Genital Tissues Research Articles

Herpesvirus surveillance in stranded striped dolphins (Stenella coeruleoalba) and bottlenose dolphins (Tursiops truncatus) from Italy with emphasis on neuropathological characterization.

Herpesvirus (HV) is widely distributed among cetacean populations, with the highest prevalence reported in the Mediterranean Sea. In this study, a comprehensive analysis was conducted, including epidemiological, phylogenetic, and pathological aspects, with particular emphasis on neuropathology, to better understand the impact of HV in these animals. Our results show a higher presence of HV in males compared to females, with males exhibiting a greater number of positive tissues. Additionally, adults were more frequently affected by HV infection than juveniles, with no infections detected in calves or neonates. The affected species were striped (Stenella coeruleoalba) and bottlenose dolphins (Tursiops truncatus). The highest positivity rates were observed in the genital system, cerebrum, and skin tissues. Phylogenetic analysis indicated a higher occurrence of Gammaherpesvirus (GHV) sequences but increased genetic diversity within Alphaherpesvirus (AHV). Key neuropathological features included astro-microgliosis (n = 4) and meningitis with minimal to mild perivascular cuffing (n = 2). The presence of concurrent infections with other pathogens, particularly cetacean morbillivirus (CeMV), underscores the complex nature of infectious diseases in cetaceans. However, the presence of lesions at the Central Nervous System (CNS) with molecular positivity for GHV, excluding the involvement of other potential neurotropic agents, would confirm the potential of this HV subfamily to induce neurological damage. Pathological examination identified lesions in other organs that could potentially be associated with HV, characterized by lymphoid depletion and tissue inflammation. These findings enhance our understanding of HV in odontocetes and highlight the need for ongoing research into the factors driving these infections and their broader implications.

Prenatal exposure to di-n-butyl phthalate promotes RIPK1-regulated necroptosis of uroepithelial cells and induces hypospadias through the epithelial-mesenchymal transition process in newborn male rats

Maternal exposure to di-n-butyl phthalate (DBP) has been linked to the induction of hypospadias; however, the underlying mechanism remains unclear. Necroptosis is reported to be implicated in developmental malformations. This study aimed to investigate the underlying mechanism of necroptosis in the development of hypospadias. DBP was dissolved in corn oil, and pregnant rats were administered a precisely measured dose of DBP (750mg/kg/day) via gastric intubation from gestation day 14 to 18. Control rats received only corn oil. The day of birth was considered postnatal day (PND) 1. Male hypospadias rats were identified on PND 7. Genital tubercle tissues were collected and stored at −80°C for subsequent PCR analysis, cryopreserved in liquid nitrogen for western blot, or fixed in formalin for immunohistochemistry (IHC) staining. IHC staining and western blot analysis revealed increased expression of RIPK1 and necroptosis markers in genital tubercle (GT) tissue compared to the control group. Additionally, higher levels of EMT and impaired androgen receptor expression were observed in GT tissue. Exposure to increased DBP concentrations in rat primary uroepithelial cells (PUCs) led to elevated ROS production. Necroptosis markers and EMT expression levels were upregulated in PUCs following DBP incubation. Notably, treatment with DBP combined with necrostatin-1, a necroptosis inhibitor, reduced the expression of EMT markers and ROS production compared to DBP treatment alone. In vitro studies further revealed that DBP-induced necroptosis promoted the degradation of E-cadherin through the ubiquitin-proteasome pathway in PUCs. Our findings suggest that maternal exposure to DBP promotes necroptosis in uroepithelial cells by elevating ROS level and EMT status. Thus, necroptosis may play an essential role in the development of hypospadias.

Effects of Platelet-Activating Factor (PAF) on the Mechanical Activities of Lower Urinary Tract and Genital Smooth Muscles.

Since its first discovery as a bioactive phospholipid inducing potent platelet aggregation, platelet-activating factor (PAF) has been shown to be involved in a wide variety of inflammatory and allergic disease states. Many pharmacological studies in the 1980s and 1990s also showed that PAF induces endothelium-dependent vascular relaxation and contraction of various smooth muscles (SMs), including those in the airway, gastrointestinal organs, and uterus. However, since the late 1990s, there have been few reports on the SM contractions induced by PAF. The lower urinary tract (LUT), particularly the urinary bladder (UB) has attracted recent attention in SM pharmacology research because patients with LUT dysfunctions including overactive bladder are increasing as the population ages. In addition, recent clinical studies have implicated the substantial role of PAF in the inflammatory state in LUT because its production increases with smoking and with cancer. However, the effects of PAF on mechanical activities of LUT SMs including UBSM have not been investigated to date. Recently, we found that PAF very strongly increased mechanical activities of UBSM in guinea pigs and mice, and partly elucidated the possible mechanisms underlying these actions of PAF. In this review, we describe the effects of PAF on LUT SMs by introducing our recent findings obtained in isolated UBSMs and discuss the physiological and pathophysiological significance. We also introduce our data showing the effects of PAF on the SM mechanical activities of genital tissues (prostate and vas deferens).

A group B streptococcal type VII-secreted LXG toxin mediates interbacterial competition and colonization of the murine female genital tract.

Group B Streptococcus (GBS) asymptomatically colonizes the vagina but can opportunistically ascend to the uterus and be transmitted vertically during pregnancy, resulting in neonatal pneumonia, bacteremia, and meningitis. GBS is a leading etiologic agent of neonatal infection and understanding the mechanisms by which GBS persists within the polymicrobial female genital mucosa has the potential to mitigate subsequent transmission and disease. Type VIIb secretion systems (T7SSb) are encoded by Bacillota and often mediate interbacterial competition using LXG toxins that contain conserved N-termini important for secretion and variable C-terminal toxin domains that confer diverse biochemical activities. Our recent work characterized a role for the GBS T7SSb in vaginal colonization and ascending infection but the mechanisms by which the T7SSb promotes GBS persistence in this polymicrobial niche remain unknown. Herein, we investigate the GBS T7SS in interbacterial competition and GBS niche establishment in the female genital tract. We demonstrate GBS T7SS-dependent inhibition of mucosal pathobiont Enterococcus faecalis both in vitro using predator-prey assays and in vivo in the murine genital tract and found that a GBS LXG protein encoded within the T7SS locus (herein named group B streptococcal LXG Toxin A) contributes to these phenotypes. We identify BltA as a T7SS substrate that is toxic to E. coli and S. aureus upon induction of intracellular expression along with associated chaperones. Finally, we show that BltA and its chaperones contribute to GBS vaginal colonization. Altogether, these data reveal a role for a novel T7b-secreted toxin in GBS mucosal persistence and competition.IMPORTANCECompetition between neighboring, non-kin bacteria is essential for microbial niche establishment in mucosal environments. Gram-positive bacteria encoding T7SSb have been shown to engage in competition through the export of LXG-motif-containing toxins, but these have not been characterized in group B Streptococcus (GBS), an opportunistic colonizer of the polymicrobial female genital tract. Here, we show a role for GBS T7SS in competition with mucosal pathobiont Enterococcus faecalis, both in vitro and in vivo. We further find that a GBS LXG protein contributing to this antagonism is exported by the T7SS and is intracellularly toxic to other bacteria; therefore, we have named this protein group B streptococcal LXG Toxin A (BltA). Finally, we show that BltA and its associated chaperones promote persistence within female genital tract tissues, in vivo. These data reveal previously unrecognized mechanisms by which GBS may compete with other mucosal opportunistic pathogens to persist within the female genital tract.

NADPH oxidase 5: Where are we now and which way to proceed?

Since the incorporation of mitochondria in early eukaryotes cells struggle to keep the deleterious effects of reactive oxygen species (ROS), mainly originating from the respiratory chain, at bay. Evolutionary adaptation to ROS burden went so far that by acting as messenger and effector molecules, ROS became important in maintaining homeostasis. The evolutionary success of this phenomenon is underscored by the arising of professional ROS-generating enzymes, namely the family of NADPH oxidases (NOXes). NOXes, by shaping ROS levels at different subcellular locations and in extracellular space, are involved in such fundamental functions as proliferation, differentiation, apoptosis, host defense, fertilization, and hormone biosynthesis. NOX5, being a calcium-regulated professional ROS source exerts its function at the crossroad of these two fundamental but potentially deleterious intracellular signaling pathways (i.e. Ca2+ and ROS). The expression of NOX5 in the adult human body under unchallenged conditions is restricted to very few sites, among which the two major tissue groups are genital organs (mainly testis) and immune tissues (mainly spleen). In cases of increased cellular proliferation and protein synthesis (e.g., diverse tumors, cultured primary cells, or sites of tissue damage) the expression and activity of NOX5 is often upregulated in various tissues. This and the evolutionary conserved nature of NOX5 would imply a very fundamental role for this enzyme, but intriguingly the genomes of rodents essentially lack the NOX5 gene. The latter fact had been a major obstacle in determining the physiological roles of NOX5 in normal tissues until the very recent generation of a NOX5-deficient rabbit model.

Bacterial vaginosis-driven changes in vaginal T cell phenotypes and their implications for HIV susceptibility.

Bacterial vaginosis (BV) is a dysbiosis of the vaginal microbiome that is prevalent in reproductive-age women worldwide. Adverse outcomes associated with BV include an increased risk of sexually acquired Human Immunodeficiency Virus (HIV), yet the immunological mechanisms underlying this association are not well understood. To investigate BV driven changes to cervicovaginal tract (CVT) and circulating T cell phenotypes, participants with or without BV provided vaginal tract (VT) and ectocervical (CX) tissue biopsies and peripheral blood mononuclear cells (PBMC). Immunofluorescence analysis of genital mucosal tissues revealed a reduced density of CD3+CD4+CCR5+ cells in the VT lamina propria of individuals with compared to those without BV (median 243.8 cells/mm2 BV- vs 106.9 cells/mm2 BV+, p=0.043). High-parameter flow cytometry of VT biopsies revealed an increased frequency in individuals with compared to those without BV of dysfunctional CD39+ conventional CD4+ T cells (Tconv) (median frequency 15% BV- vs 30% BV+, padj=0.0331) and tissue-resident CD69+CD103+ Tconv (median frequency 24% BV- vs 38% BV+, padj=0.0061), previously reported to be implicated in HIV acquisition and replication. Our data suggests that BV elicits diverse and complex VT T cell alterations and expands on potential immunological mechanisms that may promote adverse outcomes including HIV susceptibility.

(098) LOW INTENSITY SHOCKWAVE THERAPY FOR WOMEN WITH SEXUAL DYSFUNCTION

Abstract Introduction Low intensity shockwave therapy (LiSWT) has been used to treat sexual dysfunction by inducing genital tissue mechanotransduction. Mechanotransduction is the ability of a cell to actively convert contraction/expansion mechanical stimuli induced by exposure to the shockwave energy into biochemical signals with physiological effects including synthesis of nitic oxide, upregulation of vascular endothelial growth factors, recruitment of endothelial progenitor cells, vasodilation, and expression of tumor necrosis factor alpha mRNA with anti-inflammatory activity. The greater the shockwave energy absorbed in genital tissues, the greater the opportunity for mechanotransduction physiologic effects to occur and improve sexual function. Therefore, effectiveness of LiSWT depends, in part, on applied energy (mJ/mm2) and number of applications (total shocks). In 2019, our sexual medicine clinic began utilizing the SoftWave (Urogold 100™) LiSWT device, FDA cleared for pain amelioration, connective tissue activation, wound healing, and improved blood flow, to treat individuals with vulvas with sexual dysfunction. Objective To review the various sexual health concerns in individuals with vulvas effectively treated by LiSWT. Methods A chart review was performed of individuals with vulvas with various sexual dysfunctions, including genital pain and genito-pelvic dysesthesia (GPD), who received LiSWT between April 2019 and June 2023. In general, 2400 shocks were applied at energy flux density 0.05-0.08 mJ/mm2, Hz = 3, membrane pressure = 1. The Patient Global Impression of Improvement (PGI-I) was used to assess post-LiSWT outcome; improvement was positive if the post-LiSWT PGI-I ranged from 1-3. Results 189 women, mean age 32 (range 18–67) years, with sexual dysfunction underwent LiSWT treatments in the 38 months. 18 women with neuroproliferative vestibulodynia (NPV) who underwent vestibulectomy received 3-6 post-operative vestibular LiSWT to reduce post-operative pain and improve wound healing; 83% reported improved PGI-I. 24 patients with hormone-mediated vestibulodynia who elected not to use hormone treatment, underwent vestibular LiSWT; 54% reported improved PGI-I. 25 menopausal patients with persistent symptoms of Genito-Urinary Syndrome of Menopause (GSM) received multiple vestibular LiSWT treatments; 64% improved PGI-I, including reduction of urinary incontinence. 36 women with PGAD/GPD from lumbosacral annular tear-and/or Tarlov cyst-induced sacral radiculopathy who were not candidates for minimally invasive spine surgery or continued to have symptoms after recovering from spine surgery, underwent multiple treatments of lumbosacral spine LiSWT; 58% reported improved PGI-I. 86 patients with neuroproliferative vestibulodynia (NPV) underwent 2-3 pre-operative sessions of vestibular LiSWT immediately prior to complete subepithelial vestibulectomy surgery; they used significantly fewer opioids post-op than those without pre-operative LiSWT based on historical data prior to 2019. All patients received multiple LiSWT sessions to maximize mechanotransduction effects on genital tissues. There were no significant adverse events noted with LiSWT use. Conclusions Multiple LiSWT sessions are utilized in individuals with vulvas with sexual dysfunction to maximize mechanotransduction. LiSWT is associated with improved sexual health symptoms ranging from 54% - 75%, not associated with any side effects. More research is needed regarding the role of LiSWT for management of various sexual dysfunctions. Disclosure Any of the authors act as a consultant, employee or shareholder of an industry for: Softwave TRT.

(070) SUCCESSFUL TREATMENT OF NEUROPROLIFERATIVE VESTIBULODYNIA IN MENOPAUSAL PATIENTS

Abstract Introduction As pain symptoms from neuroproliferative vestibulodynia (NPV) often arise at initial attempt at tampon insertion or penetrative sex, most vestibulodynia patients present in their 20’s or 30’s. Consequently, individuals who reached this age prior to 2004-when Dr. Bornstein and colleagues delineated the excess mast cell and nerve pathology-had limited opportunity for successful NPV diagnosis and treatment. Menopausal patients often experience vulvar pain with genitourinary syndrome of menopause (GSM) due to the paucity of sex steroid hormonal support of their genital tissues. While symptoms of painful penetration are similar in both of these conditions, NPV is diagnosed when severe allodynia/hyperalgesia is still present with a normal or corrected hormonal milieu. A treatment for NPV is complete vestibulectomy surgery, performed in premenopausal eugonadal patients. However, in hormonally depleted patients with GSM, the vaginal tissue is thinner and less robust making it less optimal for reconstructive surgery. Objective To examine the intersection of genitourinary syndrome of menopause and neuroproliferative vestibulodynia symptoms in a subgroup of at-risk patients and to report techniques for optimizing management of this patient population. Methods We reviewed charts of two menopausal patients with NPV and report a clinical strategy for successful management. Results Two patients, ages 58 and 63 reported histories consistent with NPV during their reproductive years-one with lifelong entrance pain with any insertion and one with pain-free penetration prior to experiencing severe burning upon use of topical miconazole for candidiasis. At presentation, both had entry pain symptoms consistent with GSM and NPV. Since NPV is a diagnosis of exclusion, each was treated for GSM with intravaginal DHEA and vestibular topical compounded testosterone/estradiol cream. After 6 months, vestibular pallor and erythema resolved, however, cotton-tip swab testing scores did not improve. A positive improvement with vestibular anesthesia testing was consistent with NPV. Each underwent a complete vestibulectomy with vaginal advancement flap with successful wound healing and no adverse events despite their age and prior GSM diagnosis. Both patients report “very much better” symptoms on post-op Patient Global Impression of Improvement. Immunohistochemical staining of excised vestibular tissue confirmed a high density of CD117 and PGP9.5 staining consistent with an NPV diagnosis. Each patient has continued their menopause treatments post-op. Conclusions We report on two menopausal patients with NPV who had classic symptoms of severe allodynia/hyperalgesia in their reproductive years but who were not managed as NPV patients due to lack of knowledge at that time. Treating the GSM first with hormone replacement may restore health to vaginal tissues, thereby allowing the excised vestibular region to be covered with a vaginal advancement flap. Managing GSM symptoms prior and then undergoing vestibulectomy allowed them to have pain-free penetration. Disclosure No.

A group B streptococcal type VII secreted LXG toxin mediates interbacterial competition and colonization of the female genital tract.

Group B Streptococcus (GBS) asymptomatically colonizes the vagina but can opportunistically ascend to the uterus and be transmitted vertically during pregnancy, resulting in neonatal pneumonia, bacteremia and meningitis. GBS is a leading etiologic agent of neonatal infection and understanding the mechanisms by which GBS persists within the polymicrobial female genital mucosa has potential to mitigate subsequent transmission and disease. Type VIIb secretion systems (T7SSb) are encoded by Firmicutes and often mediate interbacterial competition using LXG toxins that contain conserved N-termini important for secretion and variable C-terminal toxin domains that confer diverse biochemical activities. Our recent work characterized a role for the GBS T7SSb in vaginal colonization and ascending infection but the mechanisms by which the T7SSb promotes GBS persistence in this polymicrobial niche remain unknown. Herein, we investigate the GBS T7SS in interbacterial competition and GBS niche establishment in the female genital tract. We demonstrate GBS T7SS-dependent inhibition of mucosal pathobiont Enterococcus faecalis both in vitro using predator-prey assays and in vivo in the murine genital tract and found that a GBS LXG protein encoded within the T7SS locus (herein named group B streptococcal LXG Toxin A) that contributes to these phenotypes. We identify BltA as a T7SS substrate that is toxic to E. coli and S. aureus upon induction of expression along with associated chaperones. Finally, we show that BltA and its chaperones contribute to GBS vaginal colonization. Altogether, these data reveal a role for a novel T7b-secreted toxin in GBS mucosal persistence and competition.

Infections of reproductive organs in female rabbits

Symptomatic infertility is a temporary or permanent disturbance in reproductive function wherein the animal cannot become pregnant due to diseases of genital organs or systemic diseases. Since female rabbits, due to their precociousness and fertility, are often used for reproduction, the leading causes of symptomatic infertility in them are the absence of estrous cycles or their deficiency and inflammation of tissues of the reproductive system. Infertility is observed in female rabbits developing such infectious diseases as pasteurellosis, spirochetosis, and myxomatosis. It is due to these diseases that obstetric and gynecological pathology (vulvitis, vulvovaginitis, metritis, pyometra, mastitis), long-term infertility, and death of animals are recorded most often. External genitalia (vulva) and mammary glands are the main routes of infection in female rabbits. The most common microorganisms causing inflammatory processes in reproductive organs are non-specific, such as Staphylococcus and Pasteurella, and specific microbes, such as toxoplasma. Spirochetes, Listeria, and Salmonella are much less common. It has been found that on farms of different forms of ownership in the Dnipropetrovsk region, the most common infectious diseases in rabbits are myxomatosis (20 %), viral hemorrhagic disease (13 %), eimeriosis (30 %), pasarulosis (10 %), and spirochetosis (10 %). Inflammation of genital tissues, especially inflammatory processes in the uterus, is the most common disease in female rabbits. The general frequency of uterine diseases increases with age, reaching more than 50% in aged female rabbits. Endometritis was the most frequent inflammatory process diagnosed in 10 % of cases and was potentially purulent, catarrhal, lymphoplasmacytic, or enterohemorrhagic. Metritis was observed in 5 % of cases and pyometra in 3 %. Autopsy of forcibly slaughtered or dead animals revealed such uterine diseases as hydrometer and mucometer. Hydrometers and mucometers comprised 8 % of uterine diseases, and the average age of sick female rabbits was 3 years. The etiology remains unclear but is potentially related to endometrial hyperplasia or endometritis. However, in some cases, no underlying uterine disease was found. During acute infection of the organs of the reproductive system of female rabbits, a serous mucocatarrhal or mucopurulent exudate is released from the genital slit. Chronic inflammatory processes of the genital organs often occur without signs of disease or are manifested by decreased fertility or abortion.

Irgm proteins attenuate inflammatory disease in mouse models of genital Chlamydia infection.

In response to genital Chlamydia infection, the immune system mounts a proinflammatory response to resist the pathogen, yet inflammation must be tightly controlled to avoid collateral damage and scarring to host genital tissue. Variation in the human IRGM gene is associated with susceptibility to autoinflammatory diseases but its role in ameliorating inflammatory diseases caused by infections is poorly defined. Here, we use mice deficient for all three murine Irgm paralogs to demonstrate that Irgm proteins not only provide host resistance to Chlamydia infections but also limit associated inflammation in the female genital tract. In particular, we find that murine Irgm expression prevents granulomatous inflammation, which parallels inflammatory diseases associated with variants in human IRGM. Our findings therefore establish genital Chlamydia infection as a useful model to study the roles for Irgm proteins in both promoting protective immunity and limiting pathogenic inflammation.

(124) An Open-Label, Vulvoscopic Pilot Study of Intravaginal Prasterone in Menopausal Women with Dyspareunia Examining Vestibular Tissue Changes

Abstract Introduction Prasterone (DHEA), a daily 6.5 mg intravaginal insert, is approved in the US and Canada for moderate to severe dyspareunia, a symptom of vulvovaginal atrophy from menopause. Prasterone converts intracellularly in vaginal epithelial cells into active androgens and estrogens without affecting systemic hormone blood levels. Although prasterone is a vaginal insert, the significant reduction of pain in the phase 3 registration trials suggests improvement of the vestibular endodermal genital tissue health associated with dyspareunia in androgen and estrogen deficient states like menopause. To the best of our knowledge, there have not been previous prospective studies documenting visible changes to the vestibule with daily administration of 6.5 mg prasterone in menopausal women with vulvovaginal atrophy and moderate to severe dyspareunia. Objective It is the aim of this study to investigate whether an intravaginal therapy, prasterone, could affect the vestibular tissue, assessing for changes in visual appearance, pain, and responses on sexual event diaries over the 20-week administration of intravaginal 6.5 mg prasterone. Methods This 20-week, open-label, prospective, pilot study included 11 menopausal women (median age = 56 y) treated daily with 6.5 mg prasterone intravaginal inserts and assessed every 4 weeks. During vulvoscopy, vestibular pain was assessed by cotton-tipped swab testing and vestibular and vaginal health were independently assessed using the Visual Scale (VS). In addition, vulvoscopic photographs were obtained and assessed using the Vulvoscopic Genital Tissue Appearance (VGTA) scale to evaluate overall genital tissue health. Mean changes from baseline for genital tissue health and pain assessments were analyzed by repeated measures one-way analysis of variance, followed by Dunnett’s post-hoc test. Sexual event diaries were completed, and adverse events recorded. Results Eleven participants completed the study, with a total of 1607 vulvoscopic photographs, showing reduction in vestibular erythema and pallor at end of study. Total VGTA scores at week 20 showed improvement from baseline (mean change = -7.9, p = 0.0016). Changes in VS scores were significantly reduced from baseline after active treatment by week 4 and maintained through week 20 for the vestibule (3.00, p = 0.004) and vagina (4.00, p = 0.002) respectively), suggesting improved vestibular and vaginal tissue health. Cotton-tipped swab test scores were significantly reduced from baseline after week 8 through week 20 (mean change = (-7.27, p = 0.019), suggesting decreased pain. Diary results showed decrease in pain from 81.3% during the first month to 8.3% during the last month of the study, with sexual activities discontinued due to discomfort reduced from 45.8% to 6.3% respectively. No prasterone related adverse events were reported. Conclusions This is the first prospective vulvoscopic study showing that intravaginal hormone therapy significantly improves total VGTA, VS and cotton-tipped swab test scores. This observation may explain the robust effect of prasterone in lowering dyspareunia in menopausal women with moderate to severe dyspareunia. Our findings suggest that intravaginal prasterone exerts biologic activity on the androgenic endodermal vestibule, as the medication passes from vagina to vestibule, resulting in amelioration of pain associated with sexual activity. Disclosure Yes, this is sponsored by industry/sponsor: AMAG Pharmaceuticals, Inc. Clarification: Industry funding only - investigator initiated and executed study.

Comparison between a deep-learning and a pixel-based approach for the automated quantification of HIV target cells in foreskin tissue

The availability of target cells expressing the HIV receptors CD4 and CCR5 in genital tissue is a critical determinant of HIV susceptibility during sexual transmission. Quantification of immune cells in genital tissue is therefore an important outcome for studies on HIV susceptibility and prevention. Immunofluorescence microscopy allows for precise visualization of immune cells in mucosal tissues; however, this technique is limited in clinical studies by the lack of an accurate, unbiased, high-throughput image analysis method. Current pixel-based thresholding methods for cell counting struggle in tissue regions with high cell density and autofluorescence, both of which are common features in genital tissue. We describe a deep-learning approach using the publicly available StarDist method to count cells in immunofluorescence microscopy images of foreskin stained for nuclei, CD3, CD4, and CCR5. The accuracy of the model was comparable to manual counting (gold standard) and surpassed the capability of a previously described pixel-based cell counting method. We show that the performance of our deep-learning model is robust in tissue regions with high cell density and high autofluorescence. Moreover, we show that this deep-learning analysis method is both easy to implement and to adapt for the identification of other cell types in genital mucosal tissue.

Aging dysregulates neutrophil extracellular trap formation in response to HIV in blood and genital tissues.

Women acquire HIV through sexual transmission, with increasing incidence in women >50 years old. Identifying protective mechanisms in the female genital tract (FGT) is important to prevent HIV-acquisition in women as they age. Human genital and blood neutrophils inactivate HIV by releasing neutrophil extracellular traps (NETs), an innate protective mechanism against HIV-infection. However, how NET formation is triggered by HIV in different tissues and whether this mechanism is affected by aging remain unknown. We demonstrate that the mechanisms that trigger NET release in response to HIV are different in blood and genital tissues, and that NET release decreases with aging. In blood neutrophils, HIV stimulation independently activated calcium pathways and endosomal TLR8, but aging reduced calcium responses, resulting in delayed NET release. In contrast, calcium responses were absent in genital neutrophils and NET release was triggered preferentially through TLR8 activation, but aging impaired this pathway. HIV induced NET formation through non-lytic pathways in blood and FGT neutrophils, except for a small subset of NETs that incorporated annexin V and lactoferrin predominantly in blood, suggesting proinflammatory and lytic NET release. Our findings demonstrate that blood neutrophils cannot model genital neutrophil responses which has important implications to understanding protection against HIV acquisition.

Knowledge of and Attitudes to Female Genital Mutilation Among users of the Gynaecology and Obstetrics Service at the Reference Health Center of Commune I of Bamako, Mali

Female genital mutilation is harmful to girls and women in many ways. First and foremost, it is painful and traumatic, and the removal of normal, healthy genital tissue interferes with the body's natural functioning. They have a variety of immediate and long-term health consequences. The aim of this study was to investigate the knowledge and attitudes of female genital mutilation users (women) in the obstetrics and gynaecology department of the commune I reference health centre in the Bamako district. Methodology: This was a cross-sectional, prospective, descriptive study designed to investigate users' (women's) knowledge of and attitudes towards female genital mutilation. It took place from 1 November 2019 to 30 April 2020, a period of 6 months, in the gynaecology and obstetrics department of the reference health centre in Commune I of the Bamako district. We included all women admitted to the department for gynaecological or obstetric consultation after informed consent had been obtained. Result: Out of a total of 188 female users, 176 of them were excised, a frequency of 93.6%. Female users reported having been excised as children in 93.9% of cases. Most users (59.6%) want to excise their daughters for traditional and religious reasons. Female genital mutilation (FGM), although practised by traditional excisers, is still being medicalised. The reason for FGM was unknown to 25.0% of the women surveyed. In 44.8% of cases, the users (women) had suffered a perineal tear during childbirth.

The Role of Extracellular Traps in HIV Infection.

Human immunodeficiency virus (HIV) infection is still an important public health problem, which justifies the research of new therapies to combat it. Recent studies show that Extracellular Traps (ETs) are cellular mechanisms useful in the capture and destruction of some viruses, such as the HIV. Here, we show that neutrophils from peripheral blood, genital tissues, and placenta are activated when exposed to human immunodeficiency virus type 1 (HIV-1) and release Neutrophil Extracellular Traps (NETs). The NETs can capture, neutralize, and inactivate the virus and, also, protect other target cells from HIV infection, as long as the DNA and other constituents of the NETs remain intact. Further, the review indicates that the immunoprotective role of NETs in the context of HIV-1 infection is a promising finding for the development of new antiviral therapies. It is necessary, however, the development of studies that evaluate the tissue injury that NETs can cause and the biological relationships with other cells to improve them as therapeutic targets.

Boosting of vaginal HSV-2-specific B and T cell responses by intravaginal therapeutic immunization results in diminished recurrent HSV-2 disease.

Boosting herpes simplex virus (HSV)-specific immunity in the genital tissues of HSV-positive individuals to increase control of HSV-2 recurrent disease and virus shedding is an important goal of therapeutic immunization and would impact HSV-2 transmission. Experimental therapeutic HSV-2 vaccines delivered by a parenteral route have resulted in decreased recurrent disease in experimental animals. We used a guinea pig model of HSV-2 infection to test if HSV-specific antibody and cell-mediated responses in the vaginal mucosa would be more effectively increased by intravaginal (Ivag) therapeutic immunization compared to parenteral immunization. Therapeutic immunization with HSV glycoproteins and CpG adjuvant increased glycoprotein-specific IgG titers in vaginal secretions and serum to comparable levels in Ivag- and intramuscular (IM)-immunized animals. However, the mean numbers of HSV glycoprotein-specific antibody secreting cells (ASCs) and IFN-γ SCs were greater in Ivag-immunized animals demonstrating superior boosting of immunity in the vaginal mucosa compared to parenteral immunization. Therapeutic Ivag immunization also resulted in a significant decrease in the cumulative mean lesion days compared to IM immunization. There was no difference in the incidence or magnitude of HSV-2 shedding in either therapeutic immunization group compared to control-treated animals. Collectively, these data demonstrated that Ivag therapeutic immunization was superior compared to parenteral immunization to boost HSV-2 antigen-specific ASC and IFN-γ SC responses in the vagina and control recurrent HSV-2 disease. These results suggest that novel antigen delivery methods providing controlled release of optimized antigen/adjuvant combinations in the vaginal mucosa would be an effective approach for therapeutic HSV vaccines. IMPORTANCE HSV-2 replicates in skin cells before it infects sensory nerve cells where it establishes a lifelong but mostly silent infection. HSV-2 occasionally reactivates, producing new virus which is released back at the skin surface and may be transmitted to new individuals. Some HSV-specific immune cells reside at the skin site of the HSV-2 infection that can quickly activate and clear new virus. Immunizing people already infected with HSV-2 to boost their skin-resident immune cells and rapidly control the new HSV-2 infection is logical, but we do not know the best way to administer the vaccine to achieve this goal. In this study, a therapeutic vaccine given intravaginally resulted in significantly better protection against HSV-2 disease than immunization with the same vaccine by a conventional route. Immunization by the intravaginal route resulted in greater stimulation of vaginal-resident, virus-specific cells that produced antibody and produced immune molecules to rapidly clear virus.

OR2H2 Activates CAMKKβ-AMPK-Autophagy Signaling Axis and Suppresses Senescence in VK2/E6E7 Cells.

Olfactory receptors are expressed in multiple extra-nasal tissues and these ectopic olfactory receptors mediate tissue-specific functions and regulate cellular physiology. Ectopic olfactory receptors may play key roles in tissues constantly exposed to odorants, thus the functionality of these receptors in genital tissues is of particular interest. The functionality of ectopic olfactory receptors expressed in VK2/E6E7 human vaginal epithelial cells was investigated. OR2H2 was the most highly expressed olfactory receptor expressed in VK2/E6E7 cells, and activation of OR2H2 by aldehyde 13-13, a ligand of OR2H2, increased the intracellular calcium and cAMP concentrations. Immunoblotting demonstrated that activation of OR2H2 by aldehyde 13-13 stimulated the CAMKKβ-AMPK-mTORC1-autophagy signaling axis, and that these effects were negated by OR2H2 knockdown. AMPK is known to regulate senescence; consequently, we investigated further the effect of aldehyde 13-13 on senescence. In H2O2-induced senescent cells, activation of OR2H2 by aldehyde 13-13 restored proliferation, and reduced the expression of senescence markers, P16 and P19. Additionally, aldehyde 13-13 induced apoptosis of H2O2-induced senescent cells, compared with non-senescent normal cells. In vivo, aldehyde 13-13 increased the lifespan of Caenorhabditis elegans and budding yeast. These findings demonstrate that OR2H2 is a functional receptor in VK2/E6E7 cells, and that activation of OR2H2 activates the AMPK-autophagy axis, and suppresses cellular aging and senescence, which may increase cellular health.

Tissue specific trisomy 15 mosaicism associated with urogenital malformations

We describe a boy born with hypospadias and later diagnosed with vesicoureteral reflux and mild cognitive disability. Routine diagnostic investigation by karyotyping, chromosomal microarray (CMA) and trio analysis with whole exome sequencing was normal. However, later CMA performed on DNA from genital tissue showed trisomy 15, which prompted further analysis.Fluorescent in situ hybridization was performed to verify the CMA result and delineate the mosaic rate. Methylation specific MLPA was performed to investigate the parent of origin of the extra chromosome 15.Further medical examination of the boy identified fine Blaschko's lines, indicative of mosaicism, but earlier unnoticed. CMA on genital tissue showed 80% mosaicism for trisomy 15. Bladder mucosa and muscle showed a high degree of trisomy 15 (56% and 45% respectively), while buccal mucosa and abdominal skin showed low-grade or no trisomy 15. The extra chromosome 15 was of maternal origin.This case report describes a boy with two different malformations in the same organ region that carries a high degree of trisomy 15 mosaicism. Hence, the clinical implication is that there is no recurrence risk for sibs, but the boy in his turn risks producing gametes with an extra chromosome 15. Tissue restricted mutations are not commonly described but may cause congenital malformations that affects the information to the family.

Aging modifies endometrial dendritic cell function and unconventional double negative T cells in the human genital mucosa

BackgroundImmune function in the genital mucosa balances reproduction with protection against pathogens. As women age, genital infections, and gynecological cancer risk increase, however, the mechanisms that regulate cell-mediated immune protection in the female genital tract and how they change with aging remain poorly understood. Unconventional double negative (DN) T cells (TCRαβ + CD4-CD8-) are thought to play important roles in reproduction in mice but have yet to be characterized in the human female genital tract. Using genital tissues from women (27–77 years old), here we investigated the impact of aging on the induction, distribution, and function of DN T cells throughout the female genital tract.ResultsWe discovered a novel site-specific regulation of dendritic cells (DCs) and unconventional DN T cells in the genital tract that changes with age. Human genital DCs, particularly CD1a + DCs, induced proliferation of DN T cells in a TFGβ dependent manner. Importantly, induction of DN T cell proliferation, as well as specific changes in cytokine production, was enhanced in DCs from older women, indicating subset-specific regulation of DC function with increasing age. In human genital tissues, DN T cells represented a discrete T cell subset with distinct phenotypical and transcriptional profiles compared to CD4 + and CD8 + T cells. Single-cell RNA and oligo-tag antibody sequencing studies revealed that DN T cells represented a heterogeneous population with unique homeostatic, regulatory, cytotoxic, and antiviral functions. DN T cells showed relative to CD4 + and CD8 + T cells, enhanced expression of inhibitory checkpoint molecules and genes related to immune regulatory as well as innate-like anti-viral pathways. Flow cytometry analysis demonstrated that DN T cells express tissue residency markers and intracellular content of cytotoxic molecules. Interestingly, we demonstrate age-dependent and site-dependent redistribution and functional changes of genital DN T cells, with increased cytotoxic potential of endometrial DN T cells, but decreased cytotoxicity in the ectocervix as women age, with implications for reproductive failure and enhanced susceptibility to infections respectively.ConclusionsOur deep characterization of DN T cell induction and function in the female genital tract provides novel mechanistic avenues to improve reproductive outcomes, protection against infections and gynecological cancers as women age.