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.

Advances and challenges in studying the tissue-resident T cell compartment in the human female reproductive tract.

Tissue-resident memory T cells (TRM ) are considered to be central to maintaining mucosal barrier immunity and tissue homeostasis. Most of this knowledge stems from murine studies, which provide access to all organs. These studies also allow for a thorough assessment of the TRM compartment for each tissue and across tissues with well-defined experimental and environmental variables. Assessing the functional characteristics of the human TRM compartment is substantially more difficult; thus, notably, there is a paucity of studies profiling the TRM compartment in the human female reproductive tract (FRT). The FRT is a mucosal barrier tissue that is naturally exposed to a wide range of commensal and pathogenic microbes, including several sexually transmitted infections of global health significance. We provide an overview of studies describing T cells within the lower FRT tissues and highlight the challenges of studying TRM cells in the FRT: different sampling methods of the FRT greatly affect immune cell recovery, especially of TRM cells. Furthermore, menstrual cycle, menopause, and pregnancy affect FRT immunity, but little is known about changes in the TRM compartment. Finally, we discuss the potential functional plasticity of the TRM compartment during inflammatory episodes in the human FRT to maintain protection and tissue homeostasis, which are required to ensure reproductive fitness.

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.

Human papillomavirus genotyping in high-grade vaginal intraepithelial neoplasia: A multicentric Italian study.

This study aimed to analyze the human papillomavirus (HPV) genotype distribution in a large cohort of high-grade vaginal intraepithelial neoplasia (VaIN) (vaginal HSIL, VaIN2/3) patients from two Italian referral centers. We included all patients with histologically confirmed VaIN2/3 from the Department of Surgical Sciences, Sant'Anna Hospital, University of Torino, Torino, Italy, and Ospedale Maggiore della Carità, Novara, Italy, between 2003 and 2022. After the histological evaluation of formalin-fixed paraffin-embedded samples, we performed HPV genotyping with VisionArray HPV Chip 1.0. We detected HPV DNA in 94.4% of VaIN2/3 (168/178), with HPV 16 as the most prevalent genotype, accounting for 51.8% of all infections, 41.2% of VaIN2 and 77.6% of VaIN3 cases. Other frequent genotypes were HPV 58 (8.3%, 10.9% of VaIN2 and 2.0% of VaIN3), HPV 73 (5.4%, 5.0% of VaIN2 and 6.1% of VaIN3), and HPV 31 (5.4%, 6.7% of VaIN2 and 2.0% of VaIN3). 73.2% of VaIN2/3 had a single HPV genotype infection and 26.8% a multiple infection (20.8% a double infection, 4.8% a triple infection, and 1.2% a quadruple infection). Single infection was more frequently present in VaIN3 than VaIN2 (81.6% vs. 69.8%). 69.1% of single infections and 73.3% of multiple infections had one or more genotypes covered by nine-valent HPV vaccine. HPV vaccination is expected to have a large impact on reducing the incidence of vaginal intraepithelial neoplasia.

Gastric-type glandular lesions of the female genital tract excluding the cervix: emerging pathological entities.

In the last two decades or so, a spectrum of benign, premalignant and malignant cervical glandular lesions exhibiting gastric differentiation has been described, with gastric-type adenocarcinoma representing the most common human papillomavirus (HPV)-independent cervical adenocarcinoma. More recently, limited literature has reported a variety of gastric-type glandular lesions at other sites within the female genital tract and, as in the cervix (the most common site for these lesions), a spectrum of benign, premalignant and malignant lesions has been proposed. We provide an update and review of the emerging spectrum of gastric-type glandular lesions at female genital tract sites other than the cervix. In the endometrium, putative gastric-type glandular lesions include mucinous metaplasia of gastric-type, atypical mucinous proliferation of gastric-type and gastric-type adenocarcinoma. Similarly in the vagina, gastric-type adenosis, atypical adenosis and adenocarcinoma have been described. There have also been occasional reports of gastric-type lesions involving the ovary and fallopian tube. We provide guidance on how to recognise gastric-type lesions morphologically and immunophenotypically and stress that sometimes these lesions occur at more than one site within the female genital tract (synchronous/multifocal gastric-type lesions of the female genital tract), sometimes in association with Peutz-Jeghers syndrome.

Increased Interferon Signaling in Vaginal Tissue of Patients With Primary Sjögren Syndrome.

OBJECTIVE: Vaginal dryness is an important factor influencing sexual function in women with primary Sjögren syndrome (pSS). Previous studies showed a higher degree of inflammation in vaginal biopsies from patients with pSS compared to non-pSS controls. However, the molecular pathways that drive this inflammation remain unclear. Therefore, the aim of this study was to investigate inflammatory pathway activity in the vaginal tissue of patients with pSS. METHODS: Vaginal biopsies of 8 premenopausal patients with pSS experiencing vaginal dryness and 7 age-matched non-pSS controls were included. Expression of genes involved in inflammation and tissue homeostasis was measured using NanoString technology and validated using TaqMan Real-Time PCR. Vaginal tissue sections were stained by immunohistochemistry for myxovirus resistance protein 1 (MxA) and CD123 (plasmacytoid dendritic cells [pDCs]). RESULTS: The most enriched pathway in vaginal biopsies from patients with pSS compared to non-pSS controls was the interferon (IFN) signaling pathway (P < 0.01). Pathway scores for Janus kinase and signal transducer and activator of transcription (JAK-STAT) and Notch signaling were also higher (P < 0.01 for both pathways). Conversely, transforming growth factor-ß signaling and angiogenesis pathway scores were lower in pSS (P = 0.02 and P = 0.04, respectively). Differences in IFN signaling between patients with pSS and non-pSS controls were confirmed by PCR and MxA tissue staining. No CD123+ pDCs were detected in vaginal biopsies. IFN-stimulated gene expression levels correlated positively with CD45+ cell numbers in vaginal biopsies and serum anti-SSA/Ro positivity. CONCLUSION: Upregulation of IFN signaling in vaginal tissue of women with pSS, along with its association with tissue pathology, suggests that IFNs contribute to inflammation of the vaginal wall and potentially also to clinical symptomatology (ie, vaginal dryness).

Denonvilliers' fascia as a potential nerve-course marker for the female urinary bladder.

OBJECTIVES: This study investigated the relationship between Denonvilliers' fascia (DF) and the pelvic plexus branches in women and explored the possibility of using the DF as a positional marker in nerve-sparing radical hysterectomy (RH). METHODS: This study included eight female cadavers. The DF, its lateral border, and the pelvic autonomic nerves running lateral to the DF were dissected and examined. The pelvis was cut into two along the mid-sagittal line. The uterine artery, deep uterine veins, vesical veins, and nerve branches to the pelvic organs were carefully dissected. RESULTS: The nerves ran sagitally, while the DF ran perpendicularly to them. The rectovaginal ligament was continuous with the DF, forming a single structure. The DF attached perpendicularly and seamlessly to the pelvic plexus. The pelvic plexus branches were classified into a ventral part branching to the bladder, uterus, and upper vagina and a dorsal part branching to the lower vagina and rectum as well as into four courses. Nerves were attached to the rectovaginal ligament and ran on its surface to the bladder ventral to the DF. The uterine branches split from the common trunk of these nerves. The most dorsal branch to the bladder primarily had a common trunk with the uterine branch, which is the most important and should be preserved in nerve-sparing Okabayashi RH. CONCLUSION: The DF can be used as a marker for nerve course, particularly in one of the bladder branches running directly superior to the DF, which can be preserved in nerve-sparing Okabayashi RH.

Advancements in three-dimensional bioprinting for reproductive medicine: a systematic review.

Reproductive failure due to age, genetics and disease necessitates innovative solutions. While reproductive tissue transplantation has advanced, ongoing research seeks superior approaches. Biomaterials, bioengineering and additive manufacturing, such as three-dimensional (3D) bioprinting, are harnessed to restore reproductive function. 3D bioprinting uses materials, cells and growth factors to mimic natural tissues, proving popular for tissue engineering, notably in complex scaffold creation with cell distribution. The versatility which is brought to reproductive medicine by 3D bioprinting allows more accurate and on-site applicability to various problems that are encountered in the field. However, in the literature, there is a lack of studies encompassing the valuable applications of 3D bioprinting in reproductive medicine. This systematic review aims to improve understanding, and focuses on applications in several branches of reproductive medicine. Advancements span the restoration of ovarian function, endometrial regeneration, vaginal reconstruction, and male germ cell bioengineering. 3D bioprinting holds untapped potential in reproductive medicine.

Vaginal morphology and position associated with prolapse recurrence after vaginal surgery: A secondary analysis of the DEMAND study.

OBJECTIVE: To identify vaginal morphology and position factors associated with prolapse recurrence following vaginal surgery. DESIGN: Secondary analysis of magnetic resonance images (MRI) of the Defining Mechanisms of Anterior Vaginal Wall Descent cross-sectional study. SETTING: Eight clinical sites in the US Pelvic Floor Disorders Network. POPULATION OR SAMPLE: Women who underwent vaginal mesh hysteropexy (hysteropexy) with sacrospinous fixation or vaginal hysterectomy with uterosacral ligament suspension (hysterectomy) for uterovaginal prolapse between April 2013 and February 2015. METHODS: The MRI (rest, strain) obtained 30-42 months after surgery, or earlier for participants with recurrence who desired reoperation before 30 months, were analysed. MRI-based prolapse recurrence was defined as prolapse beyond the hymen at strain on MRI. Vaginal segmentations (at rest) were used to create three-dimensional models placed in a morphometry algorithm to quantify and compare vaginal morphology (angulation, dimensions) and position. MAIN OUTCOME MEASURES: Vaginal angulation (upper, lower and upper-lower vaginal angles in the sagittal and coronal plane), dimensions (length, maximum transverse width, surface area, volume) and position (apex, mid-vagina) at rest. RESULTS: Of the 82 women analysed, 12/41 (29%) in the hysteropexy group and 22/41 (54%) in the hysterectomy group had prolapse recurrence. After hysteropexy, women with recurrence had a more laterally deviated upper vagina (p = 0.02) at rest than women with successful surgery. After hysterectomy, women with recurrence had a more inferiorly (lower) positioned vaginal apex (p = 0.01) and mid-vagina (p = 0.01) at rest than women with successful surgery. CONCLUSIONS: Vaginal angulation and position were associated with prolapse recurrence and suggestive of vaginal support mechanisms related to surgical technique and potential unaddressed anatomical defects. Future prospective studies in women before and after prolapse surgery may distinguish these two factors.

Müllerian anomalies in girls with congenital solitary kidney.

BACKGROUND: The prevalence of Müllerian anomalies (MA) among patients with congenital solitary functioning kidney (SFK) is not well defined. A delay in diagnosis of obstructive MA can increase the risk of poor clinical outcomes. This study describes the prevalence of MA in patients with congenital SFK. METHODS: A retrospective review was performed of patients within the Nationwide Children's Hospital system with ICD9 or ICD10 diagnostic codes for congenital SFK defined as either unilateral renal agenesis (URA) or multicystic dysplastic kidney (MCDK) and confirmed by chart review. Patients with complex urogenital pathology were excluded. Renal anomaly, MA, reason for and type of pelvic evaluation, and age of diagnosis of anomalies were evaluated. RESULTS: Congenital SFK occurred in 431 girls due to URA (209) or MCDK (222). Pelvic evaluation, most commonly by ultrasound for evaluation of abdominal pain or dysmenorrhea, occurred in 115 patients leading to MA diagnosis in 60 instances. Among 221 patients ages 10 years and older, 104 underwent pelvic evaluation and 52 were diagnosed with an MA of which 20 were obstructive. Isolated uterine or combined uterine and vaginal anomalies were the most common MA. MA were five-fold more common in patients with URA compared to MCDK. In 75% of patients, the SFK was diagnosed prior to the MA. CONCLUSIONS: The prevalence of MA in patients with congenital SFK was 24% among those age 10 years or older, and 38% were obstructive. This justifies routine screening pelvic ultrasound in girls with congenital SFK to improve early diagnosis.

TRImodal DEfinitive invasive vagiNal carcinoma Treatment (TRIDENT protocol): how a standardized approach may change prognostic outcomes.

OBJECTIVE: Vaginal carcinoma is a rare malignancy accounting for 1-2% of all gynecological cancers. Surgery has a limited role, while definitive radiotherapy-chemotherapy followed by interventional radiotherapy is considered a valid alternative. The aim of the TRIDENT (TRImodal DEfinitive invasive vagiNal carcinoma Treatment) pilot study was to report the results of a modern standardized trimodal protocol treatment consisting of image guided definitive radiotherapy-chemotherapy followed by image guided interventional radiotherapy in terms of safety and efficacy. METHODS: Between January 2019 and December 2021, we analyzed 21 consecutive patients with primary vaginal cancer who had received radiotherapy-chemotherapy followed by interventional radiotherapy. The primary study endpoint was local control, and secondary endpoints were metastasis free survival, overall survival, and rate and severity of acute and late toxicities. RESULTS: 14 patients had FIGO (International Federation of Gynecology and Obstetrics) stage II, five patients had stage III, and two had stage IVB disease. Median total external beam radiotherapy dose for the tumor was 45 Gy. Median total dose on positive nodes was 60 Gy. Median total dose for interventional radiotherapy was 28 Gy over four high dose rate fractions to achieve between 85 and 95 Gy equivalent dose, in 2 Gy fractions (EQD2)α/ß10, to the high risk clinical target volume, and 60 Gy EQD2α/ß10 to the intermediate risk clinical target volume. All patients received weekly platinum based chemotherapy. Median follow-up was 20 months (range 10-56 months). Two year actuarial local control, metastasis free survival, and overall survival rate were 79.4%, 90.5%, and 79.4%, respectively. In terms of acute toxicity, there were no grade 4 events and only one acute grade (G) 3 toxicity (skin). Only vaginal stenosis (G3) was documented 12 months after therapy due to late toxicity. CONCLUSIONS: In this study, definitive radiotherapy-chemotherapy followed by interventional radiotherapy was a safe and effective treatment modality for primary vaginal cancer.

Comparison of the Vaginal and Labial Dimensions of Ethnic Chinese and Western Nullipara.

INTRODUCTION AND HYPOTHESIS: Vaginal dimensions have clinical and surgical implications. We sought to quantify the differences between vaginal and labial dimensions in healthy ethnic Chinese and Western women with normal pelvic organ support. METHODS: This is a cross-sectional study of a convenience sample of ethnic Chinese nulliparas (n = 33) and Western nulliparas (n = 33) recruited for research purposes. For each subject, magnetic resonance imaging was used to quantify the vaginal and labial dimensions. Specifically, we identified the anterior and posterior vaginal wall, the outline of the cervix in the mid-sagittal and coronal planes, and the distance from the labia majora to the hymenal ring at the urethral meatus. RESULTS: There were significant differences in age and weight between groups. Substantial variation in vaginal and labial dimensions was found within each group. The vaginal and labial dimensions of ethnic Chinese women ranged from 9-21% smaller than those of Western women; In the ethnic Chinese group, increasing weight and BMI correlated with greater labial distance (r = 0.66 and r = 0.63 respectively); as did height and the distance from the vaginal opening to the cervical os (r = 0.5). In the Western group, only weight correlated with the labial distance (r = 0.51). CONCLUSIONS: Significant group differences in vaginal and labial dimensions were found, with the dimensions of Chinese nulliparas being up to 21% smaller than those of Western nulliparas.

Shaping Microbiota During the First 1000 Days of Life.

Given that the host-microbe interaction is shaped by the immune system response, it is important to understand the key immune system-microbiota relationship during the period from conception to the first years of life. The present work summarizes the available evidence concerning human reproductive microbiota, and also, the microbial colonization during early life, focusing on the potential impact on infant development and health outcomes. Furthermore, we conclude that some dietary strategies including specific probiotics and other-biotics could become potentially valuable tools to modulate the maternal-neonatal microbiota during this early critical window of opportunity for targeted health outcomes throughout the entire lifespan.

Vaginal malignant peripheral nerve sheath tumor treated with complete surgical resection and postoperative radiation therapy.

Malignant peripheral nerve sheath tumors (MPNSTs) are neoplasms originating from or differentiating into nerve sheaths of peripheral nerves. Vaginal origin is rare, with only six vaginal primary cases reported to date. A 55-year-old woman presented to our hospital with a 7 cm vulvar mass. Tumor biopsy results were suspicious of sarcoma, and pelvic magnetic resonance imaging and hysterofiberscopy showed that the tumor originated from the lower vagina. The mass was transvaginally excised, and histological examination confirmed the diagnosis of a vaginal MPNST with negative surgical margins. The patient underwent radiotherapy because the risk of recurrence was high, owing to the large tumor size and high mitotic index. The patient remained recurrence-free for 1 year after the primary treatment. This is the first case of a high-risk vaginal MPNST that avoided early disease recurrence with additional radiotherapy after complete tumor resection.

Temporal disposition of pelvic girdle, vagina and vulva in relation to urovagina in cycling and pregnant-postpartum cows.

Urovagina (UV) is less studied in cows. The vaginal contents, constrictor vestibule muscle activity, circulating progesterone, pelvic girdle, vagina and vulva angles were compared on Days 0 and 14 within and between UV (UV group; n = 36) and normal (N group; n = 36) cows. The oestrous duration was compared among the groups. Parameters for various UV severities were also compared. Another set of pregnant-postpartum cows (P-PP group; n = 31) underwent monthly evaluations for UV and the angles from the third month of pregnancy until the second month postpartum. The effect of age, parity and body condition score on UV severities on Day 0, and parity on angles in the P-PP group was evaluated. Different variables were correlated in different groups. The UV group was repeat breeder, exhibited prolonged oestrus and reduced progesterone on Day 14. The latter increased with UV severity on Day 0. On Day 14, severe form of UV was more prevalent. The UV severity increased with parity. In the UV group, a cranioventral pelvic girdle on Day 0 became more cranioventral on Day 14 and was correlated with the vagina moving from a caudodorsal position on Day 0 to a cranioventral position on Day 14. In the P-PP group, the pelvic girdle and vagina remained caudodorsal and UV was undetectable throughout the study. Vagina and vulva displayed a parity-by-month interaction in the P-PP group. To summarize, the UV characteristics were influenced by the oestrous cycle stage, pelvic girdle angles, and parity.

Reproductive Tract Microbial Transitions from Late Gestation to Early Postpartum Using 16S rRNA Metagenetic Profiling in First-Pregnancy Heifers.

Studies in recent years indicate that reproductive tract microbial communities are crucial for shaping mammals' health and reproductive outcomes. Following parturition, uterine bacterial contamination often occurs due to the open cervix, which may lead to postpartum uterine inflammatory diseases, especially in primiparous individuals. However, investigations into spatio-temporal microbial transitions in the reproductive tract of primigravid females remain limited. Our objective was to describe and compare the microbial community compositions in the vagina at late gestation and in the vagina and uterus at early postpartum in first-pregnancy heifers. Three swab samples were collected from 33 first-pregnancy Holstein Friesian heifers: one vaginal sample at gestation day 258 ± 4, and vaginal and uterine samples at postpartum day 7 ± 2. Each sample underwent 16S rRNA V4 region metagenetic analysis via Illumina MiSeq, with bioinformatics following Mothur MiSeq SOP. The reproductive tract bacterial communities were assigned to 1255 genus-level OTUs across 30 phyla. Dominant phyla, accounting for approximately 90% of the communities, included Proteobacteria, Firmicutes, Actinobacteria, Bacteroidetes, and Fusobacteria. However, the results revealed distinct shifts in microbial composition between the prepartum vagina (Vag-pre), postpartum vagina (Vag-post), and postpartum uterus (Utr-post). The Vag-pre and Utr-post microbial profiles were the most distinct. The Utr-post group had lower relative abundances of Proteobacteria but higher abundances of Bacteroidetes, Fusobacteria, and Tenericutes compared to Vag-pre, while Vag-post displayed intermediate values for these phyla, suggesting a transitional profile. Additionally, the Utr-post group exhibited lower bacterial richness and diversity compared to both Vag-pre and Vag-post. The unsupervised probabilistic Dirichlet Multinomial Mixtures model identified two distinct community types: most Vag-pre samples clustered into one type and Utr-post samples into another, while Vag-post samples were distributed evenly between the two. LEfSe analysis revealed distinct microbial profiles at the genus level. Overall, specific microbial markers were associated with anatomical and temporal transitions, revealing a dynamic microbial landscape during the first pregnancy and parturition. These differences highlight the complexity of these ecosystems and open new avenues for research in reproductive biology and microbial ecology.

Reconstruction of Cloacal Defect: Switch Operation - A New Approach.

Introduction: Reconstruction of a cloacal defect in a girl is often difficult and complicated.[1] This is most often done either by the sacro-perineal route or through the posterior sagittal route.[2] The procedures may involve total mobilization of the cloaca with or without the creation of a lower vagina with the help of a loop of vascularized bowel.[3] In our approach, such defects can be corrected using the lower anorectum for the creation of a lower vagina and abdominoperineal pull-through of the proximal divided bowel. This method can be used both in short and long common channel cloaca. Materials and Methods: We have used this procedure in one patient of posterior cloaca and four patients of anterior cloaca. These operations were done in patients of 1 year and 6 months to 4 years. Only one patient is waiting for the closure of the colostomy. Results: In all the cases, the urethra, the vagina, and the anus were successfully separated. Conclusion: This operation restores normal pelvic anatomy and physiology with minimal mutilation of the pelvic floor.

Glycogen-Degrading Activities of Catalytic Domains of α-Amylase and α-Amylase-Pullulanase Enzymes Conserved in Gardnerella spp. from the Vaginal Microbiome.

Gardnerella spp. are associated with bacterial vaginosis in which normally dominant lactobacilli are replaced with facultative and anaerobic bacteria, including Gardnerella spp. Co-occurrence of multiple species of Gardnerella is common in the vagina, and competition for nutrients such as glycogen likely contributes to the differential abundances of Gardnerella spp. Glycogen must be digested into smaller components for uptake, a process that depends on the combined action of glycogen-degrading enzymes. In this study, the ability of culture supernatants of 15 isolates of Gardnerella spp. to produce glucose, maltose, maltotriose, and maltotetraose from glycogen was demonstrated. Carbohydrate-active enzymes (CAZymes) were identified bioinformatically in Gardnerella proteomes using dbCAN2. Identified proteins included a single-domain α-amylase (EC 3.2.1.1) (encoded by all 15 isolates) and an α-amylase-pullulanase (EC 3.2.1.41) containing amylase, carbohydrate binding modules, and pullulanase domains (14/15 isolates). To verify the sequence-based functional predictions, the amylase and pullulanase domains of the α-amylase-pullulanase and the single-domain α-amylase were each produced in Escherichia coli. The α-amylase domain from the α-amylase-pullulanase released maltose, maltotriose, and maltotetraose from glycogen, and the pullulanase domain released maltotriose from pullulan and maltose from glycogen, demonstrating that the Gardnerella α-amylase-pullulanase is capable of hydrolyzing α-1,4 and α-1,6 glycosidic bonds. Similarly, the single-domain α-amylase protein also produced maltose, maltotriose, and maltotetraose from glycogen. Our findings show that Gardnerella spp. produce extracellular amylase enzymes as "public goods" that can digest glycogen into maltose, maltotriose, and maltotetraose that can be used by the vaginal microbiota. IMPORTANCE Increased abundance of Gardnerella spp. is a diagnostic characteristic of bacterial vaginosis, an imbalance in the human vaginal microbiome associated with troubling symptoms, and negative reproductive health outcomes, including increased transmission of sexually transmitted infections and preterm birth. Competition for nutrients is likely an important factor in causing dramatic shifts in the vaginal microbial community, but little is known about the contribution of bacterial enzymes to the metabolism of glycogen, a major food source available to vaginal bacteria. The significance of our research is characterizing the activity of enzymes conserved in Gardnerella species that contribute to the ability of these bacteria to utilize glycogen.

Peripheral and central neuroplasticity in a mouse model of endometriosis.

Chronic pelvic pain (CPP) is the most debilitating symptom of gynaecological disorders such as endometriosis. However, it remains unclear how sensory neurons from pelvic organs affected by endometriosis, such as the female reproductive tract, detect and transmit nociceptive events and how these signals are processed within the central nervous system (CNS). Using a previously characterized mouse model of endometriosis, we investigated whether the increased pain sensitivity occurring in endometriosis could be attributed to (i) changes in mechanosensory properties of sensory afferents innervating the reproductive tract, (ii) alterations in sensory input from reproductive organs to the spinal cord or (iii) neuroinflammation and sensitization of spinal neural circuits. Mechanosensitivity of vagina-innervating primary afferents was examined using an ex vivo single-unit extracellular recording preparation. Nociceptive signalling from the vagina to the spinal cord was quantified by phosphorylated MAP kinase ERK1/2 immunoreactivity. Immunohistochemistry was used to determine glial and neuronal circuit alterations within the spinal cord. We found that sensory afferents innervating the rostral, but not caudal portions of the mouse vagina, developed mechanical hypersensitivity in endometriosis. Nociceptive signalling from the vagina to the spinal cord was significantly enhanced in mice with endometriosis. Moreover, mice with endometriosis developed microgliosis, astrogliosis and enhanced substance P neurokinin-1 receptor immunoreactivity within the spinal cord, suggesting the development of neuroinflammation and sensitization of spinal circuitry in endometriosis. These results demonstrate endometriosis-induced neuroplasticity occurring at both peripheral and central sites of sensory afferent pathways. These findings may help to explain the altered sensitivity to pain in endometriosis and provide a novel platform for targeted pain relief treatments for this debilitating disorder.

The voltage-gated sodium channel NaV 1.7 underlies endometriosis-associated chronic pelvic pain.

Chronic pelvic pain (CPP) is the primary symptom of endometriosis patients, but adequate treatments are lacking. Modulation of ion channels expressed by sensory nerves innervating the viscera has shown promise for the treatment of irritable bowel syndrome and overactive bladder. However, similar approaches for endometriosis-associated CPP remain underdeveloped. Here, we examined the role of the voltage-gated sodium (NaV ) channel NaV 1.7 in (i) the sensitivity of vagina-innervating sensory afferents and investigated whether (ii) NaV 1.7 inhibition reduces nociceptive signals from the vagina and (iii) ameliorates endometriosis-associated CPP. The mechanical responsiveness of vagina-innervating sensory afferents was assessed with ex vivo single-unit recording preparations. Pain evoked by vaginal distension (VD) was quantified by the visceromotor response (VMR) in vivo. In control mice, pharmacological activation of NaV 1.7 with OD1 sensitised vagina-innervating pelvic afferents to mechanical stimuli. Using a syngeneic mouse model of endometriosis, we established that endometriosis sensitised vagina-innervating pelvic afferents to mechanical stimuli. The highly selective NaV 1.7 inhibitor Tsp1a revealed that this afferent hypersensitivity occurred in a NaV 1.7-dependent manner. Moreover, in vivo intra-vaginal treatment with Tsp1a reduced the exaggerated VMRs to VD which is characteristic of mice with endometriosis. Conversely, Tsp1a did not alter ex vivo afferent mechanosensitivity nor in vivo VMRs to VD in Sham control mice. Collectively, these findings suggest that NaV 1.7 plays a crucial role in endometriosis-induced vaginal hyperalgesia. Importantly, NaV 1.7 inhibition selectively alleviated endometriosis-associated CPP without the loss of normal sensation, suggesting that selective targeting of NaV 1.7 could improve the quality of life of women with endometriosis.