vitro and in vivo. Using a targeted AAV-based depletion strategy, we demonstrate that a reduction of PitMacs decreases serum gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), in female mice. PitMacs are transcriptomically distinct from other tissue-resident macrophages and harbor a unique translational program that reflects the pituitarys endocrine identity, including active translation of growth hormone (Gh) and prolactin (Prl). Cytokine profiling identified CXCL5 and IFN-{gamma} as key PitMac-derived mediators of gonadotropin regulation. Mechanistically, CXCL5 signals through CXCR2 to activate the MAPK pathway, converging with Gonadotropin-Releasing Hormone (GnRH) signaling in a time-dependent manner to regulate LH secretion and GnRH receptor surface expression. These findings establish PitMacs as essential endocrine-immune integrators, opening new avenues for understanding inflammation-driven reproductive disorders. One Sentence SummaryPituitary macrophages are unique hormone-producing immune cells that regulate hormone secretion via cytokine signaling.

Lamins are the major structural components of the nuclear lamina with a variety of roles in development and organogenesis. However, the function of lamins during trophoblast development, the first lineage to differentiate during mouse embryogenesis, remains unexplored. By utilizing an in vitro trophoblast stem cell differentiation model in a lamin null setting, we uncover that lamins maintain expression of genes related to trophoblast differentiation, while repressing genes involved in maintaining trophoblast stem cell stemness and off-lineage development. By deleting different combinations of lamins in mice, we show that both lamin triple-knockout and lamin-A and -B1 (lamin-A/B1) double-knockout result in placental defects, including reduced placenta size and disrupted placental organogenesis at embryonic day (E)9.5. At this stage, lamin-A/B1 are expressed in trophoblast giant cells of the placenta, and lamin-A/B1 loss leads to their impaired maturation in vivo. Lamin-A/B1 double knockout trophoblast giant cells exhibit reduced nuclear size along with a reduction of DNA damage signaling foci, suggesting a role for lamins in supporting trophoblast giant cell polyploidization. Similar to the transcriptional dysregulation observed during differentiation of lamin triple knockout trophoblast stem cells in vitro, lamin-A/B1 knockout in vivo results in downregulation of genes related to trophoblast giant cell function and upregulation of off-lineage genes. Our results suggest lamins are required for placental organogenesis by maintaining polyploidization and lineage-associated transcriptional programs in trophoblast giant cells.

Extravillous trophoblasts (EVTs) are a trophoblast subpopulation critical for feto-maternal tolerance during early pregnancy, primarily using HLA-G to exert immunomodulatory effect, and possessing N-glycomic profiles distinct from other trophoblast subpopulations. However, whether the N-glycosylation confers distinct immunological properties to EVTs remains poorly understood. To investigate this, we employed JEG-3, a human choriocarcinoma cell line having the capacity to produce pregnancy-related hormones and expressing both HLA-C and HLA-G resembling placental EVTs, as an in vitro EVT model, alongside cell line JAR which exhibits villous trophoblast phenotypes distinct from JEG-3. Both cell lines were treated with kifunensine or swainsonine, inhibitors of -mannosidases, to remodel their N-glycosylation patterns. This led to significant remodelling of their N-glycomic profiles, with JEG-3 cells showing an increased level of polylactosamine chains and decreased levels of -2,6-sialylation and core -1,6-fucosylation. Western blot analysis showed that inhibiting -mannosidases altered only the composition of N-glycans on cell-surface HLA-G, without affecting the overall abundance of cell-surface HLA-G. In kifunensine-treated JEG-3 cells that predominantly express oligomannose type N-glycans, an intracellular accumulation of unfolded HLA-G fragments, increased hCG secretion, and down-regulations of EVT markers GATA3 and KRT7 were observed compared to untreated control, while swainsonine treatment did not impact N-glycan expression. Cytotoxicity assays using NK-92 as effector cells showed that the de-sialylation of JEG-3 by neuraminidase treatment led to increased NK-92 mediated killing. JEG-3 cell sustained its EVT immunological properties through generating bioequivalent N-glycans, exemplified by NK-92 cells pre-conditioned with used culture media of kifunensine-treated JEG-3 cells displaying reduced cytotoxicity toward NK-sensitive lymphoblast cell line K562, an effect not observed with swainsonine-treated JEG-3 cells. This model suggests that EVTs immunological properties are dependent on specific N-glycomic profiles that are maintained by unique N-glycosylation homeostasis, and overall improves our understanding of how EVTs maintain their immunomodulatory effect at the maternal-fetal interface.

Menstrual symptoms vary across the cycle, yet most research assumes a normative 28-day cycle with fixed phase durations, obscuring the physiological relevance of natural cycle variation. Using the mcPHASES dataset, we characterised cycle and phase length variation across 96 menstrual cycles from 37 participants, with ovulation timing estimated from daily urinary luteinizing hormone measurements using a Bayesian hierarchical model, and examined associations with daily symptoms in a subset of 64 cycles from 35 participants with complete symptom data. Twelve physical, mental, and behavioural symptom domains were modelled using Bayesian ordinal regression, with posterior uncertainty in phase-length predictors propagated via a measurement error framework. Total cycle length was not associated with daily symptom burden, except sleep disturbances. By contrast, phase length decomposition revealed systematic associations across multiple domains: longer menstrual phase length was broadly associated with greater symptom intensity spanning physical, gastrointestinal, affective, and sleep domains; longer luteal phase duration was associated with greater fatigue and more frequent headaches, but lower sore breast intensity and lower stress; and longer follicular phase duration and later ovulation were each associated with greater sore breast intensity and more frequent mood swings. These associations require knowledge of actual ovulation timing and cannot be recovered from cycle length alone, indicating that the common assumption of a fixed 14-day luteal phase introduces systematic misclassification of hormonal exposure. Daily symptom intensity was also predominantly person-specific, with cycle phase explaining little of the between-person variance across most symptoms. These findings indicate that calendar-based phase assignment is insufficient for research and clinical assessment of hormone-sensitive conditions, and that person-specific baselines, rather than population-level phase averages, are needed for clinically meaningful symptom monitoring.

Abstract. Introduction: Preterm birth drives adverse perinatal maternal and infant health outcomes through heterogeneous symptoms, severity, and etiologies. Delivery prior to reaching 37 weeks of gestation may result from medically indicated intervention for pregnancy complications or spontaneously in the absence of prior symptoms. Placental tissue collected following preterm birth exhibits differential DNA methylation compared to full-term placentas and may indicate pregnancy health during gestation. Placental DNA currently has limited utility for assessing health of ongoing pregnancy, as sampling placental tissue during gestation increases the risk of infection and miscarriage. Risks associated with placental sampling during pregnancy limit the use of DNA methylation in clinical preterm birth prediction. Assessing preterm birth risk during gestation requires non-invasive methods for characterizing placental DNA methylation. Results: We quantified genome-wide DNA methylation patterns of hypermethylated cell-free DNA in pregnant (n = 99) and non-pregnant (n = 93) plasma using cell-free methylated DNA immunoprecipitation sequencing (cfMeDIP-seq). In each sample, we assessed DNA methylation status in 300-bp genomic windows, examining both sequencing read counts and calculated absolute molar DNA amount. Known hypermethylated placental regions, including RASSF1, STAT5A, and ERG promoters showed significantly increased odds of detection in pregnant samples, suggesting enrichment of cell-free placental DNA. Of the 536,444 300-bp windows examined, 173,071 (32%) showed significant enrichment in pregnant plasma. Linear modeling identified 107,505 differentially methylated regions (DMRs) associated with pregnancies later diagnosed with intrauterine growth restriction (IUGR) (n = 22). Alu elements showed increased representation in these DMRs than expected, while other repetitive elements exhibited underrepresentation. Discussion: These results demonstrate cfMeDIP-seq's ability to enrich for cell-free placental DNA and characterize cell-free DNA methylation signatures of pregnancies complicated by IUGR. Enrichment of cell-free placental DNA enables non-invasive profiling of placental DNA methylation from maternal plasma. Detectable epigenetic signatures in maternal plasma may identify pregnancies at elevated risk for preterm birth before clinical symptoms appear. Our findings further highlight the potential of cell-free placental DNA for monitoring pregnancy health.

Preeclampsia (PE) is a hypertensive pregnancy-specific disorder and a leading cause of maternal and fetal mortality. A common feature of PE placentas and maternal plasma is dyslipidemia, or abnormal lipid levels, which can increase oxidative stress and endothelial dysfunction. However, the precise transcriptional, post-transcriptional, and epigenetic mechanisms underlying these abnormalities remain poorly characterized. Identifying such changes may clarify disease mechanisms and identify lipid-related PE biomarkers. We conducted a large-scale meta-analysis integrating public placental datasets from NCBI GEO, comprising four DNA methylation (DNAm) datasets (n = 172), three RNA-sequencing datasets (n = 92), and an independent RNA microarray validation cohort (n =146). We evaluated differential DNAm (limma), gene expression (DESeq2), transcript-level shifts (Swish), and alternative splicing (rMATS) in PE versus control placentas, with all analyses stratified by fetal sex via an interaction term model. We also performed placental cell-type deconvolution to quantify PE-associated cell-type proportion changes. Our results demonstrated that lipid-related regulation changes in PE placentas occur primarily at the gene and transcript level, with DNAm showing no changes. We also identified significant isoform switching in PE that were undetected by differential gene expression analysis, and primarily driven by alternative transcription initiation and termination sites rather than alternative splicing. A subset of these isoform switches mapped to pathways dysregulated in PE and were predicted to cause functional protein changes. An interaction term model identified several sex-specific differentially expressed genes (DEGs) in PE, including a subset of male-specific downregulated genes involved in oxidative metabolism. However, many of the remaining sex-specific DEGs across both sexes were previously uncharacterized in the literature. These findings suggest that transcriptional and isoform-level regulation play a role in PE-associated dyslipidemia, with certain regulatory pathways displaying fetal sex-specific patterns. Highlights- Preeclampsia-associated dyslipidemia manifests at the gene and transcript level - Reciprocal isoform switches were missed by standard gene-level analyses - Alternative transcript initiation and termination drove isoform switching - Sex-interaction modeling identified sex-specific transcriptional shifts in PE

Background & Aims Dynamic cell cycle control is critical for intestinal crypt maintenance and injury responses, yet genetic regulators driving these changes remain poorly defined. As reserve intestinal stem cells (rISCs) are often considered to be slowly-cycling and can resist replication-dependent injury, factors that restrain proliferation may confer cytoprotection. Here, we define SOX9 as a regulator of intestinal stem cell (ISC) cycling and injury resistance. Methods Primary human ISCs were engineered to tune SOX9 levels, visualize cell cycle state, and manipulate cell cycle regulators. Using this system, we tested how SOX9 dosage impacts stemness, differentiation, proliferative recovery after SOX9 washout, and survival after 5-FU-mediated injury. Transcriptional analyses identified candidate links between SOX9 levels and cell cycle control, which were functionally tested using inducible INK4A (CDKN2A) and Cyclin D2 (CCND2) ISC lines. Results SOX9 induction lengthens the cell cycle in a dose-dependent manner largely by elongating G1 phase through the INK4A-Rb pathway. The effects of high SOX9 levels repressing proliferation and stem cell activity are reversible. SOX9 induction protects against 5-FU toxicity. This protection is mimicked by INK4A overexpression or pharmacological G1 phase arrest and repressed by CCND2 induction. Conclusions These findings identify SOX9-mediated G1 elongation as a reversible cytoprotective program that confers key functional properties associated with rISCs: proliferative restraint, retained stem cell potential, and resistance to replication-dependent injury. This positions G1 length as a potential determinant of which crypt cells survive injury to act as reserve stem cells.

The prevalence of neurodevelopmental disorders (NDDs) has increased dramatically, with growing evidence linking prenatal maternal stress exposure to NDDs. Across diverse maternal stressors, immune dysregulation emerges as a common feature, suggesting that fetal microglia may detect changes in the intrauterine environment and influence neurodevelopment. Accordingly, we utilized a mouse model of prenatal maternal cold stress to investigate the impact of maternal stress during pregnancy on fetal microglia morphology, cellular interactions, and phagocytic behaviors. Pregnant mice were exposed to cold stress from embryonic day 11.5 (E11.5) to E15.5 and fetal hypothalamic tissue was assessed from both male and female embryos. By adapting the morphology analysis toolset MicrogliaMorphology to assess fetal microglia, we demonstrate regional differences in microglial morphology in the fetal hypothalamus at baseline, with hypothalamic nuclei such as the paraventricular nucleus (PVN) containing fewer rod-like microglia compared to the broader hypothalamus. Interestingly, prenatal maternal cold stress induced a male-specific shift in microglial morphology from ameboid to ramified within the E15.5 PVN. Male embryos also displayed increased microglial-arginine vasopressin (AVP) neuronal interactions and microglial phagocytosis within the E15.5 PVN, but these changes were unique to microglia with a ramified morphology and were not observed when microglia with an ameboid or rod-like morphology were assessed. Using pHrodo bioparticles and flow cytometry, we further illustrate that prenatal maternal cold stress drives increased phagocytic activity in the E15.5 hypothalamus of male embryos, but not females. Together, these data demonstrate that prenatal maternal cold stress alters microglia morphology and drives morphology-dependent microglial interactions and phagocytic behaviors in male embryos which are unique to the hypothalamic PVN--a nuclei critical for social behaviors. Our findings also suggest that specific hypothalamic nuclei such as the PVN may be more sensitive to prenatal maternal stress, which has the potential to provide a cellular basis underlying the sex differences in microglia-dependent social deficits that were previously reported for this model.