Reproduction

Several different mechanisms of regeneration have been proposed to explain repair of the endometrial luminal epithelium (LE) following menstrual/estrous cyclicity, parturition or other damaging insults to the uterine mucosa. These include activity of an as-yet unidentified epithelial stem cell population, mesenchymal transdifferentiation, or integration of bone marrow-derived cells. Here, we used a novel Cag-Flox-STOP-Flox-Pgrmc2-3XFlag (Pgrmc2-3XFlag) reporter mouse coupled with a recently developed GE-specific cre recombinase mouse (Prss29-Cre) to determine if and to what extent the GE contributes to regeneration of the LE following parturition and during aging in nulliparous mice. To initiate regeneration in the endometrium, control (Pgrmc2-3XFlag) and reporter (Prss29-Cre;Pgrmc2-3XFlag) mice were placed with males for one pregnancy. Postpartum (pp) uterine tissues were then collected 5 days (1x5dpp), 3-weeks (1x3wpp) and 7-weeks (1x7wpp) later. A fourth set of uterine tissues was collected 3-weeks after three pregnancies (3x3wpp). The number of glands did not change at any time point. Indelibly marked FLAG+ GE cells were detected by immunohistochemistry and immunofluorescence in GE as expected. As demonstrated in serial uterine sections of uteri from all four treatment groups, GE cells migrated toward the LE and formed junctional points (JP) where GE cells transitioned from cuboidal GE cells to pseudostratified columnar LE cells. Transitioning cells at junctional points expressed markers of both GE (i.e., FOXA2) and LE (i.e., CALB1) tissues. Following transition, GE-derived LE cells expressed PGR and ESR1 consistent with surrounding developmentally-derived LE cells. About 17 FLAG+ LE cell islands were also observed in 1x5dpp 4 mm longitudinal sections at sites distant from GE-LE junctional points. Interestingly, the number of islands did not change from 1x5dpp to 1x7wpp. However, the total number of GE-derived FLAG+ LE cells observed in each island decreased from 1x5dpp to 1x3wpp, but then tended (p=0.07) to increase by 1x7wpp. There was no difference in the number of GE-LE junctional points or GE-derived FLAG+ LE islands between 1x3wpp and 3x3wpp tissues. However, a significant increase in the total (p=0.02) and average (p=0.02) number of FLAG+ LE cells was observed in 3x3wpp LE islands highlighting the proliferative expansion of these cells over the intervening ~80 days. GE-LE junctional points were also observed in 5-month old nulliparous Prss29-Cre;Pgrmc2-3XFlag mice at diestrus, but not in younger 6-week old mice. Collectively, these findings establish the GE as a quantifiable cellular source for LE regeneration during postpartum involution and as part of normal LE cell turnover during the estrous cycle in nulliparous mice. We propose a mechanism whereby GE cells within glandular structures are not terminally differentiated, but rather are stably differentiated to perform glandular function. However, when needed and based on the level of endometrial damage, GE cells can dedifferentiate, become integrated in to LE tissue and then reprogram to take on an LE phenotype as part of the regenerative process following parturition and during estrous cyclicity over time.

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.

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.

In mammals, ovarian follicle development is a highly coordinated process that underlies female fertility. Granulosa cells expressing anti-Mullerian hormone (AMH) are widely used as a marker of growing follicles. However, the in vivo roles of granulosa cells in follicular development and female fertility remain unclear. Here, we analyzed AMH-toxin receptor-mediated cell knockout (AMH-TRECK) transgenic (Tg) mice on a NOG background, in which AMH-expressing granulosa cells are specifically depleted by diphtheria toxin (DT). We first found that, after a single DT injection into postnatal AMH-TRECK Tg females, AMH-expressing granulosa cells in primary and secondary follicles exhibited cleaved caspase-3 signals 1 day later and were depleted 4 days later. Second, after repeated DT injections weekly from 1 to 7 weeks of age in AMH-TRECK Tg females, antral follicles and corpora lutea were rarely observed, and the numbers of primordial, primary, and secondary follicles were decreased. Following PMSG and hCG stimulation, repeated DT-injected Tg females exhibited a reduced number of ovulated oocytes with a low proportion of mature oocytes, resulting in reduced IVF rates and fertility. Further, after a cessation of repeated DT treatment, ovarian weight and follicular development recovered: the numbers of primary, secondary, and antral follicles were recovered, whereas the primordial follicle pool remains reduced. We conclude that selective depletion of AMH-expressing granulosa cells in vivo impairs follicular development and fertility. Our model enables assessment of the in vivo effects of granulosa cell depletion and may provide a useful platform for future transplantation-based studies to understand complex follicular dynamics.

Progesterone governs the establishment and maintenance of pregnancy in ruminants, acting through the nuclear progesterone receptor (PGR) and, potentially, through membrane progesterone receptors (mPRs) of the PAQR family. The cellular distribution of mPRs in ruminant reproductive tissues, however, remains undefined. We generated and validated rabbit polyclonal antibodies against the intracellular C-terminal peptides of bovine PAQR6 (mPR{delta}) and PAQR8 (mPR{beta}), and used them, with quantitative PCR of the uterus and conceptus, to map these receptors in the ovine uterus and conceptus and in the bovine ovary. Both antisera showed high ELISA titer against their immunizing peptides, negligible preimmune reactivity, and complete loss of tissue signal after peptide pre-absorption. By qPCR, PAQR6, PAQR8 and PGR transcript levels did not change significantly across the peri-implantation period in either the conceptus or the endometrium; in contrast, the progesterone-responsive gene ZBTB16 increased markedly in the conceptus (up to ~45-fold by day 21, p < 0.05 at days 17, 19 and 21), whereas FKBP5 showed a numerical increase without reaching statistical significance. Both genes remained relatively stable in the endometrium, indicating conceptus-specific changes in progesterone-responsive gene expression. At the protein level, PAQR6 and PAQR8 localized to the luminal and glandular epithelium of the endometrium across the cycle and early pregnancy, and to follicular (granulosa) and luteal cells of the ovary. Dual immunofluorescence in the ovine endometrium showed that both PAQR6 and PAQR8 are enriched at the apical surface of the luminal epithelium and are spatially distinct from the basolateral adherens-junction marker E-cadherin. These results provide a protein-level map of PAQR6 and PAQR8 in the ruminant reproductive tract, identify the apical epithelial surface as a site of receptor enrichment, and show that the endometrial epithelium retains membrane progesterone receptors while progesterone-responsive gene expression changes dynamically in the conceptus during the peri-implantation period. The validated ruminant-reactive antibodies provide a resource for future functional studies.

Embryo implantation remains a major limitation of assisted reproductive technology, with failure occurring in approximately 30% of euploid embryo transfers. Implantation requires a synchronized dialogue between the blastocyst and receptive endometrium during the window of implantation (WOI), yet minimally invasive approaches to characterize the structural and molecular features of receptivity remain limited. We analyzed paired sonohysterogram images and uterine lavage samples collected during the proliferative and mid-secretory phases from subjects with regular ovulatory cycles and proven fertility. Endometrial folds were quantified, and lavage samples were analyzed by Luminex multiplex immunoassay. Folds were present in both phases but were significantly more abundant during the mid-secretory WOI, independent of imaging view and endometrial thickness. Folding correlated strongly with circulating estradiol level during the proliferative phase but not the mid-secretory phase, and folding patterns between phases were not correlated, suggesting distinct regulatory mechanisms. Consistent with these structural patterns, uterine lavage demonstrated phase-specific differences in expression of factors associated with endometrial receptivity and implantation, with glandular epithelium, and myeloid-lineage cells emerging as major contributors. Together, these findings identify coordinated structural and secretory processes during the WOI and support further evaluation of endometrial folding and uterine lavage as complementary, minimally invasive markers of endometrial receptivity.

Capacitation is a post-ejaculatory maturation process by which sperm become hyperactive and undergo the acrosome reaction, both of which are required to fertilize an oocyte. Two key features of capacitation are increased intracellular calcium via the calcium channel CatSper and changes in mitochondrial function. In mouse spermatozoa, CatSper-dependent calcium entry promotes mitochondrial calcium uptake and mitochondrial activation, contributing to hyperactivated motility and fertilization ability. Whether this mechanism is conserved in human spermatozoa was unknown. Here, we assessed mitochondrial function and mitochondrial calcium in paired non-capacitated and capacitated human spermatozoa from normozoospermic donors. Capacitation increased mitochondrial respiratory control ratio, coupling efficiency, mitochondrial membrane potential, and mitochondrial calcium. The increase in mitochondrial calcium required extracellular calcium and was prevented by pharmacological inhibition of CatSper or the mitochondrial calcium uniporter (MCU). Inhibition of CatSper or mitochondrial calcium uniporter also attenuated the capacitation-associated increase in mitochondrial membrane potential and reduced hyperactivated motility without affecting progressive or total motility. Pharmacological disruption of mitochondrial function reduced capacitation-associated hyperactivated motility without affecting the acrosome reaction. Together, these findings support a conserved pathway in human and mouse spermatozoa in which CatSper-mediated calcium entry promotes MCU-dependent mitochondrial calcium uptake and mitochondrial activation, contributing to the acquisition of hyperactivated motility during capacitation. These findings identify mitochondrial calcium handling as a potential target for modulating human spermatozoa function.

Study question: Can adeno-associated viruses (AAVs) efficiently and safely transduce human primary granulosa cells and human ovarian stromal fibroblasts to modulate the expression of genes involved in follicle recruitment and ovarian microenvironment regulation? Summary answer: AAVs can efficiently transduce human primary granulosa cells and human ovarian stromal cells with low toxicity, with the serotype DJ (AAV-DJ) demonstrating superior performance. AAVs enable targeted modulation of ovarian genes such as AMHR2 in human primary granulosa cells and TGF{beta}1 in human ovarian stromal cells, supporting the feasibility of gene-based approaches to improve ovarian function. What is known already: Infertility affects approximately 15% of couples, with diminished oocyte quality and quantity being major contributors, particularly with advanced reproductive age. Assisted reproductive technologies rely primarily on hormonal stimulation and do not directly target molecular pathways governing folliculogenesis or the ovarian microenvironment. Animal studies suggest that gene therapy using AAVs offers a promising strategy for tissue-specific and durable gene modulation, yet its application in human ovarian cells remains largely unexplored. Study design, size, duration: This was an experimental laboratory study using primary human granulosa cells from patients undergoing in vitro fertilization (IVF) and ovarian stromal cells obtained from post-pubertal patients who donated surplus ovarian tissue from ovarian tissue cryopreservation procedures for research. Sixteen AAV serotypes were evaluated for transduction efficiency, toxicity, and expression dynamics, followed by functional gene knockdown studies using AAV-DJ-based shRNA vectors. Participants/materials, setting, methods: Primary human granulosa cells and ovarian stromal cells were isolated from follicular aspirates and ovarian tissue, respectively. Cells were transduced with GFP-expressing AAV serotypes at varying multiplicities of infection (MOIs), incubation times, and culture days to assess transduction efficiency and cytotoxicity. Lead serotypes were further evaluated in dose-response studies. Functional gene modulation was assessed using AAV-DJ-mediated shRNA knockdown of AMHR2 in granulosa cells and TGF{beta}1 in stromal cells, quantified by RT-qPCR, immunofluorescence, and western blotting. Main results and the role of chance: Six of sixteen AAV serotypes (AAV2, AAV3, AAV6, AAV9, AAV-DJ, and AAV-X1.1) achieved greater than 10% transduction efficiency in human primary granulosa cells. Among these, AAV-DJ demonstrated the highest efficiency across all MOIs tested, reaching up to 48% transduction at 100,000 MOI, significantly outperforming AAV2 and AAV9. Toxicity increased with viral dose for all serotypes but remained comparable across vectors, with AAV-DJ exhibiting higher efficiency with comparable toxicity. The AAV-DJ-shRNA-AMHR2 construct produced a significant reduction in expression of AMHR2 mRNA by ~81% (p<0.05) in human primary granulosa cells as well as at the protein level in HEK293T-AMHR2-ZsGreen engineered cells (~85% knockdown, p<0.01). In human ovarian stromal cells, AAV-DJ preferentially transduced EMILIN1 and ACTA2-positive fibroblast and myofibroblast-like cells and enabled robust knockdown of TGF{beta}1 at both transcript (~89% knockdown, p<0.05) and protein levels (~67% knockdown, p<0.0001) using AAV-DJ-shRNA-TGF{beta}1 construct. Limitations, reasons for caution: These experiments were performed in vitro using primary human ovarian cells, which may not fully recapitulate the in vivo ovarian environment. Functional reproductive outcomes were not assessed, and future studies will be required to evaluate the effects of gene modulation on folliculogenesis and fertility. Wider implications of the findings: These findings establish AAV-DJ as a promising vector for targeted gene modulation in human ovarian cells. This approach introduces a novel framework for addressing infertility and ovarian function by directly targeting molecular pathways involved in follicle recruitment and ovarian stromal signaling, potentially enhancing current assisted reproductive technologies.

Uterine leiomyomas, commonly known as fibroids, are benign tumors of the female reproductive tract. They are the most common tumor of the female reproductive system and the leading cause of hysterectomy in the United States. Progesterone, estrogen, and their receptors are important drivers for the development and growth of leiomyomas. How estrogen drives leiomyoma growth remains unclear. We have previously shown that simvastatin can inhibit leiomyoma growth by modulating intracellular calcium. This suggests that calcium signaling is important for leiomyoma growth. We therefore hypothesized that estrogen may drive leiomyoma growth by stimulating calcium release through the membrane estrogen receptor GPR30 (also known as GPER). We found that estrogen dose-dependently induced calcium release in leiomyoma cells. The GPR30-specific agonist G-1 also induced calcium release, indicating that GPR30 mediates estrogen-mediated calcium release. The GPR30 antagonist G-15 blocked estrogen-induced calcium release and significantly reduced leiomyoma proliferation. Our results demonstrate that GPR30 is a major driver of estrogen-dependent growth in leiomyoma cells by modulating intracellular calcium. These findings may have high therapeutic relevance to leiomyomas and other estrogen-dependent tumors.

Extracellular matrix (ECM) remodeling and angiogenesis are essential processes underlying endometrial decidualization during embryo implantation. Fibrillar collagens, the major structural components of the ECM, form the architectural framework of the decidua and undergo dynamic reorganization during this process. However, the functional role of type V collagen--a key regulator of collagen fibrillogenesis-- remains undefined. Here, we demonstrate that Col5a1 is highly expressed in decidual stromal and endothelial cells of the mouse uterus. Conditional deletion of Col5a1 leads to progressive uterine hemorrhage beginning at gestation day 8, culminating in complete embryo resorption and pregnancy loss by day 12. Col5a1-deficient decidua exhibits severe structural distortion, marked disorganization of fibrillar collagen, shallow and misdirected embryo invasion, and profound disruption of decidual angiogenesis and vascular network formation. Transcriptomic profiling further reveals distinct gene expression signatures and signaling pathways regulated by COL5A1 in the decidua. Collectively, these findings identify type V collagen as a critical ECM regulator required for maintaining decidual integrity, supporting angiogenesis, and ensuring successful pregnancy.

Mammalian spermatozoa face various challenges during their long march to the destination. As a specialized membrane-bound organelle in the sperm head, the acrosome is particularly sensitive to changes in ionic and osmotic conditions, yet how acrosomal ion homeostasis is maintained remains largely unknown. Here, we show that LRRC37A is required for acrosomal ion homeostasis. Loss of Lrrc37a causes complete male infertility, accompanied by pronounced enlargement and deformation of the acrosome in the sperm head. Acrosome biogenesis was not affected by Lrrc37a knockout during the early stages of spermiogenesis, whereas acrosomal enlargement emerged at later stages and became more pronounced in the epididymis. LRRC37A localized to the outer acrosomal region and interacted with the Na/K-ATPase -subunit isoforms ATP1A1 and ATP1A4. Loss of LRRC37A disrupted the spatial distribution of both isoforms and was accompanied by increased Na within the acrosomal region. In addition, pharmacological perturbation of Na/K-ATPase activity or cellular ion homeostasis induced acrosomal swelling. These findings establish an essential role for LRRC37A in acrosomal ion homeostasis, suggesting that its deficiency may be associated with a distinct form of teratozoospermia characterized by acrosomal enlargement.

Background Isotretinoin (ISO) is a systemic retinoid widely used for the treatment of acne vulgaris, yet its potential effects on male reproductive function remain unclear with previous studies reporting inconsistent findings. Methods and finding We conducted a multi-model study combining data from the Danish Young Men Study (DYMS) cohort with ex vivo cultured human testis tissue and an in vitro Retinoic Acid Receptor alpha (RAR) reporter gene assay. In the DYMS cohort semen parameters and reproductive hormone concentrations assessed on a specific examination day were compared between ISO-users (n=322) and non-users (n=4,065). Ex vivo human testis tissue was exposed to ISO and RA for 96 hours to assess changes in hormone secretion and germ cell dynamics, while the in vitro RAR assay evaluated ISO for effects on RA-induced RAR-signaling. In the DYMS cohort, ISO use was associated with reduced FSH concentrations, higher inhibin B/FSH ratio, and elevated estradiol concentrations, along non-significant tendencies of lower sperm counts. These associations persisted even after cessation of ISO use but were not evident in men first initiating ISO use 1 year after their examination date. In the ex vivo testis tissue, ISO exposure altered hormone secretion, including increased testosterone, but did not affect germ cell proliferation or apoptosis. ISO also inhibited RA-induced RAR-signaling in vitro in a competitive manner. Conclusions These findings suggests that ISO-use may influence male reproductive function, potentially with persistent effects. Further randomized clinical trials are needed to validate these effects.

Background: Ion channels regulate mammalian sperm function, yet chloride channels have received less attention than their cation counterparts. Herein, we investigated the presence, localization, and function of voltage-gated chloride channels (ClC-1, ClC-2, and ClC-K) and calcium-activated channels (ANO1 and ANO2) during sperm capacitation and the acrosome reaction. Results: Using the pig as an animal model, we localized these channels in different sperm regions: ClC-1 in the equatorial and post-acrosomal regions; ClC-2 in the connecting and principal pieces; ClC-K in the acrosome, connecting, and principal pieces; ANO1 in the principal piece; and ANO2 in the mid-piece and post-acrosomal sheath region. Following this, we addressed the roles of these channels in sperm capacitation and acrosome reaction by blocking them with different inhibitors: 9AC, specific for ClCs; BBR, specific for CaCCs; and NPPB, a broad-spectrum blocker that inhibits ClCs, CaCCs, and other chloride channels. Blocking CaCCs with BBR led to a significant reduction in sperm motility and mitochondrial membrane potential, with effects similar to those of the non-selective inhibitor NPPB or to a combination of BBR and NPPB. Moreover, although none of the three inhibitors affected intracellular calcium levels or acrosomal integrity, a significant reduction in protein tyrosine phosphorylation was observed in sperm treated with the combination of NPPB and BBR. Finally, membrane lipid disorder was increased when ClCs were blocked with 9AC and when sperm were incubated with a combination of BBR and NPPB. Conclusions: Our results suggest that CaCCs play a major role in sperm capacitation compared to ClCs. Further research to address the regulatory mechanisms involved and the potential relationship with other ion channels is warranted.

Understanding how to maintain and direct spermatogenesis in vitro is central to advancing reproductive biotechnologies in aquaculture species where the ability to generate gametes outside the organism could facilitate selective breeding, genetic modification, and germline preservation. However, current culture systems remain poorly defined in farmed fish species. In Atlantic salmon (Salmo salar), progress has been further limited by the absence of a comprehensive reference atlas of testicular cell types, making it difficult to determine how cells maintained in culture relate to their native counterparts. To address this, we first established a single-cell RNA atlas of the Atlantic salmon testis from freshly isolated tissue, resolving somatic and germ cell populations across all major stages of spermatogenesis. Primary testicular cells were then cultured under distinct conditions designed to promote either proliferation or differentiation for 14 days and subsequently subjected to single-cell RNA sequencing. To assign cell identities in cultured samples, the transcriptional profiles of cultured cells were computationally mapped onto the atlas, allowing direct comparison of cultured and native cell states. This approach revealed pronounced, condition-specific shifts in cellular composition. Proliferation medium supplemented with epidermal growth factors (EGF) and insulin-like growth factor (IGF) enriched spermatogonial populations, indicating preferential support of undifferentiated and actively dividing germ cells. In contrast, basal medium favoured the preferential survival of Sertoli cells in the absence of defined growth cues. A differentiation medium containing hormones that stimulate male gonad development (gonadotropins and androgens) failed to robustly promote meiotic progression. Further, comparative analysis of Sertoli cells across different conditions (in vivo and in vitro) revealed a loss of canonical identity markers and induction of stress-associated transcriptional programs in vitro compared to in vivo, indicating a shift away from specialised somatic function. Together, these findings establish the first single-cell reference atlas of Atlantic salmon testis and provide a framework for evaluating and optimising testis culture systems in salmonids. While early germ cell populations could be maintained and enriched in vitro, progression through later stages of spermatogenesis remained limited, indicating that important biological requirements of the native testicular environment are not yet fully recapitulated under current culture conditions.

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.

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.

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.

BackgroundPremature ovarian insufficiency (POI) is a condition affecting female fertility e.g., in the context of autoimmune disease. A recent proof-of-concept study showed that B-cell depletion with rituximab (RTX) temporarily restored ovarian function in patients with autoimmune POI. However, the underlying immunological mechanisms behind this response is unknown. ObjectivesTo characterize B- and T-cell phenotypes before and after RTX therapy in the above-mentioned patients and associations with autoantibody levels and treatment response. MethodsCryopreserved cell samples were available from six autoimmune patients with POI, four of whom were positive for autoantibodies against steroidogenic enzymes (21OH, 17OH and/or SCC). Patients received RTX and blood samples were collected at baseline, second infusion (2 weeks), 3, 8 and 12 months. Peripheral B- and T-cell phenotypes were analysed using spectral flow cytometry and correlated with autoantibody levels. ResultsAt baseline, patients displayed signs of B-cell activation by alteration in subset composition compared to matched controls. Upon closer analysis, patients positive for steroidogenic enzyme autoantibodies had significantly increased frequency of CXCR3+switched memory (SWM) B-cells than autoantibody-negative patients, which correlated positively with anti-17OH and-SCC autoantibody levels. In parallel, increased Th1 cell frequencies were also observed in the same patient group. Longitudinal analyses showed expected B-cell depletion followed by repopulation after RTX. Changes in T-cell subsets were observed at 8 months with reduced frequencies of Th1 and Tfh cells. ConclusionAutoimmune POI displayed altered B- and T-cell subsets implicating cell crosstalk, immune cell infiltration to sites of inflammation such as ovaries, and presence of Th1-driven immunopathogenesis. Key messagesO_LIIncreased CXCR3+SWM B cells and Th1 cells were found in patients with autoimmune POI who were positive for autoantibodies against steroidogenic enzymes. C_LIO_LICXCR3 expression on SWM B cells positively associated with anti-17OH and anti-SCC autoantibody levels. C_LIO_LIRTX induced rapid B-cell depletion followed by repopulation and a delayed T-cell effect at 8 months with reduced Th1 and Tfh cell frequencies. C_LI Capsule summaryRTX has recently been shown to restore fertility in autoimmune POI. Lymphocyte profiling identified Th1 bias accompanied by CXCR3+memory B cells that correlate with autoantibody levels. B-cell depletion appears to temporarily alleviate ovarian autoimmunity and ameliorate physiological function.

Background: Endometriosis is an estrogen-driven, inflammatory disorder affecting ~10% of menstruators, causing severe pain. Current treatments that reduce estrogen or inflammation have inconsistent efficacy and poor tolerability. Therapies identified via drug-repositioning methods, which target patient-specific pathways, offer a promising alternative. Simvastatin and primaquine were identified as potential treatments, strongly reversing endometriosis-associated gene expression pathways, and behavioral testing in an animal model showed that these drugs diminish endometriosis-associated pain. This paper examines their effects in human assembloids to provide proof-of-concept validation and insights into their mechanisms of action relevant to endometriosis. Methods: We established human endometrial assembloids using immortalized epithelial (12Z) and stromal fibroblast (iEc) cell lines, as well as primary tissue from patients. Assembloids were generated in 96-well agarose molds with both cell types. We tested ibuprofen, fenoprofen, primaquine, and simvastatin. Following a 24-hour exposure, assembloids and monolayer cultures were harvested and stranded mRNA-seq libraries were sequenced on an Illumina NovaSeqX Plus System. Results: Gene set enrichment analysis showed key pathway reversals: Primaquine reversed the chemical carcinogenesis ROS pathway in assembloids. Simvastatin reversed cytokine-cytokine receptor interaction in both epithelial and stromal cell lines, and the cytoskeleton in muscle cells pathway in stromal cell lines. Fenoprofen reversed the calcium signaling pathway in stromal cell lines. Conclusion: Simvastatin induced the most significant gene expression changes, notably reversing cytokine-cytokine receptor interactions in both cell lines, mirroring findings from our rat model. Despite the small sample size limitation, these experiments highlight the promise of assembloid models to test therapeutic candidates for endometriosis.

Objective Sodium valproate (SV) is a widely used anti-epileptic drug with well-established reproductive and teratogenic effects, yet its impact on the developing prepubertal reproductive system remains poorly understood. This study investigated the effects of prepubertal SV exposure on gonadal development, including folliculogenesis, testicular architecture and steroidogenic gene expression in male and female mice in vivo. Methods CD1 mouse pups received intraperitoneal injections of saline (control), low or high dose SV (50 or 100 mg/kg, respectively) on postnatal days (PND) 6, 8, and 10. On PND17 the animals were culled and gonads dissected. Ovaries and testes were assessed histologically using haematoxylin and eosin staining. Ovarian follicle number, stage and health were quantified. Testicular tubule morphology and key testicular cell numbers (germ, Sertoli, spermatogonial stem cells, and interstitial/Leydig cells) were evaluated using immunofluorescence with automated image analysis. Expression of key steroidogenic genes (CYP11A1, STAR, CYP19A1 in ovaries; INSL3, STAR, CYP11A1 in testes) was measured by RT-qPCR. Results SV exposure did not significantly affect ovarian follicle number, distribution or health. Testicular morphology and the density of Sertoli, spermatogonial stem cells, and interstitial cells were likewise unaffected. However, a dose-dependent trend toward reduced germ cell density was observed in males at a high dose SV, though this did not reach statistical significance (P = 0.058). Steroidogenic gene expression was unaffected by SV exposure in both ovaries and testes across all treatment groups. Significance This study provides the first in vivo assessment of short-term prepubertal SV exposure on gonadal development in both sexes. The overall findings suggest that brief SV exposure during the prepubertal window does not cause overt gonadotoxicity. Nonetheless, the trend toward reduced testicular germ cell density warrants further investigation with longer exposure durations and functional fertility endpoints, to better inform clinical risk assessment in paediatric patients receiving SV.