Preconception Blood Pressure and Adverse Pregnancy Outcomes: A Population-Based Cohort Study.

BACKGROUND: Adverse pregnancy outcomes (APOs) share clinical features and risk factors with cardiovascular disease and there is an increasing prevalence of hypertension among reproductive women. However, the associations between maternal preconception blood pressure (BP) and APOs remain controversial and inconclusive. METHODS: This population-based cohort study used data of 567 127 mother-neonate-father triads from the National Free Preconception Checkup Project in Guangdong Province, China. Maternal BP levels within 1 year before pregnancy were classified using the American College of Obstetricians and Gynecologists definition of hypertension. The primary outcome was a composite of APOs, including preterm birth, small for gestational age, and perinatal infant death. Log-binomial and marginal structural binomial regressions were employed to estimate adjusted risk ratios and absolute risk differences, respectively. RESULTS: Compared with women with normal BP, women with elevated BP (adjusted risk ratio, 1.07 [95% CI, 1.05-1.09]; absolute risk difference, 1.03% [95% CI, 0.72%-1.29%]), hypertension (adjusted risk ratio, 1.25 [95% CI, 1.18-1.32]; and absolute risk difference, 3.42% [95% CI, 1.97%-5.42%]) had a higher risk of a composite of APOs. Compared with women with normal BP, women with elevated BP and hypertension had higher risks of multiple APOs, preterm birth, small for gestational age, and perinatal infant death. However, these associations attenuated with increasing duration of pregnancy preparation and were not statistically significant beyond 90 days of pregnancy preparation. CONCLUSIONS: Women with elevated BP or hypertension before pregnancy were associated with an increased risk of APOs. Preconception hypertension screening and control among women should not be ignored by policymakers, clinicians, and the general population.

Association of maternal preconception blood pressure with preterm birth: a population-based cohort study.

The association between maternal preconception blood pressure (BP) and preterm birth (PTB) is still unclear. The purpose of this study was to investigate the association between maternal preconception BP and PTB. This population-based cohort study included 715 984 Chinese women aged 20-49 years who participated in the National Free Preconception Health Examination Project and successfully had a singleton livebirth during 2014-2019 in Guangdong Province, China. Maternal preconception BP were measured by trained health workers. Multivariate logistic regression models and restricted cubic spline regressions were used to examine the association and dose-response relationship between maternal preconception BP and PTB, respectively. Maternal preconception hypertension was associated with the increased risk of PTB (adjusted odds ratios (aOR): 1.24; 95% CI: 1.14-1.34). Compared to women with normal preconception BP, the aORs for PTB were 1.09 (95% CI: 1.06-1.12), 1.24 (95% CI: 1.13-1.36), and 1.43 (95% CI: 1.15-1.79) for women with preconception elevated BP (120-139/ 80-89 mmHg, stage-1 hypertension (140-159/ 90-99 mmHg, and stage-2 hypertension (160-179/100-109 mmHg), respectively. According to the 2017 American College of Cardiology/American Heart Association criteria, maternal preconception elevated BP and hypertension were also significantly associated with an increased risk of PTB. Preconception systolic and diastolic BP showed a U-shaped (χ2 = 40.54; nonlinear P < 0.001) and linear (χ2 = 6.62; nonlinear P = 0.085) dose-response relationship with PTB, respectively. The association was modified by maternal age and preconception body mass index. These findings identify maternal preconception elevated BP and hypertension as a modifiable risk factor for PTB, providing evidence for future research studies, public health and clinical interventions.

Deficiency in AK9 causes asthenozoospermia and male infertility by destabilising sperm nucleotide homeostasis.

BACKGROUND: Asthenozoospermia is the primary cause of male infertility; however, its genetic aetiology remains poorly understood. Adenylate kinase 9 (AK9) is highly expressed in the testes of humans and mice and encodes a type of adenosine kinase that is functionally involved in cellular nucleotide homeostasis and energy metabolism. We aimed to assess whether AK9 is involved in asthenozoospermia. METHODS: One-hundred-and-sixty-five Chinese men with idiopathic asthenozoospermia were recruited. Whole-exome sequencing (WES) and Sanger sequencing were performed for genetic analyses. Papanicolaou staining, Haematoxylin and eosin staining, scanning electron microscopy, and transmission electron microscopy were used to observe the sperm morphology and structure. Ak9-knockout mice were generated using CRISPR-Cas9. Sperm adenosine was detected by liquid chromatography-mass spectrometry. Targeted sperm metabolomics was performed. Intracytoplasmic sperm injection (ICSI) was used to treat patients. FINDINGS: We identified five patients harbouring bi-allelic AK9 mutations. Spermatozoa from men harbouring bi-allelic AK9 mutations have a decreased ability to sustain nucleotide homeostasis. Moreover, bi-allelic AK9 mutations inhibit glycolysis in sperm. Ak9-knockout male mice also presented similar phenotypes of asthenozoospermia. Interestingly, ICSI was effective in bi-allelic AK9 mutant patients in achieving good pregnancy outcomes. INTERPRETATION: Defects in AK9 induce asthenozoospermia with defects in nucleotide homeostasis and energy metabolism. This sterile phenotype could be rescued by ICSI. FUNDING: The National Natural Science Foundation of China (82071697), Medical Innovation Project of Fujian Province (2020-CXB-051), open project of the NHC Key Laboratory of Male Reproduction and Genetics in Guangzhou (KF202004), Medical Research Foundation of Guangdong Province (A2021269), Guangdong Provincial Reproductive Science Institute Innovation Team grants (C-03), and Outstanding Young Talents Program of Capital Medical University (B2205).

Prevalence of preconception TORCH infections and its influential factors: evidence from over 2 million women with fertility desire in southern China.

BACKGROUND: TORCH (Toxoplasma gondii [TOX], Cytomegalovirus [CMV], Rubella virus [RV], and Herpes simplex virus [HSV]) represents pathogens known to traverse the maternal-fetal barrier and cause severe neonatal anomalies. We aimed to assess the prevalence of preconception TOX, CMV, and RV infections among women with fertility desire in southern China, and identify related risk factors. METHODS: Data were obtained from a population-based cross-sectional study conducted as part of the National Free Preconception Health Examination Project. Women planning to conceive within the next 6 months in Guangdong Province were enrolled between 2014 and 2019. Information on sociodemographic, gynecological, and obstetric characteristics was collected. Sera were analyzed for TOX IgG, CMV IgG, and RV IgG antibodies using an enzyme-linked immunosorbent assay. Descriptive, univariate, and multivariate logistic regression analyses were performed to assess the association between TORCH infections and related factors. RESULTS: Among 2,409,137 participants, the prevalence of IgG antibodies for TOX, CMV, and RV was 3.20% (95% CI: 3.18-3.22%), 77.67% (95% CI: 77.62-77.71%) and 76.03% (95% CI: 75.98-76.07%), respectively. Of all participants, 141,047 women (5.85%, 95% CI:5.83-5.88%) reported a history of immunization for RV. Women living in the Pearl River Delta, a more developed region, have significantly lower vaccination rates than those living in other regions. The seropositivity of TOX IgG was highest among women aged 35 years and above, with primary or lower education levels, and rural registration. Factors such as being older, having a higher educational level, and being of other ethnicities were associated with a higher prevalence of naturally acquired CMV and RV infections. Women living in the Pearl River Delta showed a higher risk of TOX, CMV, and RV infections, with aORs of 2.21, 4.45, and 1.76, respectively. A history of pregnancy, gynecological diseases, and sexually transmitted infections were potentially associated with TORCH infections, but this association varied across pathogens. CONCLUSION: The findings of this study update the baseline of preconception TORCH infections among women with fertility desire in southern China, helping to estimate the risk of congenital infection and guide the development and implementation of effective prevention measures for preconception TORCH infections.

Maternal NAT10 orchestrates oocyte meiotic cell-cycle progression and maturation in mice.

In mammals, the production of mature oocytes necessitates rigorous regulation of the discontinuous meiotic cell-cycle progression at both the transcriptional and post-transcriptional levels. However, the factors underlying this sophisticated but explicit process remain largely unclear. Here we characterize the function of N-acetyltransferase 10 (Nat10), a writer for N4-acetylcytidine (ac4C) on RNA molecules, in mouse oocyte development. We provide genetic evidence that Nat10 is essential for oocyte meiotic prophase I progression, oocyte growth and maturation by sculpting the maternal transcriptome through timely degradation of poly(A) tail mRNAs. This is achieved through the ac4C deposition on the key CCR4-NOT complex transcripts. Importantly, we devise a method for examining the poly(A) tail length (PAT), termed Hairpin Adaptor-poly(A) tail length (HA-PAT), which outperforms conventional methods in terms of cost, sensitivity, and efficiency. In summary, these findings provide genetic evidence that unveils the indispensable role of maternal Nat10 in oocyte development.

Single-cell RNA sequencing reveals the local cell landscape in mouse epididymal initial segment during aging.

BACKGROUND: Morphological and functional alterations in aging reproductive organs result in decreased male fertility. The epididymis functions as the transition region for post-testicular sperm maturation. And we have previously demonstrated that the epididymal initial segment (IS), a region of the reproductive tract essential for sperm maturation and capacitation, undergoes considerable histological changes and chronic immune activation in mice during aging. However, the local aging-associated cellular and molecular changes in the aged epididymal IS are poorly understood. RESULTS: We conducted single-cell RNA sequencing analysis on the epididymal IS of young (3-month-old) and old (21-month-old) mice. In total, 10,027 cells from the epididymal IS tissues of young and old mice were obtained and annotated. The cell composition, including the expansion of a principal cell subtype and Ms4a4bHiMs4a6bHi T cells, changed with age. Aged principal cells displayed multiple functional gene expression changes associated with acrosome reaction and sperm maturation, suggesting an asynchronous process of sperm activation and maturation during epididymal transit. Meanwhile, aging-related altered pathways in immune cells, especially the "cell chemotaxis" in Cx3cr1Hi epididymal dendritic cells (eDCs), were identified. The monocyte-specific expression of chemokine Ccl8 increased with age in eDCs. And the aged epididymal IS showed increased inflammatory cell infiltration and cytokine secretion. Furthermore, cell-cell communication analysis indicated that age increased inflammatory signaling in the epididymal IS. CONCLUSION: Contrary to the general pattern of lower immune responses in the male proximal genital tract, we revealed an inflammaging status in mouse epididymal initial segment. These findings will allow future studies to enable the delay of male reproductive aging via immune regulation.

Preconception syphilis seroprevalence and association with duration of marriage and age among married individuals in Guangdong Province, China: A population-based cross-sectional study.

BACKGROUND: Duration of marriage (DoM) and age are important characteristics of married individuals, who are the critical population for eliminating mother-to-child transmission (MTCT) of syphilis. A deep understanding of the preconception syphilis seroprevalence (PSS) and its distribution among this population may be able to help to eliminate MTCT. However, few population-based epidemiological studies have been focused on this group, and the association of DoM and age with PSS remains unclear. METHODOLOGY/PRINCIPAL FINDINGS: This study used data from 4,826,214 married individuals aged 21-49 years who participated in the National Free Preconception Health Examination Project in Guangdong Province, China, between 2014 and 2019. Syphilis was screened using the rapid plasma reagin (RPR) test. The seroprevalence time series, seroprevalence map, and hot spot analysis (HSA) were employed to visualize the spatiotemporal distribution. The restricted cubic spline (RCS) based on multivariate logistic regression was used to model the association of DoM and age with PSS. The interactions on the additive scale of DoM and age were also assessed. The PSS was 266.61 per 100,000 persons (95% CI: 262.03-271.24) and the burden was higher in economically underdeveloped area within the province. A strong J-shaped non-linearity association was observed between age and PSS. Specifically, the risk of seropositivity was relatively flat until 27 years of age among men and increased rapidly afterwards, with an adjusted odds ratio (aOR) of 1.13 (95% CI: 1.12-1.13) per unit. Among women, the risk of seropositivity was relatively flat until 25 years of age and increased rapidly afterwards with an aOR of 1.08 (95% CI: 1.08-1.09) per unit. DoM was negatively associated with PSS among married individuals. Moreover, the combined effects of age and DoM appeared to be synergistic. CONCLUSIONS/SIGNIFICANCE: Our findings suggest that attention should be paid to preventing syphilis in underdeveloped areas and that syphilis screening in newly married individuals who are in their late 20s or older should be recommended. Additionally, early syphilis prevention strategies should be implemented among young people as early as possible.

Novel mutations of PMFBP1 in a man with acephalic spermatozoa defects.

BACKGROUND: Acephalic spermatozoa (AS) is a serious but rare reproductive genetic disorder that causes infertility in men. To date, only a few genes associated with AS defects have been identified, including the polyamine modulated factor 1 binding protein 1 (PMFBP1) gene. Consistent with this, PMFBP1 localizes to the head-neck connection, which bridges the implantation fossa and basal body. METHODS: A male patient was diagnosed as having an AS defect. Blood samples from all family members and a sample of the patient's semen were collected to determine the genetic causes of his infertility. RESULTS: Compound heterozygote mutation in the PMFBP1 gene, which is associated with AS defects in the present case: two loss-of-function mutations, with one a nonsense mutation c.361C > T p.Gln121Ter, and another a splice donor mutation c.414 + 1G > T. The current study, together with previous studies, suggests that the nonsense mutation is responsible for a truncated PMFBP1 protein during its formation; a splice donor mutation c.414 + 1G > T might lead to new open reading frames, from which the dysfunction of an abnormal PMFBP1 protein might be predicted. Additionally, the expression of outer dense fiber 1 (ODF1) and ODF2 proteins has been experimentally shown to be regulated by the truncated PMFBP1 protein. CONCLUSION: We herein present a case with AS defects associated with heterozygote mutations of PMFBP1, which have been shown to be rare and pathogenic; the association with an AS defect is a monogenic disorder with a recessive inherited pattern in the patient's family.

Comprehensive Analysis of circRNAs, miRNAs, and mRNAs Expression Profiles and ceRNA Networks in Decidua of Unexplained Recurrent Spontaneous Abortion.

The diagnosis and treatment of unexplained recurrent spontaneous abortion (URSA) are subject to debate, because the exact underlying mechanisms remain unclear. To address this issue, we elucidated the expression profiles of dysregulated circRNAs, miRNAs, and mRNAs and constructed circRNA-associated competitive endogenous RNA (ceRNA) networks by comparing the decidua of URSA with that of normal early pregnancy (NEP) using RNA-sequencing. In total, 550 mRNAs, 88 miRNAs, and 139 circRNAs were differentially expressed (DE) in decidua of URSA. Functional annotation revealed that DE mRNAs as well as potential target genes of DE miRNAs and DE circRNAs are mainly involved in immunologic function, such as antigen processing and presentation, allograft rejection, and T cell receptor signaling pathway. In addition, the top hub genes, including CCL4, DDX58, CXCL10, CXCL9, MX1, CD44, RPS2, SOCS3, RPS3A, and CXCL11, were identified. The mRNAs involved in ceRNA network were enriched in complement and coagulation cascades and protein processing in the endoplasmic reticulum. We found that circRNAs in the ceRNA network, which acted as decoys for hsa-miR-204-5p, were positively correlated with MFGE8 expression. Collectively, the results demonstrated that circRNAs, miRNAs, and mRNAs were aberrantly expressed in the decidua of patients with URSA and played a potential role in the development of URSA. Thus, the establishment of the ceRNA network may profoundly affect the diagnosis and therapy of URSA in the future.

WDFY1, a WD40 repeat protein, is not essential for spermatogenesis and male fertility in mice.

The mouse WD repeat and FYVE domain containing 1 (Wdfy1) gene is located in chromosome 1qC4 and spans over 73.7 kilobases. It encodes a protein of 410-amino acid protein that shares 97.8% amino acid sequence identity with the human WDFY1 protein. However, the expression pattern of WDFY1 in reproductive organs and its function in male fertility remain unknown. In this study, we generated transgenic mice expressing FLAG-Wdfy1-mCherry cDNA driven by the Wdfy1 promoter to clarify the expression of WDFY1. The results showed that WDFY1 is highly expressed in mouse testes and located in the cytoplasm of late pachytene spermatocytes to elongated spermatids. Interestingly, the global Wdfy1 knockout (KO) male mice displayed normal growth, development, and fertility. Further histological analysis of Wdfy1 knockout mouse testes revealed that all spermatogenic cells are present in Wdfy1 KO seminiferous tubules. Together, our data demonstrate that WDFY1 is dispensable for mouse spermatogenesis and male fertility.

Homozygous mutation in DNALI1 leads to asthenoteratozoospermia by affecting the inner dynein arms.

Asthenozoospermia is the most common cause of male infertility. Dynein protein arms play a crucial role in the motility of sperm flagella and defects in these proteins generally impair the axoneme structure and affect sperm flagella function. In this study, we performed whole exome sequencing for a cohort of 126 infertile patients with asthenozoospermia and identified homozygous DNALI1 mutation in one patient from a consanguineous family. This identified homozygous mutation was verified by Sanger sequencing. In silico analysis showed that this homozygous mutation is very rare, highly pathogenic, and very conserved. Sperm routine analysis confirmed that the motility of the spermatozoa from the patient significantly decreased. Further sperm morphology analysis showed that the spermatozoa from the patient exhibited multiple flagella morphological defects and a specific loss in the inner dynein arms. Fortunately, the patient was able to have his child via intracytoplasmic sperm injection treatment. Our study is the first to demonstrate that homozygous DNALI1 mutation may impair the integration of axoneme structure, affect sperm motility and cause asthenoteratozoospermia in human beings.

Tex13a Optimizes Sperm Motility via Its Potential Roles in mRNA Turnover.

mRNAs have been found to undergo substantial selective degradation during the late stages of spermiogenesis. However, the mechanisms regulating this biological process are unknown. In this report, we have identified Tex13a, a spermatid-specific gene that interacts with the CCR4-NOT complex and is implicated in the targeted degradation of mRNAs encoding particular structural components of sperm. Deletion of Tex13a led to a delayed decay of these mRNAs, lowered the levels of house-keeping genes, and ultimately lowered several key parameters associated with the control of sperm motility, such as the path velocity (VAP, average path velocity), track speed (VCL, velocity curvilinear), and rapid progression.

Bioprinting of a Blue Light-Cross-Linked Biodegradable Hydrogel Encapsulating Amniotic Mesenchymal Stem Cells for Intrauterine Adhesion Prevention.

Intrauterine adhesion (IUA) is a common and prevailing complication after uterine surgery, which can lead to clinical symptoms such as a low menstrual volume, amenorrhea, periodic lower abdominal pain, infertility, and so on. Placing a three-dimensional printing hydrogel between the injured site and the adjacent tissue is considered to be a physical barrier to prevent adhesion, which can isolate the damaged area during the healing process. In this work, a tissue hydrogel with various proportions of a methacrylated gelatin (GelMA) and methacrylated collagen (ColMA) composite hydrogel loaded with amniotic mesenchymal stem cells (AMSCs) was constructed by using three-dimensional biological printing technology. Compared with the single GelMA hydrogel, the composite antiadhesion hydrogel (GelMA/ColMA) showed an appropriate swelling ratio, enhanced mechanical properties, and impressive stability. Meanwhile, the microstructure of the GelMA/ColMA composite hydrogel showed a denser and interconnected microporous structure. In addition, the cytotoxicity study indicated that the GelMA/ColMA hydrogel has a cytocompatibility nature toward AMSCs. Finally, the fabrication of stem cell encapsulation hydrogels was studied, and the cells could be released continuously for more than 7 days with the normal cell function. The results of in vivo experiments indicated that the GelMA/ColMA/hAMSC (human amnion mesenchymal stem cell) hydrogel can prevent cavity adhesion in a rat IUA model. Therefore, bioprinting a biodegradable hydrogel cross-linked by blue light has satisfactory anticavity adhesion effects with excellent physical properties and biocompatibility, which could be used as a preventive barrier for intrauterine adhesion.

Towards Post-Meiotic Sperm Production: Genetic Insight into Human Infertility from Mouse Models.

Declined quality and quantity of sperm is currently the major cause of patients suffering from infertility. Male germ cell development is spatiotemporally regulated throughout the whole developmental process. While it has been known that exogenous factors, such as environmental exposure, diet and lifestyle, et al, play causative roles in male infertility, recent progress has revealed abundant genetic mutations tightly associated with defective male germline development. In mammals, male germ cells undergo dramatic morphological change (i.e., nuclear condensation) and chromatin remodeling during post-meiotic haploid germline development, a process termed spermiogenesis; However, the molecular machinery players and functional mechanisms have yet to be identified. To date, accumulated evidence suggests that disruption in any step of haploid germline development is likely manifested as fertility issues with low sperm count, poor sperm motility, aberrant sperm morphology or combined. With the continually declined cost of next-generation sequencing and recent progress of CRISPR/Cas9 technology, growing studies have revealed a vast number of disease-causing genetic variants associated with spermiogenic defects in both mice and humans, along with mechanistic insights partially attained and validated through genetically engineered mouse models (GEMMs). In this review, we mainly summarize genes that are functional at post-meiotic stage. Identification and characterization of deleterious genetic variants should aid in our understanding of germline development, and thereby further improve the diagnosis and treatment of male infertility.

Baking of methionine-choline deficient diet aggravates testis injury in mice.

Dietary pattern and cooking methods are important factors to determine the nutrients supplementation for male reproduction. Methionine and choline are two methyl donors in daily diet, which could mediate the lipid metabolism, but their effects on the sperms are not clear. In this study, we fed the mice with methionine-choline deficient (MCD) diet or the baked MCD diet for 6 weeks to evaluate this dietary pattern and the appended high temperature cooking on the spermatogenesis. The results have shown that MCD diet induced testis degradation and the damage of spermatocytes, reduced sperm vitality, motility, but elevated sperm deformity. Additionally, baking of MCD diet aggravated the testis injury, further reduced sperm density, sperm motility, and decreased normal sperm morphology dramatically. These changes were not related to the blood-testis barrier nor the Leydig cells dysfunction, but related to spermatocytes lost and apoptosis. The spermatocyte apoptosis was mediated by reticulum stress, including GRP78, XBP-1 and CHOP gene expression. Our study has shown the importance of methionine and choline in diet, and emphasized the crucial role of cooking condition, which are dietary factors to influence the quality of sperms.

Novel Gene Regulation in Normal and Abnormal Spermatogenesis.

Spermatogenesis is a complex and dynamic process which is precisely controlledby genetic and epigenetic factors. With the development of new technologies (e.g., single-cell RNA sequencing), increasingly more regulatory genes related to spermatogenesis have been identified. In this review, we address the roles and mechanisms of novel genes in regulating the normal and abnormal spermatogenesis. Specifically, we discussed the functions and signaling pathways of key new genes in mediating the proliferation, differentiation, and apoptosis of rodent and human spermatogonial stem cells (SSCs), as well as in controlling the meiosis of spermatocytes and other germ cells. Additionally, we summarized the gene regulation in the abnormal testicular microenvironment or the niche by Sertoli cells, peritubular myoid cells, and Leydig cells. Finally, we pointed out the future directions for investigating the molecular mechanisms underlying human spermatogenesis. This review could offer novel insights into genetic regulation in the normal and abnormal spermatogenesis, and it provides new molecular targets for gene therapy of male infertility.

Triptonide is a reversible non-hormonal male contraceptive agent in mice and non-human primates.

There are no non-hormonal male contraceptives currently on the market despite decades of efforts toward the development of "male pills". Here, we report that triptonide, a natural compound purified from the Chinese herb Tripterygium Wilfordii Hook F displays reversible male contraceptive effects in both mice and monkeys. Single daily oral doses of triptonide induces deformed sperm with minimal or no forward motility (close to 100% penetrance) and consequently male infertility in 3-4 and 5-6 weeks in mice and cynomolgus monkeys, respectively. Male fertility is regained in ~4-6 weeks after cessation of triptonide intake in both species. Either short- or long-term triptonide treatment causes no discernable systematic toxic side effects based on histological examination of vital organs in mice and hematological and serum biochemical analyses in monkeys. Triptonide appears to target junction plakoglobin and disrupts its interactions with SPEM1 during spermiogenesis. Our data further prove that targeting late spermiogenesis represents an effective strategy for developing non-hormonal male contraceptives.

Epigenetic Regulation of Spermatogonial Stem Cell Homeostasis: From DNA Methylation to Histone Modification.

Spermatogonial stem cells(SSCs)are the ultimate germline stem cells with the potential of self-renewal and differentiation, and a dynamic balance of SSCs play an essential role in spermatogenesis. During the gene expression process, genomic DNA and nuclear protein, working together, contribute to SSC homeostasis. Recently, emerging studies have shown that epigenome-related molecules such as chromatin modifiers play an important role in SSC homeostasis through regulating target gene expression. Here, we focus on two types of epigenetic events, including DNA methylation and histone modification, and summarize their function in SSC homeostasis. Understanding the molecular mechanism during SSC homeostasis will promote the recognition of epigenetic biomarkers in male infertility, and bring light into therapies of infertile patients.Graphical Abstract.

High Throughput scRNA-Seq Provides Insights Into Leydig Cell Senescence Induced by Experimental Autoimmune Orchitis: A Prominent Role of Interstitial Fibrosis and Complement Activation.

Leydig cells (Lc), located in the interstitial space of the testis between seminiferous tubules, produce 95% of testosterone in male individuals, which is pivotal for male sexual differentiation, spermatogenesis, and maintenance of the male secondary sex characteristics. Lc are prone to senescence in aging testes, resulting in compromised androgen synthesis capability upon aging. However, little is known about whether Lc undergo senescence in a chronic inflammatory environment. To investigate this question, mouse models of experimental autoimmune orchitis (EAO) were used, and Lc were analyzed by high throughput scRNA-Seq. Data were screened and analyzed by correlating signaling pathways with senescence, apoptosis, androgen synthesis, and cytokine/chemokine signaling pathways. EAO did induce Lc senescence, and Lc senescence in turn antagonized androgen synthesis. Based on the correlation screening of pathways inducing Lc senescence, a plethora of pathways were found to play potential roles in triggering Lc senescence during EAO, among which the Arf6 and angiopoietin receptor pathways were highly correlated with senescence signature. Notably, complement and interstitial fibrosis activated by EAO worsened Lc senescence and strongly antagonized androgen synthesis. Furthermore, most proinflammatory cytokines enhanced both senescence and apoptosis in Lc and spermatogonia (Sg) during EAO, and proinflammatory cytokine antagonism of the glutathione metabolism pathway may be key in inducing cellular senescence during EAO.