Molecular Human Reproduction

BackgroundMitochondrial dysfunction is a leading contributor to the decline in oocyte quality associated with maternal aging. Prior investigations of mitochondrial function in the ovarian follicle have largely treated the mitochondrial pool as a homogeneous population, reporting aggregate values that may obscure biologically meaningful differences between distinct mitochondrial subpopulations. The present study addresses this limitation by characterizing mitochondrial subpopulation dynamics in oocytes and cumulus granulosa cells at single-organelle resolution using fluorescence-activated mitochondria sorting (FAMS). ResultsAnalysis of the aggregate mitochondrial population in mouse oocytes revealed no significant age-associated differences in mitochondrial DNA copy number or membrane potential, a result that would previously have been interpreted as evidence of minimal age-related mitochondrial change. Subpopulation analysis revealed this conclusion to be incomplete: aged oocytes showed significantly elevated mitochondrial DNA copy number specifically within the high membrane potential and small mitochondrial subpopulations, with no significant differences in the low membrane potential or large subpopulations. NMN supplementation normalized mitochondrial DNA copy number in the high membrane potential and small subpopulations toward young levels while producing an opposing effect in large mitochondria, demonstrating subpopulation-specific rather than uniform rejuvenation. In cumulus cells, significant age-associated changes were detectable at the aggregate level, including a reduction in mitochondrial DNA copy number and an elevation in membrane potential, and subpopulation analysis further resolved these findings. The age-associated reduction in cumulus cell mitochondrial DNA copy number was driven predominantly by the high membrane potential subpopulation. NMN supplementation exerted opposing effects on small and large cumulus cell mitochondrial subpopulations, increasing mitochondrial DNA copy number above both young and aged levels in small mitochondria while further reducing it below aged levels in large mitochondria. ConclusionsViewing the mitochondrial pool as a heterogeneous mixture of functionally distinct subpopulations rather than a uniform population reveals age-associated alterations in oocytes and cumulus cells that are undetectable by aggregate analysis. NMN supplementation exerts subpopulation-specific effects in both cell types, identifying specific mitochondrial subtypes as more precise targets for future mechanistic investigation of age-associated infertility than the mitochondrial pool considered in aggregate.

BACKGROUNDDuring spermiogenesis, histones are exchanged by protamines (PRMs) in spermatids, which results in DNA hypercondensation and protection. Rodents and primates express two PRMs (PRM1 and PRM2) in a species-specific ratio. Maintaining this ratio is necessary for functional chromatin reorganization and alteration is associated with sub- or infertility in mice and humans. Prm1 and Prm2 deficient mice are infertile, while Prm1+/- males are subfertile showing a severely altered PRM ratio. Prm2+/- males are fertile and display a protamine ratio comparable to WT. OBJECTIVESHere, we addressed the question whether loss of one allele of Prm1 and one allele of Prm2 affects fertility. MATERIAL AND METHODSDouble heterozygous (dHET) mice lacking one allele of Prm1 and one allele of Prm2 were generated and analyzed RESULTSdHET males were infertile with sperm showing retention of histones and TNPs, high levels of PRM2 precursor and decreased levels of mature PRM2. In mature sperm the PRM ratio and the total PRM content was not altered. However, CMA3 staining revealed incomplete protamination and sperm nuclei appeared more rounded and slightly bigger, suggesting impaired DNA-hypercondensation. In dHET sperm, DNA degradation was apparent, but to a lower level compared to sperm from Prm1 and Prm2 deficient males. Increased 8-OHdG levels suggested oxidative stress in the epididymis of dHET mice. However, a fraction of dHET sperm were capable of fertilization, with embryonic development up to 8-cell stage. DISCUSSION AND CONCLUSIONThese results suggest, that male factor infertility might not be reliably detected by measuring PRM1/PRM2 ratio but rather by determining the level of protamination by e.g. CMA3 analysis and pre-PRM2 retention.

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

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

In vitro fertilization (IVF) is key for genetic improvement programs in bovine. However, embryos produced through IVF have lower developmental competence than those produced under in vivo conditions. Conventional sperm preparation for IVF typically relies on heparin for sperm capacitation but fails to replicate the finely tuned molecular environment of the oviduct, resulting in compromised embryonic competence. Here, we evaluated the effect of HyperBull, a novel capacitation technology, on bovine IVF outcomes using unsorted cryopreserved semen. In a split-sample design, 528 cumulus-oocyte complexes were co-incubated with either control or HyperBull capacitated spermatozoa from the same bull. While overall blastocyst rates were not significantly different between groups (34.21% HyperBull vs. 28.63% control, p=0.148), the proportion of hatched embryos was significantly higher in the HyperBull group (15.82% vs. 9.13%, p=0.016). These findings suggest that modulating capacitation signals prior to insemination enhances embryonic developmental competence, thereby improving readiness for implantation. HyperBull may thus represent a valuable tool to increase the efficiency of IVF programs.

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

ObjectiveTo determine whether quantitative ultrasound (QUS), which characterizes tissue microstructure using radiofrequency data, can identify regional heterogeneity within seminiferous tubules that corresponds to localized spermatogenesis in men with non-obstructive azoospermia (NOA). DesignTwo-cohort study using a biological extremes cohort to establish plausibility of a QUS biomarker, followed by an independent NOA-biopsy cohort with site-matched imaging and tissue sampling. SettingAcademic male fertility referral center. PatientsThe biological extremes cohort included fertile men with presumed intact spermatogenesis (n=15) and men with NOA and subsequent negative microdissection testicular sperm extraction (mTESE; n=10). The NOA-biopsy cohort consisted of 27 men with NOA undergoing site-matched testicular biopsy via testicular sperm aspiration (TESA) or testicular sperm extraction (TESE), yielding 12 sperm-positive and 36 sperm-negative biopsy sites. InterventionsHigh-frequency testicular ultrasound (36 MHz) with acquisition of raw radiofrequency data, allowing objective, quantitative analysis of tissue scattering patterns beyond conventional grayscale imaging. Regions of interest were manually annotated and, in the NOA-biopsy cohort, spatially matched to biopsy locations. Main Outcome MeasuresAssociation between sperm presence at biopsy sites and a pre-specified QUS measure of local tissue heterogeneity: the 75th percentile of a sliding window coefficient of variation map of the Nakagami k-factor within the superficial testicular parenchyma (K_Zone1_CV). This metric reflects the upper range of local variability in ultrasound backscatter, which is influenced by the underlying organization of seminiferous tubules. ResultsIn the biological extremes cohort, K_Zone1_CV distinguished fertile controls (median 1.79, IQR 1.64-1.85) from NOA men with globally negative mTESE (median 1.51, IQR 1.42-1.58; P < 0.001), with an area under the receiver operating characteristic curve (AUC) of 0.91 (95% CI 0.79-1.00). In the independent NOA-biopsy cohort, K_Zone1_CV discriminated sperm-positive from sperm-negative biopsy sites with an AUC of 0.93 (95% CI 0.85-0.99). At a threshold of 1.60, sensitivity was 100%, specificity was 86.1%, positive predictive value was 70.6%, and negative predictive value was 100%. Serum hormone levels, testicular volumes, and biopsy technique did not differ significantly between groups. ConclusionsRegional testicular tissue heterogeneity measured by quantitative ultrasound is associated with localized spermatogenesis in men with NOA. At the selected threshold, no sperm-positive biopsy site was misclassified as negative. These findings support the hypothesis that QUS can noninvasively detect the focal seminiferous tubule heterogeneity that predicts sperm retrieval success. This imaging approach could inform future image-guided sperm retrieval strategies. Further validation in larger cohorts and assessment of intra-patient variability are needed.

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

STUDY QUESTIONAre pathogenic variants in Homeodomain-interacting protein kinase (HIPK4) associated with sperm head abnormalities causing male infertility? SUMMARY ANSWERHIPK4 is a novel candidate gene associated with sperm head defects and human male infertility. WHAT IS KNOWN ALREADYNumerous genes causing male infertility due to Multiple Morphological Abnormalities of the sperm flagella (MMAF) have been described but the genetic basis of sperm head defects is less well understood. STUDY DESIGN, SIZE, DURATIONFour infertile brothers displaying varying degrees of quantitatively and/or qualitatively impaired spermatogenesis, their parents, and their fertile brother were included in the study. Further, the Male Reproductive Genomics (MERGE) cohort comprising exome/genome sequencing data of >3,300 men was queried. PARTICIPANTS/MATERIALS, SETTING, METHODSWe performed exome sequencing in all five brothers and their parents. To characterise the sperm phenotype, standard semen analysis, immunofluorescence staining, and transmission-electron microscopy (TEM) were carried out. Further, we evaluated the impact of the HIPK4 variant in cell culture experiments using HEK293T cells. MAIN RESULTS AND THE ROLE OF CHANCEAnalysing the exome data, we could not identify a common genetic cause in all four affected brothers. However, one of the affected brothers was compound heterozygous for two loss-of-function variants in DNAH17 (c.1076_1077dup p.(Lys360*) and c.7752+2T>A p.?) associated with markedly reduced sperm motility and MMAF. The variants pathogenicity was further validated by TEM of flagellar cross-sections revealing an outer dynein arm defect and axonemal disruption. On the contrary, his three infertile brothers were homozygous for the start-loss variant c.1A>G in HIPK4. This gene is expressed during spermiogenesis and is reportedly involved in sperm head shaping in mice. Heterologous expression of (partial) HIPK4 variant cDNA elucidated the alternative use of an in frame start codon located 35 amino acids downstream, resulting in an N-terminally truncated protein p.(Met1_Glu35del). The truncated HIPK4 protein lacks parts of its kinase domain and shows reduced protein stability. In line with published mouse models, all three brothers displayed 100% abnormal sperm head morphology with variable defects. Importantly, one brother affected by HIPK4 variants fathered a child after successful intracytoplasmic sperm injection demonstrating that it is a treatment option for HIPK4-related teratozoospermia. No further men from the MERGE cohort were affected by biallelic HIPK4 variants. Taken together, HIPK4 is an autosomal-recessive candidate gene associated with sperm head defects and male infertility. LARGE SCALE DATAThe reported variants in DNAH17 and HIPK4 were submitted to ClinVar. LIMITATIONS, REASONS FOR CAUTIONIndependent replication is required to assess the phenotypic spectrum and the reproductive outcome associated with biallelic HIPK4 variants and to formally establish the gene-disease relationship for male infertility. WIDER IMPLICATIONS OF THE FINDINGSThis study raises awareness of the significant genetic heterogeneity of male infertility. The described family highlights that distinct genetic causes may underlie a seemingly similar phenotype. Exome sequencing of families is helpful to efficiently disentangle individual causes among affected family members. STUDY FUNDING/COMPETING INTEREST(S)N.N., J.R., H.O., S.L., C.F., and F.T. were supported by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) within the Clinical Research Unit Male Germ Cells (CRU326, project number 329621271). R.T.W., N.N., J.R., H.O., and F.T. were supported by the Federal Ministry of Research, Technology and Space (BMFTR) as part of the project ReproTrack.MS (grant 01GR2303). S.A.K. was supported by the DFG Clinician Scientist programme CareerS Munster (project number 493624047). A.S.G. was supported by the Medical Faculty Munster via an Innovative Medical Research (IMF) grant (GA-122104).

Boar semen contains spermatozoa and seminal plasma (SP) that carries extracellular vesicles (EVs) among other components. However, artificial insemination (AI) doses produced by AI companies are highly diluted based solely on sperm concentration. The aim of this study was to evaluate the integrity of EVs isolated from AI doses, characterize the protein and miRNA content from high-fertility (HF) and reduced-fertility (RF) boars, and evaluate their functional impact on spermatozoa after dilution by a coincubation up to 24 hours at 38 {degrees}C. Proteomics identified 108 differentially expressed proteins between HF and RF EVs (97 upregulated in HF, 11 in RF), and transcriptomics revealed 80 differentially expressed miRNAs (DEMs) in EVs, 52 in SP, and 3 in spermatozoa, showing inverse expression in various shared DEMs between fertility rates, suggesting compartment-specific regulation. Functional coincubation demonstrated that EVs remain biologically active after dilution. HF EVs improved sperm quality parameters and reduced oxidative stress, while RF EVs increased total and progressive motility. Overall, our findings show that EVs from AI doses retain structural integrity, carry fertility-associated protein and miRNA signatures, and functionally modulate sperm quality in vitro. These features highlight porcine EVs as promising biomarkers and potential tools to optimize reproductive performance in swine production.

BackgroundApproximately one-third of men having undergone gonadotoxic treatment in their childhood experience impaired testicular function for whom autologous transplantation of cryopreserved immature testicular tissue may represent the only opportunity to restore their fertility. Pre-clinical studies have demonstrated successful restoration of spermatogenesis following grafting of immature testicular tissue in various species, including non-human primates. In 2002, our institution pioneered with clinical testicular tissue banking for fertility preservation in boys and adolescents. Over time, this strategy has been increasingly implemented by numerous fertility centres worldwide for patients at high risk of treatment-induced sterility. Here, we report the first human case of autologous transplantation of frozen-thawed immature testicular tissue. PatientIn 2008, testicular tissue was cryopreserved from a pre-pubertal boy diagnosed with sickle cell disease. The procedure was performed after a three-year hydroxyurea treatment and prior to receiving conditioning therapy with busulfan and cyclophosphamide for haematopoietic stem cell transplantation. One testis was surgically removed, sectioned into small fragments, and cryopreserved. Histological analysis confirmed preserved tubular architecture and the presence of spermatogonia. During the period from 2022 to 2024, the patient consistently presented with azoospermia. In December 2024, at the time of transplantation, two abnormal sperm cells were detected after enzymatic digestion. MethodEleven testicular tissue fragments (4-21 mm3) were thawed and autologously grafted to four intra-testicular and four subcutaneous scrotal sites. Over a one-year follow-up period, graft survival, vascularization, hormone profiles, and semen parameters were monitored. One year after transplantation, all grafts were surgically retrieved. ResultsPost-operative recovery was uneventful. No significant changes in endocrine or semen parameters were observed during follow-up. Whereas the intra-testicular grafts exhibited a compact parenchyma that was distinct from the looser surrounding adult parenchyma and remained readily identifiable as graft tissue, the scrotal grafts appeared more fibrotic. Enzymatic digestion of the grafts was required to recover spermatozoa, with one spermatozoon obtained from one of the four intra-testicular grafts. Histological evaluation revealed intact tubular architecture and maturation of somatic cells across all grafts. Spermatogonial stem cells, together with evidence of active spermatogenesis, were identified in two of the four intra-testicular grafts, whereas no germ cells were detected in the subcutaneous scrotal grafts. ConclusionThese findings demonstrate that human immature testicular tissue can survive long-term cryostorage, revascularize after transplantation and establish spermatogenesis in vivo. This study provides essential proof-of-concept for fertility restoration in individuals who banked testicular tissue before puberty. FundingThis study was supported by the Research Programme of FWO Vlaanderen (Research Foundation-Flanders; G0A6U25N) and VUB strategic research program (SRP89). Trial Registration: NCT05414045

Pregnancy results in profound physiological changes driven by dynamic and precisely programmed molecular processes. Maternal peripheral blood is generally the specimen of choice for studying these processes, as it is easily accessible and essential for many aspects of maintaining a healthy pregnancy. Here, we present a high-resolution atlas of the dynamic temporal changes in the transcriptome of maternal peripheral blood in healthy human pregnancy. We generated comprehensive RNA sequencing data in 802 weekly samples from 31 healthy pregnant women from the first trimester until after delivery. Using a strict discovery and replication setup, our longitudinal analysis of gene expression identified 720 genes with robust pregnancy-specific expression patterns. Using weighted graph correlation network analysis, we identified nine pregnancy-associated transcriptional modules that reveal a strong, coordinated enrichment of innate/neutrophil and antiviral immune programs, alongside changes in adaptive immunity (T cell differentiation and signaling), erythropoiesis and hemoglobin metabolism. Cell-type deconvolution revealed that these transcriptomic shifts were accompanied by increased relative neutrophil proportions and reduced naive CD4 and CD8 T cells in pregnancy. We provide a comprehensive characterization of dynamic changes across pregnancy, highlighting maternal blood as a key systemic regulator in healthy gestation. Together, our findings establish a reference atlas of healthy pregnancy, which can be used to identify dysregulated processes and mechanisms in women with pregnancy complications. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=168 SRC="FIGDIR/small/715300v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@2a4b28org.highwire.dtl.DTLVardef@ac49d9org.highwire.dtl.DTLVardef@12468c8org.highwire.dtl.DTLVardef@15b282f_HPS_FORMAT_FIGEXP M_FIG C_FIG O_LI720 genes showed robust pregnancy specific expression patterns. C_LIO_LICo-expression analysis clustered the genes into nine modules with distinct dynamics. C_LIO_LIEnrichment in pathways involved in innate and neutrophil-mediated immunity, antiviral responses, T cell differentiation and signaling, erythropoiesis and hemoglobin metabolism. C_LIO_LICell-type deconvolution showed increases in neutrophils and decreases in naive CD4 and CD8 T cells. C_LIO_LIThe atlas of detailed longitudinal transcriptional changes provides a baseline reference for healthy pregnancy. C_LIO_LIResults for all genes and protein-protein interaction networks are made available for interactive exploration. C_LI

ObjectiveTo 1) determine the expression and distribution of all PDE4 isozymes (A-D) along the length of the anterior urethra, 2) culture fibroblasts and epithelial cells from healthy and strictured urethras, 3) investigate an in vitro model of anterior urethral stricture disease (aUSD), and 4) assess the therapeutic potential of phosphodiesterase-4 (PDE4) inhibitors and testosterone compared to paclitaxel. MethodsThe presence and relative abundance of PDE4 isozymes (A-D) was confirmed using immunohistochemistry on 5 male cadaveric urethras. Human urethral fibroblasts (FBs) were cultured from healthy control urethras of patients undergoing vaginoplasty (n=3) and from idiopathic bulbar urethral strictures (L2S1E2) of patients undergoing urethroplasty (n=3). Epithelial cells (ECs) were cultured from a healthy control urethra and two urethral strictures. To investigate a model of aUSD, Control FBs were stimulated with TGF{beta}1 and compared to Stricture FBs on assays of cell proliferation and expression of genes relevant to aUSD pathophysiology. To test therapeutics, Stricture FBs were treated with the PDE4 inhibitor, roflumilast, testosterone (T), or paclitaxel and compared to Control FBs on the previously mentioned assays and cell viability. ResultsPDE4- A, B, and D were detected along the length of the urethra. Expression levels did not differ between urethral regions. TGF{beta}1 altered proliferation and gene expression in a dose-dependent manner. Roflumilast and T preserved cell viability and proliferation and decreased expression of genes positively associated with auSD. ConclusionUrethral FBs and ECs can be cultured from healthy and strictured surgical specimens, enabling in vitro research. PDE4 inhibitors and T may be non-cytotoxic alternatives or additions to paclitaxel for aUSD. HighlightsO_LIPDE4 isozymes A, B, and D are expressed in adult anterior urethras C_LIO_LIPDE4 is expressed equally from proximal bulbar to meatal urethra C_LIO_LIEpithelial cells and fibroblasts can be cultured from healthy and stricture urethra C_LIO_LITGF{beta}1 may not be an optimal method to model aUSD in vitro C_LIO_LIUnlike paclitaxel, roflumilast and testosterone are not toxic to urethral cells C_LI

BackgroundPolycystic ovary syndrome (PCOS) is a prevalent endocrine disorder and a leading cause of female infertility, with complex genetic, metabolic, and hormonal etiologies. Long non-coding RNAs (lncRNAs) have emerged as important regulators of diverse biological processes, yet their roles in PCOS remain underexplored. Here, we identified and characterized PCOS differentially expressed gene-associated lncRNAs (PDEGAL) with an integrative approach combining expression data, genetic association, and evolutionary analysis. MethodsThirty-three PCOS-associated protein-coding genes were obtained from our prior study, and all their nearby and overlapping lncRNAs were annotated. These candidates were analyzed using UCSC Genome Browser-mapped annotations and datasets, including NCBI RefSeq, GENCODE, GTEx, GWAS SNPs, and conservation, as well as the FANTOM5 cap analysis of gene expression (CAGE) promoter data, to assess their expression, regulatory potential, genetic variant overlaps, and evolutionary conservation. ResultsTwenty-three PDEGALs (18 antisense to, and 5 sharing bidirectional promoters with, known PCOS-associated protein-coding genes) were identified. 17 PDEGALs contained GWAS SNPs with statistically significant disease associations, 9 of which were associated with PCOS-related traits. 5 PDEGALs demonstrated expression in the KGN granulosa cell model of PCOS. Key gene structure element (KGSE) analysis revealed that most PDEGALs are primate-specific. Integrating four criteria--GTEx expression, GWAS SNPs, FANTOM promoterome, and KGSE conservation--highlighted HELLPAR as the only lncRNA fulfilling all four, while five others--PGR-AS1, MTOR-AS1, ENSG00000265179, ENSG00000256218, and LOC105377276--fulfilled three of the four criteria. ConclusionsWe have systematically identified candidate PCOS regulatory lncRNAs with convergent genetic, expression, and evolutionary evidence. These results provide a framework for functional validation and highlight lncRNAs as potential biomarkers and therapeutic targets in PCOS that function by regulating their nearby and overlapping protein-coding genes.

Chronically stressing male rodents can induce stress-specific epigenetic changes in sperm that contribute to altered offspring phenotypes. Whether similar phenomena occur in men is unclear. This study addresses this knowledge gap by analyzing sperm microRNAs (miRNAs) from 51 men exposed to various levels of adult trauma including crime, disaster, and physical or sexual violence, quantified by the Trauma History Questionnaire (THQ), a measure of risk for Post-Traumatic Stress Disorder (PTSD). Four sperm miRNAs, miR-532-3p, 491-5p, 375-3p and 361-3p correlated positively with mens THQ scores, showing 4X to 130X over expression in sperm from the most highly traumatized men. These changes were independent of mens adverse childhood experiences (ACEs), which we previously linked to decreased miR-34/449 in their sperm; and sperm miR-34/449 levels were not associated with THQ scores. Injecting these 4 miRNAs into fertilized mouse oocytes at levels comparable to those found in men reporting high THQ scores yielded offspring with elevated anxiety-and depression-like phenotypes. This finding differs from the stress related phenotypes we observed in offspring of mice fertilized by sperm with reduced levels of miR-34/449. Consistent with only a small subset of men with high THQ scores developing PTSD, we observed no statistically significant increase in overall anxiety or depression among this highly traumatized group, however there were indications of increased sleeplessness, appetite and concentration difficulties and negative self-concept among this group. Nevertheless, almost all men reporting high THQ scores had elevated levels of all 4 of these miRNAs in their sperm, suggesting these trauma-induced epigenetic changes may raise mental health risks in the offspring of men with only subtle mental health problems. Since [~]20 % of men report either THQ or ACE scores in the ranges linked here and in our earlier study to changes in sperm miRNAs that in mice lead to elevated levels of stress-related behaviors, a large human population with an elevated risk of transmitting stress-related traits to their offspring likely exists.

Background: Male factor infertility is present in around 40% of couples utilising assisted reproductive technology (ART). However, it is unclear how specific causes of male infertility impact the chance of successful ART treatment, with most research either treating male infertility as single diagnostic group or being isolated smaller-scale studies focused on the treatment and outcomes of specific diagnoses. Objective: To study the impact of eleven specific aetiologies of male infertility on the chance of a clinical pregnancy in couples with known causes of male or female infertility following their first ART cycle. Material and methods: Population-based (initiated ART in Australia and New Zealand in 2020- 2022) cohort study assessing the impact of eleven male infertility diagnoses (idiopathic, Klinefelter syndrome, Y chromosome microdeletions, testis damage from cancer, testis damage from other causes, gonadotropin deficiency, congenital absence of the vas deferens/cystic fibrosis (CBAVD), other obstruction disorder, erectile dysfunction, and ejaculatory disorder) on the chance of a clinical pregnancy following a couple's first complete ART cycle (all fresh and frozen-thawed embryo transfers arising from one episode of ovarian stimulation). Adjusted risk ratios comparing a couples undergoing ART solely for treatment of male infertility with couples undergoing ART solely for treatment of tubal disease were calculated for the chance of a clinical pregnancy following a complete ART cycle and following an attempted fertilisation procedure. Results: A total of 39,053 couples were included, with male infertility present in 42.7% of cases, and the only cause of infertility in just under half of these cases. In more than three-quarters of male infertility cases the cause of infertility was unknown (idiopathic) or undiagnosed. Most couples undertaking ART for treatment of male infertility can expect similar success rates to couples seeking treatment for good prognosis female infertility diagnoses. However, those with Klinefelter syndrome and Y chromosome microdeletions had a 59.5% (aRR: 40.5% [95% CI: 16.9%-64.1%]) and 28.9% (aRR: 71.1% [95% CI: 45.5%-96.7%]) lower chance of a clinical pregnancy per initiated stimulation cycle compared to those with tubal disease as the only source of infertility. However, there was no difference once sperm was retrieved compared to other diagnoses tending to require surgical sperm retrieval, use frozen oocytes and necessitating ICSI. Discussion and Conclusion: In this population-based study most couples undergoing ART because of male infertility had similar success rates to those undergoing ART for treatment of female tubal disease, except for patients with Klinefelter syndrome and Y chromosome microdeletion who had approximately half and three-quarters the chance of a clinical pregnancy due to failed sperm retrieval/survival, but no difference (accounting for the use of surgical sperm, ICSI and potentially frozen oocytes) in outcomes once sperm were available. While these finding are reassuring for most men presenting to an ART clinic with male infertility, with more than three-quarters of male infertility cases reported as being idiopathic, there is an urgent need for greater research on the causes, diagnosis and implications of male infertility.

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

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

Primary ovarian insufficiency (POI) and related infertility, early menopause, and endocrine disorders due to hormonal deficiency are major side effects in young female cancer patients undergoing cancer therapy. Current strategies preserving the fertility and hormonal functions of the ovary remain imperfect due to concerns of feasibility, efficacy, or safety. Herein, we identified c-Jun N-terminal kinase (JNK) as a pivotal regulator of the DNA damage response (DDR) signaling in oocytes of primordial follicles in response to DNA-damaging cancer therapy. Using pharmacological JNK inhibition and a genetically modified mouse model with oocyte-specific JNK deletion, together with histological, bioinformatic, and molecular approaches, we demonstrated that JNK inhibition prevented chemotherapy-induced oocyte apoptosis and POI, and preserved long-term reproductive cycles and fertility. Mechanistically, JNK was activated in response to chemotherapy-induced DNA damage in oocytes of primordial follicles, causing activation of transcription factor TAp63 and subsequent oocyte apoptosis, ultimately resulting in diminished ovarian reserve and POI. A more clinically relevant breast cancer-bearing mouse model revealed that JNK inhibition preserved the ovarian reserve without compromising anti-cancer efficacy of chemotherapy. Together, our study identifies oocyte-intrinsic JNK as a promising target for developing ovarian protectants and safeguarding reproductive health and fertility in young female cancer survivors.

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