Deciphering the role of female reproductive tract microbiome in reproductive health: a review.

Relevant studies increasingly indicate that female reproductive health is confronted with substantial challenges. Emerging research has revealed that the microbiome interacts with the anatomy, histology, and immunity of the female reproductive tract, which are the cornerstone of maintaining female reproductive health and preventing adverse pregnancy outcomes. Currently, the precise mechanisms underlying their interaction and impact on physiological functions of the reproductive tract remain elusive, constituting a prominent area of investigation within the field of female reproductive tract microecology. From this new perspective, we explore the mechanisms of interactions between the microbiome and the anatomy, histology, and immunity of the female reproductive tract, factors that affect the composition of the microbiome in the female reproductive tract, as well as personalized medicine approaches in managing female reproductive tract health based on the microbiome. This study highlights the pivotal role of the female reproductive tract microbiome in maintaining reproductive health and influencing the occurrence of reproductive tract diseases. These findings support the exploration of innovative approaches for the prevention, monitoring and treatment of female reproductive tract diseases based on the microbiome.

Comparison of Siglec-1 protein networks and expression patterns in sperm and male reproductive tracts of mice, rats, and humans.

Background: Sialic acid-binding immunoglobulin-like lectin 1 (Siglec-1) is a transmembrane glycoprotein involved in the sialic acid (Sia)-dependent regulation of the immune system. Siglec-1 expression has recently been identified in the male reproductive tract (MRT) of several species, including humans, cattle, horses, and sheep, and may play a role in modulating fertility in a Sia-dependent manner. Materials and Methods: In this study, protein-protein interaction (PPI) analysis of Siglec-1 was conducted to identify associated network protein conservation, and the expression of Siglec-1 in the MRT of mice and rats, including their accessory sex glands and spermatozoa was determined by immunostaining. Results: Network analysis of proteins with Siglec-1 in mice and rats demonstrated significant similarity to human Siglec-1 networks, suggesting a similar conservation of network proteins between these species and, hence, a potential conservation role in immune modulation and function. Specific immunostaining patterns of mouse and rat testes, epididymis, ductus deferens, accessory sex gland tissues, and sperm were detected using human Siglec-1. These results confirmed that the human Siglec-1 antibody could cross-react with mouse and rat Siglec-1, suggesting that the specific expression patterns of Siglec-1 in the MRT and sperm of both mice and rats are similar to those observed in other species. Conclusions: The conservation of Siglec-1 expression patterns in sperm and within the MRT and the similarity of protein networks for Siglec-1 across species suggest that Siglec-1 may function in a similar manner across species. These results also suggest that rodents may serve as a valuable model system for exploring the function of Siglecs in the reproductive system across species and their potential role in modulating fertility in a Sia-dependent manner.

Torsion in Uterus: An Obstetrical and Gynaecological Emergency.

Uterine torsion is defined as torsion of the uterus along its longitudinal axis greater than 45 degrees. It is observed in all age groups of the reproductive period, in all parity groups, and at all stages of pregnancy. Torsion from 60 degrees to 720 degrees has been described. It is not possible to clarify why it occurs, but numerous abnormalities have appeared with uterine torsion. It is a rare complication that can result in placental abruption and intrauterine foetal death. Pregnancy, giant fibroids, and ovarian cysts are among the most common causes. Vague clinical attributes make diagnosis challenging pre-operatively and can be missed on routine ultrasound. Being a rare life-threatening condition, it necessitates a high level of concern for diagnosis and prompt intervention to optimise results. This review will help the healthcare worker to understand the various presentation of uterine torsion and their management by appropriately and timely diagnosing it.

Case report: Synchronous tumors of the female reproductive tract in systemic lupus erythematosus: report of two cases and review of the literature.

Background: Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that affects multiple systems. Patients with SLE are prone to a variety of malignancies, especially neoplasms of the female reproductive tract. Synchronous tumors, considered to involve multiple sites, are rare in the female reproductive tract. There are hardly any reports of SLE with synchronous reproductive tract tumors. Case presentation: We report the occurrence of two to three reproductive tract tumors in two women with SLE. A 52-year-old woman was diagnosed with vulvar cancer and cervical cancer. Another woman, aged 67, was diagnosed with concurrent vulvar cancer, vaginal cancer, and cervical cancer and also presented with a suspected lung cancer. Conclusion: The presence of synchronous tumors of the reproductive tract in patients with SLE is uncommon and can be easily disregarded. It is crucial to highlight that SLE patients with multiple primary malignancies exhibit notable late-stage presentation at the time of diagnosis, inadequate disease-free survival, poor overall survival, rapid progression rates, and mortality. Consequently, greater awareness must be raised regarding synchronous reproductive tract tumors in patients with SLE. Regular comprehensive cancer screening and management should be implemented for individuals diagnosed with SLE.

How sperm protects itself: A journey in the female reproductive system.

Sperm must pass a complex route in the female reproductive tract (FRT) to reach the fertilization site and join the oocyte. Thus, it should employ several mechanisms to survive against the female immune system, fertilize the oocyte, and successfully transmit paternal genes to the next generation. In addition to self-protection, sperm may be involved in the immune tolerance to the developing embryo and regulating the FRT for embryo implantation and subsequent pregnancy. Hence, this review intends to summarize the mechanisms that protect sperm in the FRT: including immunomodulatory factors that are carried by seminal plasma, cell-to-cell and molecular interaction of sperm with epithelial and immune cells of the FRT, high regulated secretions of inflammatory factors such as cytokines, chemokines, and growth factors, inducing immune tolerance to paternal antigens, and specialized expression of cell receptors and binding proteins. In most of these events sperm induces the FRT to protect itself by modulating immune responses for its own benefit. However, not all sperm in the semen are able to trigger the survival mechanisms and only high-quality sperm will overcome this challenge. A clear understanding of the molecular mechanisms that maintain sperm viability and function in the FRT can lead to new knowledge about infertility etiology and a new approach in assisted reproductive technologies for the preparation and selection of the best sperm based on the criteria that physiologically happen in-vivo.

MAP3K1 regulates female reproductive tract development.

Mitogen-activated protein 3 kinase 1 (MAP3K1) has a plethora of cell type-specific functions not yet fully understood. Herein, we describe a role for MAP3K1 in female reproductive tract (FRT) development. MAP3K1 kinase domain-deficient female mice exhibited an imperforate vagina, labor failure and infertility. These defects corresponded with shunted Müllerian ducts (MDs), the embryonic precursors of FRT, that manifested as a contorted caudal vagina and abrogated vaginal-urogenital sinus fusion in neonates. The MAP3K1 kinase domain is required for optimal activation of the Jun-N-terminal kinase (JNK) and cell polarity in the MD epithelium, and for upregulation of WNT signaling in the mesenchyme surrounding the caudal MD. The MAP3K1-deficient epithelial cells and MD epithelium had reduced expression of WNT7B ligands. Correspondingly, conditioned media derived from MAP3K1-competent, but not -deficient, epithelial cells activated a TCF/Lef-luciferase reporter in fibroblasts. These observations indicate that MAP3K1 regulates MD caudal elongation and FRT development, in part through the induction of paracrine factors in the epithelium that trans-activate WNT signaling in the mesenchyme.

Interferon-epsilon is a novel regulator of NK cell responses in the uterus.

The uterus is a unique mucosal site where immune responses are balanced to be permissive of a fetus, yet protective against infections. Regulation of natural killer (NK) cell responses in the uterus during infection is critical, yet no studies have identified uterine-specific factors that control NK cell responses in this immune-privileged site. We show that the constitutive expression of IFNε in the uterus plays a crucial role in promoting the accumulation, activation, and IFNγ production of NK cells in uterine tissue during Chlamydia infection. Uterine epithelial IFNε primes NK cell responses indirectly by increasing IL-15 production by local immune cells and directly by promoting the accumulation of a pre-pro-like NK cell progenitor population and activation of NK cells in the uterus. These findings demonstrate the unique features of this uterine-specific type I IFN and the mechanisms that underpin its major role in orchestrating innate immune cell protection against uterine infection.

Influence of connatural factors in shaping vaginal microflora and ensuring its health.

Vaginal canal (VC) is exposed to the external environment affected by habitual factors like hygiene and sexual behaviour as well as physiological factors like puberty, menstrual cycle, pregnancy, child birth and menopause. Healthy VC harbours beneficial microflora supported by vaginal epithelium and cervical fluid. Connatural antimicrobial peptide (AMPs) of female reproductive tract (FRT) conjunctly with these beneficial microbes provide protection from a large number of infectious diseases. Such infections may either be caused by native microbes of the VC or transitory microbes like bacteria or virus which are not a part of VC microflora. This review highlight's the role of hormones, enzymes, innate immunological factors, epithelial cells and vaginal mucus that support beneficial microbes over infectious ones thus, helping to maintain homeostasis in VC and further protect the FRT. We also discuss the prospective use of vaginal probiotics and AMPs against pathogens which can serve as a potential cure for vaginal infections.

Finishing the egg.

Gamete development is a fundamental process that is highly conserved from early eukaryotes to mammals. As germ cells develop, they must coordinate a dynamic series of cellular processes that support growth, cell specification, patterning, the loading of maternal factors (RNAs, proteins, and nutrients), differentiation of structures to enable fertilization and ensure embryonic survival, and other processes that make a functional oocyte. To achieve these goals, germ cells integrate a complex milieu of environmental and developmental signals to produce fertilizable eggs. Over the past 50 years, Drosophila oogenesis has risen to the forefront as a system to interrogate the sophisticated mechanisms that drive oocyte development. Studies in Drosophila have defined mechanisms in germ cells that control meiosis, protect genome integrity, facilitate mRNA trafficking, and support the maternal loading of nutrients. Work in this system has provided key insights into the mechanisms that establish egg chamber polarity and patterning as well as the mechanisms that drive ovulation and egg activation. Using the power of Drosophila genetics, the field has begun to define the molecular mechanisms that coordinate environmental stresses and nutrient availability with oocyte development. Importantly, the majority of these reproductive mechanisms are highly conserved throughout evolution, and many play critical roles in the development of somatic tissues as well. In this chapter, we summarize the recent progress in several key areas that impact egg chamber development and ovulation. First, we discuss the mechanisms that drive nutrient storage and trafficking during oocyte maturation and vitellogenesis. Second, we examine the processes that regulate follicle cell patterning and how that patterning impacts the construction of the egg shell and the establishment of embryonic polarity. Finally, we examine regulatory factors that control ovulation, egg activation, and successful fertilization.

Upregulation of IFNE in cervical biopsies of patients with high-risk human papillomavirus infections.

PROBLEM: Interferon epsilon (IFN-ε) is constitutively expressed in the epithelium of female reproductive tract and confers vital protection against sexually transmitted pathogens in mouse models. However, there is limited insight into the role of IFN-ε in human sexually transmitted infections such as human papillomavirus (HPV). METHOD OF STUDY: Cervical biopsies were obtained from high-risk (HR) HPV positive (n = 28) and HR-HPV negative (n = 10) women. mRNA expression of IFN-ε in cervical tissues was measured by qPCR. Expression of the IFN-ε protein was determined by Western blot analysis, immunohistochemistry and immunofluorescence staining. RESULTS: mRNA expression of IFN-ε was higher in the ectocervix than that of other IFNs, and was further upregulated in HR-HPV positive women compared with HR-HPV negative women. Expression of the IFN-ε protein was comparable between HR-HPV infected patients and healthy controls. CONCLUSIONS: These results reveal differential expression of IFN-ε mRNA between individuals with or without HR-HPV infection, and imply direct or indirect regulatory mechanisms for IFN-ε transcription by HPV. Expression of IFN-ε protein in HPV infections would require further validation.

BHLHE40 drives protective polyfunctional CD4 T cell differentiation in the female reproductive tract against Chlamydia.

The protein basic helix-loop-helix family member e40 (BHLHE40) is a transcription factor recently emerged as a key regulator of host immunity to infections, autoimmune diseases and cancer. In this study, we investigated the role of Bhlhe40 in protective T cell responses to the intracellular bacterium Chlamydia in the female reproductive tract (FRT). Mice deficient in Bhlhe40 exhibited severe defects in their ability to control Chlamydia muridarum shedding from the FRT. The heightened bacterial burdens in Bhlhe40-/- mice correlated with a marked increase in IL-10-producing T regulatory type 1 (Tr1) cells and decreased polyfunctional CD4 T cells co-producing IFN-γ, IL-17A and GM-CSF. Genetic ablation of IL-10 or functional blockade of IL-10R increased CD4 T cell polyfunctionality and partially rescued the defects in bacterial control in Bhlhe40-/- mice. Using single-cell RNA sequencing coupled with TCR profiling, we detected a significant enrichment of stem-like T cell signatures in Bhlhe40-deficient CD4 T cells, whereas WT CD4 T cells were further down on the differentiation trajectory with distinct effector functions beyond IFN-γ production by Th1 cells. Altogether, we identified Bhlhe40 as a key molecular driver of CD4 T cell differentiation and polyfunctional responses in the FRT against Chlamydia.

Qualified sperm selection based on the rheotaxis and thigmotaxis in a microfluidic system.

Microfluidic systems with the ability to mimic the female reproductive tract (FRT) and sperm features have emerged as promising methods to separate sperm with higher quality for the assistant reproductive technology. Thereby, we designed and fabricated a microfluidic system based on FRT features with a focus on rheotaxis and thigmotaxis for passive sperm separation. In this regard, four various geometries (linear, square, zigzag, and sinusoidal) were designed, and the effect of rheotaxis and thigmotaxis were investigated. Although separated sperm in all microchannels were 100% motile, non-linear geometries were more effective than linear geometry in the term of separating the progressive sperm with high quality. In the presence of upstream flow, periodical changes in the slope of walls (in non-linear geometries) give rise to the periodical facing sperm with a high flow rate in the middle of microchannels, which was a reason for the high quality of separated sperm. However, because of sharp corners in the square and zigzag microchannels that create dead zones with a lack of upstream flow, which is noticeable via simulation results, these geometries have obstacles against sperm swimming toward the outlet, which was proved by image analysis. The sinusoidal geometry showed the highest enhancement level of the designed geometries compared to the linear geometry. Separated sperm exhibited 34.7% normal morphology, 100% motility, and 100% viability in the sinusoidal geometry. Therefore, the periodic change in the position of sperm from one wall to another wall can be a strategy for separating sperm with high quality. Graphical abstract: In the present study, we used a microfluidic system for studying the combined effects of thigmotaxis and rheotaxis for sperm separation process to achieve the successful Assisted reproductive technology (ART). The designed PDMS-based microfluidic system had four various geometries, including linear, square, zigzag, and sinusoidal. The functionality of separated sperm was evaluated by sperm tracking (ImageJ), motility assay (CASA software), and morphology assay (Papanicolaou ultrafast staining). Probing various geometries revealed 100% motility. In non-linear geometries, sperm's periodic detachment from the walls gave rise to the periodic interaction with the high flow velocity in the center of the channel, resulting in the separation of high-quality sperm with progressive motility. The collected data proved the influence of thigmotaxis on the quality of separated sperm. Morphologically improvement in separated sperm from the sinusoidal geometry was significant than others, which means the sinusoidal structure would be the best candidate for the sperm separation process. Supplementary Information: The online version contains supplementary material available at 10.1007/s13534-023-00294-8.

Buffalo sperm membrane glycan-binding proteins reveal precise and preferential binding signatures with specific glycans targets on oviduct epithelium and zona pellucida-an implication in fertilization.

Sperm membrane glycan-binding proteins (lectins) interact with the counterpart glycans in the oviduct, oocytes, and vice-versa. It has already been well known that specific glycans are present on oviductal epithelium and zona pellucida (ZP) in different mammalian species. Some of these glycans are necessary for oviductal sperm reservoir formation and gamete recognition. The specific binding phenomenon of lectin-glycans is one of the vital factors for successful fertilization in mammals. We hypothesized that buffalo sperm membrane glycan-binding proteins have specific glycan targets in the oviduct and ZP supporting the fertilization event. In the present investigation, sperm membrane proteins were extracted and assessed for their binding capacity with glycans using a high-throughput glycan microarray. The most promising glycan binding signals were evaluated to confirm the sperm putative receptors for glycan targets in the oviductal epithelial cells (OEC) and on ZP using an in-vitro competitive binding inhibition assay. Based on an array of 100 glycans, we found that N-acetyllactosamine (LacNAc), Lewis-a trisaccharide, 3'-sialyllactosamine and LacdiNAc were the most promising glycans and selected for further in-vitro validation. We established an inhibitory concentration of 12 mM Lewis-a trisaccharide and 10 µg/ml Lotus tetragonolobus (LTL) lectin for the sperm-OEC binding interaction, indicating its specificity and sensitivity. We observed that 3 mM 3'-sialyllactosamine, and LacdiNAc were the most competitive inhibitory concentration in sperm-ZP binding, suggesting a specific and abundance-dependent binding affinity. The competitive binding affinity of Maackia amurensis (MAA) lectin with Neu5Ac(α2-3)Gal(ß1-4)GlcNAc further supports the abundance of 3'-sialyllactosamine on ZP responsible for sperm binding. Our findings develop the strong evidence on buffalo sperm putative receptors underlying their locking specificities with Lewis-a trisaccharide in oviduct and 3'-sialyllactosamine on ZP. The functional interaction of buffalo sperm lectins with the target glycans in OEC and ZP appears to be accomplished in an abundance-dependent manner, facilitating the fertilization event in buffaloes.

Safety of multiple administrations of spermicidal LL-37 antimicrobial peptide into the mouse female reproductive tract.

We have previously demonstrated spermicidal activity of LL-37 antimicrobial peptide on mouse/human sperm and its contraceptive effects in female mice. With its microbicidal action against Neisseria gonorrhoeae, LL-37 warrants development into a multipurpose prevention technology (MPT) agent for administering into the female reproductive tract (FRT). However, it is important to verify that multiple administrations of LL-37 do not lead to damage of FRT tissues and/or irreversible loss of fecundity. Herein, we transcervically injected LL-37 (36 µM-10× spermicidal dose) into female mice in estrus in three consecutive estrous cycles. A set of mice were sacrificed for histological assessment of the vagina/cervix/uterus 24 h after the last injection, while the second set were artificially inseminated with sperm from fertile males 1 week afterwards, and then monitored for pregnancy. Mice injected with PBS in parallel were regarded as negative controls, whereas those injected with vaginal contraceptive foam (VCF, available over the counter), containing 12.5% nonoxynol-9, served as positive controls for vaginal epithelium disruption. We demonstrated that the vagina/cervix/uterus remained normal in both LL-37-injected and PBS-injected mice, which also showed 100% resumption of fecundity. In contrast, VCF-injected mice showed histological abnormalities in the vagina/cervix/uterus and only 50% of them resumed fecundity. Similarly, LL-37 multiply administered intravaginally caused no damage to FRT tissues. While our results indicate the safety of multiple treatments of LL-37 in the mouse model, similar studies have to be conducted in non-human primates and then humans. Regardless, our study provides an experimental model for studying in vivo safety of other vaginal MPT/spermicide candidates.

The carboxypeptidase B and carbonic anhydrase genes play a reproductive regulatory role during multiple matings in Ophraella communa.

Seminal fluid proteins (SFPs) are key factors in sexual reproduction and are transferred to females during mating with sperm. SFPs have a nutritional value because they protect and activate sperm storage and release to optimize fecundity. Multiple matings promote ovipositioning in several insect species. Therefore, insects may obtain more SFP through multiple matings to maximize reproduction, but this process has not yet been clearly confirmed. Here, the relationship between multiple matings and the SFPs in Ophraella communa (Coleoptera: Chrysomelidae), a biological control agent of the common ragweed Ambrosia artemisiifolia (Asterales: Asteraceae), was studied. Multiple matings significantly increased female fecundity and ovary egg deposition. Carboxypeptidase B (OcCpb) and carbonic anhydrase (OcCa) genes were identified as putative SFP genes in O. communa and they showed strong male-biased expression. Additionally, OcCpb and OcCa expression was upregulated in the bursa copulatrix of mating females compared to that in virgin females, but their expression gradually declined after copulation. Furthermore, OcCpb and OcCa knockdown in males led to a decrease in insect fecundity compared to that in the control. The reproductive tract of females mated with dsRNA-treated males was dissected and observed and, notably, the ovaries produced significantly fewer eggs. These data suggest that OcCpb and OcCa play regulatory roles during multiple matings in O. communa.

Evolutionary Quantitative Proteomics of Reproductive Protein Divergence in Drosophila.

Reproductive traits often evolve rapidly between species. Understanding the causes and consequences of this rapid divergence requires characterization of female and male reproductive proteins and their effect on fertilization success. Species in the Drosophila virilis clade exhibit rampant interspecific reproductive incompatibilities, making them ideal for studies on diversification of reproductive proteins and their role in speciation. Importantly, the role of intraejaculate protein abundance and allocation in interspecific divergence is poorly understood. Here, we identify and quantify the transferred male ejaculate proteome using multiplexed isobaric labeling of the lower female reproductive tract before and immediately after mating using three species of the virilis group. We identified over 200 putative male ejaculate proteins, many of which show differential abundance between species, suggesting that males transfer a species-specific allocation of seminal fluid proteins during copulation. We also identified over 2000 female reproductive proteins, which contain female-specific serine-type endopeptidases that showed differential abundance between species and elevated rates of molecular evolution, similar to that of some male seminal fluid proteins. Our findings suggest that reproductive protein divergence can also manifest in terms of species-specific protein abundance patterns.

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.

Non-human primate models for understanding the impact of the microbiome on pregnancy and the female reproductive tract†.

The microbiome has been shown, or implicated to be involved, in multiple facets of human health and disease, including not only gastrointestinal health but also metabolism, immunity, and neurology. Although the predominant focus of microbiome research has been on the gut, other microbial communities such as the vaginal or cervical microbiome are likely involved in physiological homeostasis. Emerging studies also aim to understand the role of different microbial niches, such as the endometrial or placental microbial communities, on the physiology and pathophysiology of reproduction, including their impact on reproductive success and the etiology of adverse pregnancy outcomes (APOs). The study of the microbiome during pregnancy, specifically how changes in maternal microbial communities can lead to dysfunction and disease, can advance the understanding of reproductive health and the etiology of APOs. In this review, we will discuss the current state of non-human primate (NHP) reproductive microbiome research, highlight the progress with NHP models of reproduction, and the diagnostic potential of microbial alterations in a clinical setting to promote pregnancy health. NHP reproductive biology studies have the potential to expand the knowledge and understanding of female reproductive tract microbial communities and host-microbe or microbe-microbe interactions associated with reproductive health through sequencing and analysis. Furthermore, in this review, we aim to demonstrate that macaques are uniquely suited as high-fidelity models of human female reproductive pathology.

Gardnerella Revisited: Species Heterogeneity, Virulence Factors, Mucosal Immune Responses, and Contributions to Bacterial Vaginosis.

Gardnerella species are associated with bacterial vaginosis (BV) and have been investigated as etiological agents of the condition. Nonetheless, the isolation of this taxon from healthy individuals has raised important questions regarding its etiological role. Recently, using advanced molecular approaches, the Gardnerella genus was expanded to include several different species that exhibit differences in virulence potential. Understanding the significance of these different species with respect to mucosal immunity and the pathogenesis and complications of BV could be crucial to solving the BV enigma. Here, we review key findings regarding the unique genetic and phenotypic diversity within this genus, virulence factors, and effects on mucosal immunity as they stand. We also comment on the relevance of these findings to the proposed role of Gardnerella in BV pathogenesis and in reproductive health and identify key gaps in knowledge that should be explored in the future.