The cycling and aging mouse female reproductive tract at single-cell resolution.

The female reproductive tract (FRT) undergoes extensive remodeling during reproductive cycling. This recurrent remodeling and how it shapes organ-specific aging remains poorly explored. Using single-cell and spatial transcriptomics, we systematically characterized morphological and gene expression changes occurring in ovary, oviduct, uterus, cervix, and vagina at each phase of the mouse estrous cycle, during decidualization, and into aging. These analyses reveal that fibroblasts play central-and highly organ-specific-roles in FRT remodeling by orchestrating extracellular matrix (ECM) reorganization and inflammation. Our results suggest a model wherein recurrent FRT remodeling over reproductive lifespan drives the gradual, age-related development of fibrosis and chronic inflammation. This hypothesis was directly tested using chemical ablation of cycling, which reduced fibrotic accumulation during aging. Our atlas provides extensive detail into how estrus, pregnancy, and aging shape the organs of the female reproductive tract and reveals the unexpected cost of the recurrent remodeling required for reproduction.

Maternal Fecal Microbiota Transplantation in Cesarean-Born Infants Rapidly Restores Normal Gut Microbial Development: A Proof-of-Concept Study.

Infants born by vaginal delivery are colonized with maternal fecal microbes. Cesarean section (CS) birth disturbs mother-to-neonate transmission. In this study (NCT03568734), we evaluated whether disturbed intestinal microbiota development could be restored in term CS-born infants by postnatal, orally delivered fecal microbiota transplantation (FMT). We recruited 17 mothers, of whom seven were selected after careful screening. Their infants received a diluted fecal sample from their own mothers, taken 3 weeks prior to delivery. All seven infants had an uneventful clinical course during the 3-month follow-up and showed no adverse effects. The temporal development of the fecal microbiota composition of FMT-treated CS-born infants no longer resembled that of untreated CS-born infants but showed significant similarity to that of vaginally born infants. This proof-of-concept study demonstrates that the intestinal microbiota of CS-born infants can be restored postnatally by maternal FMT. However, this should only be done after careful clinical and microbiological screening.

The Central Nervous System and the Gut Microbiome.

Neurodevelopment is a complex process governed by both intrinsic and extrinsic signals. While historically studied by researching the brain, inputs from the periphery impact many neurological conditions. Indeed, emerging data suggest communication between the gut and the brain in anxiety, depression, cognition, and autism spectrum disorder (ASD). The development of a healthy, functional brain depends on key pre- and post-natal events that integrate environmental cues, such as molecular signals from the gut. These cues largely originate from the microbiome, the consortium of symbiotic bacteria that reside within all animals. Research over the past few years reveals that the gut microbiome plays a role in basic neurogenerative processes such as the formation of the blood-brain barrier, myelination, neurogenesis, and microglia maturation and also modulates many aspects of animal behavior. Herein, we discuss the biological intersection of neurodevelopment and the microbiome and explore the hypothesis that gut bacteria are integral contributors to development and function of the nervous system and to the balance between mental health and disease.

Vaginal Exposure to Zika Virus during Pregnancy Leads to Fetal Brain Infection.

Zika virus (ZIKV) can be transmitted sexually between humans. However, it is unknown whether ZIKV replicates in the vagina and impacts the unborn fetus. Here, we establish a mouse model of vaginal ZIKV infection and demonstrate that, unlike other routes, ZIKV replicates within the genital mucosa even in wild-type (WT) mice. Mice lacking RNA sensors or transcription factors IRF3 and IRF7 resulted in higher levels of local viral replication. Furthermore, mice lacking the type I interferon (IFN) receptor (IFNAR) became viremic and died of infection after a high-dose vaginal ZIKV challenge. Notably, vaginal infection of pregnant dams during early pregnancy led to fetal growth restriction and infection of the fetal brain in WT mice. This was exacerbated in mice deficient in IFN pathways, leading to abortion. Our study highlights the vaginal tract as a highly susceptible site of ZIKV replication and illustrates the dire disease consequences during pregnancy.

Krause corpuscles are genital vibrotactile sensors for sexual behaviours.

Krause corpuscles, which were discovered in the 1850s, are specialized sensory structures found within the genitalia and other mucocutaneous tissues1-4. The physiological properties and functions of Krause corpuscles have remained unclear since their discovery. Here we report the anatomical and physiological properties of Krause corpuscles of the mouse clitoris and penis and their roles in sexual behaviour. We observed a high density of Krause corpuscles in the clitoris compared with the penis. Using mouse genetic tools, we identified two distinct somatosensory neuron subtypes that innervate Krause corpuscles of both the clitoris and penis and project to a unique sensory terminal region of the spinal cord. In vivo electrophysiology and calcium imaging experiments showed that both Krause corpuscle afferent types are A-fibre rapid-adapting low-threshold mechanoreceptors, optimally tuned to dynamic, light-touch and mechanical vibrations (40-80 Hz) applied to the clitoris or penis. Functionally, selective optogenetic activation of Krause corpuscle afferent terminals evoked penile erection in male mice and vaginal contraction in female mice, while genetic ablation of Krause corpuscles impaired intromission and ejaculation of males and reduced sexual receptivity of females. Thus, Krause corpuscles of the clitoris and penis are highly sensitive mechanical vibration detectors that mediate sexually dimorphic mating behaviours.

An innate antiviral pathway acting before interferons at epithelial surfaces.

Mucosal surfaces are exposed to environmental substances and represent a major portal of entry for microorganisms. The innate immune system is responsible for early defense against infections and it is believed that the interferons (IFNs) constitute the first line of defense against viruses. Here we identify an innate antiviral pathway that works at epithelial surfaces before the IFNs. The pathway is activated independently of known innate sensors of viral infections through a mechanism dependent on viral O-linked glycans, which induce CXCR3 chemokines and stimulate antiviral activity in a manner dependent on neutrophils. This study therefore identifies a previously unknown layer of antiviral defense that exerts its action on epithelial surfaces before the classical IFN response is operative.

Mucosal Microbes Mitigate Maladies.

Understanding the factors that promote or prevent HIV transmission remains a critical component of the global battle against HIV/AIDS. Gosmann et al. (2017) reveal a putative role for the vaginal microbiome in modulating heterosexual transmission of HIV, uncovering a potential strategy for protecting women from acquisition of the virus.

Lactobacillus-Deficient Cervicovaginal Bacterial Communities Are Associated with Increased HIV Acquisition in Young South African Women.

Elevated inflammation in the female genital tract is associated with increased HIV risk. Cervicovaginal bacteria modulate genital inflammation; however, their role in HIV susceptibility has not been elucidated. In a prospective cohort of young, healthy South African women, we found that individuals with diverse genital bacterial communities dominated by anaerobes other than Gardnerella were at over 4-fold higher risk of acquiring HIV and had increased numbers of activated mucosal CD4+ T cells compared to those with Lactobacillus crispatus-dominant communities. We identified specific bacterial taxa linked with reduced (L. crispatus) or elevated (Prevotella, Sneathia, and other anaerobes) inflammation and HIV infection and found that high-risk bacteria increased numbers of activated genital CD4+ T cells in a murine model. Our results suggest that highly prevalent genital bacteria increase HIV risk by inducing mucosal HIV target cells. These findings might be leveraged to reduce HIV acquisition in women living in sub-Saharan Africa.

Therapeutic prime/pull vaccination of HSV-2-infected guinea pigs with the ribonucleotide reductase 2 (RR2) protein and CXCL11 chemokine boosts antiviral local tissue-resident and effector memory CD4+ and CD8+ T cells and protects against recurrent genital herpes.

Following acute herpes simplex virus type 2 (HSV-2) infection, the virus undergoes an asymptomatic latent infection of sensory neurons of dorsal root ganglia (DRG). Chemical and physical stress cause intermittent virus reactivation from latently infected DRG and recurrent virus shedding in the genital mucosal epithelium causing genital herpes in symptomatic patients. While T cells appear to play a role in controlling virus reactivation from DRG and reducing the severity of recurrent genital herpes, the mechanisms for recruiting these T cells into DRG and the vaginal mucosa (VM) remain to be fully elucidated. The present study investigates the effect of CXCL9, CXCL10, and CXCL11 T-cell-attracting chemokines on the frequency and function of DRG- and VM-resident CD4+ and CD8+ T cells and its effect on the frequency and severity of recurrent genital herpes in the recurrent herpes guinea pig model. HSV-2 latent-infected guinea pigs were immunized intramuscularly with the HSV-2 ribonucleotide reductase 2 (RR2) protein (Prime) and subsequently treated intravaginally with the neurotropic adeno-associated virus type 8 expressing CXCL9, CXCL10, or CXCL11 chemokines to recruit CD4+ and CD8+ T cells into the infected DRG and VM (Pull). Compared to the RR2 therapeutic vaccine alone, the RR2/CXCL11 prime/pull therapeutic vaccine significantly increased the frequencies of functional tissue-resident and effector memory CD4+ and CD8+ T cells in both DRG and VM tissues. This was associated with less virus in the healed genital mucosal epithelium and reduced frequency and severity of recurrent genital herpes. These findings confirm the role of local DRG- and VM-resident CD4+ and CD8+ T cells in reducing virus shedding at the vaginal site of infection and the severity of recurrent genital herpes and propose the novel prime-pull vaccine strategy to protect against recurrent genital herpes.IMPORTANCEThe present study investigates the novel prime/pull therapeutic vaccine strategy to protect against recurrent genital herpes using the latently infected guinea pig model. In this study, we used the strategy that involves immunization of herpes simplex virus type 2-infected guinea pigs using a recombinantly expressed herpes tegument protein-ribonucleotide reductase 2 (RR2; prime), followed by intravaginal treatment with the neurotropic adeno-associated virus type 8 expressing CXCL9, CXCL10, or CXCL11 T-cell-attracting chemokines to recruit T cells into the infected dorsal root ganglia (DRG) and vaginal mucosa (VM) (pull). We show that the RR2/CXCL11 prime-pull therapeutic vaccine strategy elicited a significant reduction in virus shedding in the vaginal mucosa and decreased the severity and frequency of recurrent genital herpes. This protection was associated with increased frequencies of functional tissue-resident (TRM cells) and effector (TEM cells) memory CD4+ and CD8+ T cells infiltrating latently infected DRG tissues and the healed regions of the vaginal mucosa. These findings shed light on the role of tissue-resident and effector memory CD4+ and CD8+ T cells in DRG tissues and the VM in protection against recurrent genital herpes and propose the prime-pull therapeutic vaccine strategy in combating genital herpes.

Compartmentalized and systemic control of tissue immunity by commensals.

The body is composed of various tissue microenvironments with finely tuned local immunosurveillance systems, many of which are in close apposition with distinct commensal niches. Mammals have formed an evolutionary partnership with the microbiota that is critical for metabolism, tissue development and host defense. Despite our growing understanding of the impact of this host-microbe alliance on immunity in the gastrointestinal tract, the extent to which individual microenvironments are controlled by resident microbiota remains unclear. In this Perspective, we discuss how resident commensals outside the gastrointestinal tract can control unique physiological niches and the potential implications of the dialog between these commensals and the host for the establishment of immune homeostasis, protective responses and tissue pathology.

Infectious hepatitis E virus excreted into the vagina.

Hepatitis E virus (HEV) persists in the male genital tract that associates with infertility. However, the presence of HEV in the female genital tract is unreported. Vaginal secretions, cervical smears, and cervix uteri were collected to explore the presence of HEV in the female genital tract. HEV RNA and/or antigens were detected in the vaginal secretions, cervical smears, and the cervix uteri of women. The infectivity of HEV excreted into vaginal secretions was further validated in vitro. In addition, HEV replicates in the female genital tract were identified in HEV-infected animal models by vaginal injection or vaginal mucosal infection to imitate sexual transmission. Serious genital tract damage and inflammatory responses with significantly elevated mucosal innate immunity were observed in women or animals with HEV vaginal infection. Results demonstrated HEV replicates in the female genital tract and causes serious histopathological damage and inflammatory responses.

Silencing of cysteine and serine rich nuclear protein 1 inhibits apoptosis, senescence and collagen degradation in human-derived vaginal fibroblasts in response to oxidative stress or DNA damage.

Pelvic organ prolapse (POP) is a group of diseases caused by extracellular matrix (ECM) degradation in pelvic supportive tissues. Cysteine and serine rich nuclear protein 1 (CSRNP1) is involved in cell proliferation and survival regulation, and reportedly facilitates collagen breakdown in human chondrocytes. The present study aimed to probe the effect of CSRNP1 on collagen metabolism in human-derived vaginal fibroblasts. High expression of CSRNP1 was found in POP patient-derived vaginal fibroblasts in comparison to normal-derived vaginal fibroblasts. Following functional experiments revealed that CSRNP1 overexpression led to proliferation inhibition, apoptosis and collagen degradation in normal vaginal fibroblasts. In line with this, silencing of CSRNP1 inhibited hydrogen peroxide (H2O2)-triggered apoptosis, ROS generation and collagen loss in normal vaginal fibroblasts. Silencing of CSRNP1 also reduced the expression of cell senescence markers p21 and γ-H2Ax (the histone H2Ax phosphorylated at Ser139), as well as curbed collagen breakdown in normal vaginal fibroblasts caused by a DNA damage agent etoposide. Transcriptomic analysis of vaginal fibroblasts showed that differentially expressed genes affected by CSRNP1 overexpression were mainly enriched in the Wnt signaling pathway. Treatment with a Wnt pathway inhibitor DKK1 blocked CSRNP1 knockdown-caused collagen deposition. Mechanistically, CSRNP1 was identified to be a target of Snail family transcriptional repressor 2 (SNAI2). Forced expression of CSRNP1 reversed the anti-apoptotic, anti-senescent and anti-collagen loss effects of SNAI2 in normal vaginal fibroblasts exposed to H2O2 or etoposide. Our study indicates that the SNAI2/CSRNP1 axis may be a key driver in POP progression, which provides a potential therapeutic strategy for POP.

Migrant memory B cells secrete luminal antibody in the vagina.

Antibodies secreted into mucosal barriers serve to protect the host from a variety of pathogens, and are the basis for successful vaccines1. In type I mucosa (such as the intestinal tract), dimeric IgA secreted by local plasma cells is transported through polymeric immunoglobulin receptors2 and mediates robust protection against viruses3,4. However, owing to the paucity of polymeric immunoglobulin receptors and plasma cells, how and whether antibodies are delivered to the type II mucosa represented by the lumen of the lower female reproductive tract remains unclear. Here, using genital herpes infection in mice, we show that primary infection does not establish plasma cells in the lamina propria of the female reproductive tract. Instead, upon secondary challenge with herpes simplex virus 2, circulating memory B cells that enter the female reproductive tract serve as the source of rapid and robust antibody secretion into the lumen of this tract. CD4 tissue-resident memory T cells secrete interferon-γ, which induces expression of chemokines, including CXCL9 and CXCL10. Circulating memory B cells are recruited to the vaginal mucosa in a CXCR3-dependent manner, and secrete virus-specific IgG2b, IgG2c and IgA into the lumen. These results reveal that circulating memory B cells act as a rapidly inducible source of mucosal antibodies in the female reproductive tract.

The Integrative Human Microbiome Project.

The NIH Human Microbiome Project (HMP) has been carried out over ten years and two phases to provide resources, methods, and discoveries that link interactions between humans and their microbiomes to health-related outcomes. The recently completed second phase, the Integrative Human Microbiome Project, comprised studies of dynamic changes in the microbiome and host under three conditions: pregnancy and preterm birth; inflammatory bowel diseases; and stressors that affect individuals with prediabetes. The associated research begins to elucidate mechanisms of host-microbiome interactions under these conditions, provides unique data resources (at the HMP Data Coordination Center), and represents a paradigm for future multi-omic studies of the human microbiome.

Microbial mismanagement: how inadequate treatments for vaginal dysbiosis drive the HIV epidemic in women.

Women and girls represent a key population driving new HIV infections and persistence of the HIV pandemic. A key determinant of HIV susceptibility is the composition of the vaginal microbiome, which can influence the local immune cell population, inflammation status, and HIV prevention drug levels. While a low-diversity composition dominated by Lactobacillus crispatus is associated with a decreased risk of HIV acquisition, high diversity environments associated with bacterial vaginosis increase risk of HIV. Given the important role of the vaginal microbiome in determining HIV susceptibility, altering the microbiome towards a Lactobacillus-dominated state is an attractive complementary strategy to reduce HIV incidence rates. Here, we provide an overview of the mechanisms by which the vaginal microbiome may contribute to HIV acquisition risk. Furthermore, we address the advantages and limitations of historical treatments and emerging technologies under investigation to modify the vaginal microbiome, including: antibiotics, bacteriophages, probiotics, topicals, and engineered bacteria. By addressing the current state of vaginal microbiome knowledge and strategies for manipulation, we hope to amplify the growing calls for increased resources and research into vaginal microbial health, which will be essential to accelerating preventative efforts amongst the world's most vulnerable populations.

High incidence of imperforate vagina in ADGRA3-deficient mice.

BACKGROUND: Ten percent of the female population suffers from congenital abnormalities of the vagina, uterus, or oviducts, with severe consequences for reproductive and psychological health. Yet, the underlying causes of most of these malformations remain largely unknown. ADGRA3 (GPR125) is involved in WNT signaling and planar cell polarity, mechanisms vital to female reproductive tract development. Although ADGRA3 is a well-established spermatogonial stem cell marker, its role within the female urogenital system remains unclear. RESULTS: In this study, we found Adgra3 to be expressed throughout the murine female urogenital system, with higher expression pre-puberty than after sexual maturation. We generated a global Adgra3-/- mouse line and observed imperforate vagina in 44% of Adgra3-/- females, resulting in distension of the reproductive tract and infertility. Ovarian morphology, plasma estradiol, ovarian Cyp19a1, and vaginal estrogen receptor α (Esr1) expression were unaffected. However, compared to controls, a significantly lower bone mineral density was found in Adgra3-/- mice. Whereas vaginal opening in mice is an estrogen-dependent process, 17ß-estradiol treatment failed to induce vaginal canalization in Adgra3-/- mice. Furthermore, a marked reduction in vaginal and ovarian progesterone receptor expression was observed concomitant with an upregulation of apoptotic regulators Bcl2, Bid, and Bmf in adult Adgra3-/- females with a closed vagina. CONCLUSIONS: Our collective results shed new insights into the complex mechanisms by which the adhesion receptor ADGRA3 regulates distal vaginal tissue remodeling during vaginal canalization via altered sex hormone responsiveness and balance in apoptotic regulators. This highlights the potential of ADGRA3 as a target in diagnostic screening and/or therapy for obstructive vaginal malformations in humans.

Increased type 1 inflammation in gynecologic cervicovaginal samples in patients with APS-1.

BACKGROUND: Inborn errors of immunity offer important insights into mucosal immunity. In autoimmune polyendocrine syndrome type-1 (APS-1), chronic mucocutaneous candidiasis has been ascribed to neutralizing IL-17 autoantibodies. Recent evidence implicates excessive T-cell IFN-γ secretion and ensuing epithelial barrier disruption in predisposition to candidiasis, but these results remain to be replicated. Whether IL-17 paucity, increased type I inflammation, or their combination underlies susceptibility to chronic mucocutaneus candidiasis in APS-1 is debated. OBJECTIVE: Our aim was to characterize the immunologic features in the cervicovaginal mucosa of females with APS-1. METHODS: Vaginal fluid was collected with a flocked swab from 17 females with APS-1 and 18 controls, and cytokine composition was analyzed using Luminex (Luminex Corporation, Austin, Tex). Cervical cell samples were obtained with a cervix brush from 6 patients and 6 healthy controls and subjected to transcriptome analysis. RESULTS: The vaginal fluid samples from patients with APS-1 had IFN-γ concentrations comparable to those of the controls (2.6 vs 2.4 pg/mL) but high concentrations of the TH1 chemokines CXCL9 and CXCL10 (1094 vs 110 pg/mL [P < .001] and 4033 vs 273 pg/mL [P = .001], respectively), whereas the IL-17 levels in the samples from the 2 groups were comparable (28 vs 8.8 pg/mL). RNA sequencing of the cervical cells revealed upregulation of pathways related to mucosal inflammation and cell death in the patients with APS-1. CONCLUSION: Excessive TH1 cell response appears to underlie disruption of the mucosal immune responses in the genital tract of patients with APS-1 and may contribute to susceptibility to candidiasis in the genital tract as well.

Prevotella timonensis Bacteria Associated With Vaginal Dysbiosis Enhance Human Immunodeficiency Virus Type 1 Susceptibility Of Vaginal CD4+ T Cells.

Dysbiosis of the vaginal microbiome poses a serious risk for sexual human immunodeficiency virus type 1 (HIV-1) transmission. Prevotella spp are abundant during vaginal dysbiosis and associated with enhanced HIV-1 susceptibility; however, underlying mechanisms remain unclear. Here, we investigated the direct effect of vaginal bacteria on HIV-1 susceptibility of vaginal CD4+ T cells. Notably, pre-exposure to Prevotella timonensis enhanced HIV-1 uptake by vaginal T cells, leading to increased viral fusion and enhanced virus production. Pre-exposure to antiretroviral inhibitors abolished P timonensis-enhanced infection. Our study shows that the vaginal microbiome directly affects mucosal CD4+ T-cell susceptibility, emphasizing importance of vaginal dysbiosis diagnosis and treatment.

Effect of Vaginal Microecological Disorders on the Increased Risk of Abnormal Cervical Cytology Among Women With Human Immunodeficiency Virus in China.

BACKGROUND: Abnormal cervical cytology is commonly observed in women with human immunodeficiency virus (WWH). METHODS: A cross-sectional study was conducted with 130 WWH and 147 age-matched healthy controls, who underwent gynecological examinations at Beijing Ditan Hospital. The presence of abnormal cervical cytology in WWH was predicted after performing a logistic regression analysis. RESULTS: Multivariate logistic regression revealed 3 independent factors, among which CD4 cell count ≥350 cells/µL was the protective factor, while human papillomavirus infection and abnormal vaginal pH were the risk factors. CONCLUSIONS: Vaginal microecological disorders can increase the risk of abnormal cervical cytology in WWH.

Gardnerella Species and Their Association With Bacterial Vaginosis.

BACKGROUND: Bacterial vaginosis (BV) is a condition marked by high vaginal bacterial diversity. Gardnerella vaginalis has been implicated in BV but is also detected in healthy women. The Gardnerella genus has been expanded to encompass 6 validly named species and several genomospecies. We hypothesized that particular Gardnerella species may be more associated with BV. METHODS: Quantitative polymerase chain reaction (PCR) assays were developed targeting the cpn60 gene of species groups including G. vaginalis, G. piotii/pickettii, G. swidsinskii/greenwoodii, and G. leopoldii. These assays were applied to vaginal swabs from individuals with (n = 101) and without BV (n = 150) attending a sexual health clinic in Seattle, Washington. Weekly swabs were collected from 42 participants for up to 12 weeks. RESULTS: Concentrations and prevalence of each Gardnerella species group were significantly higher in participants with BV; 91.1% of BV-positive participants had 3 or more Gardnerella species groups detected compared to 32.0% of BV-negative participants (P < .0001). BV-negative participants with 3 or more species groups detected were more likely to develop BV within 100 days versus those with fewer (60.5% vs 3.7%, P < .0001). CONCLUSIONS: These results suggest that BV reflects a state of high Gardnerella species diversity. No Gardnerella species group was a specific marker for BV.