Cervico-Vaginal Immunoglobulin G Levels Increase Post-Ovulation Independently of Neutrophils

The prevalence of sexually transmitted infections (STIs) is often higher in females than in males. Although the reproductive cycle profoundly modulates local immunity in the female reproductive tract (FRT) system, significant gaps in our knowledge of the immunobiology of the FRT still exist. An intriguing and frequently observed characteristic of the FRT is the predominant presence of immunoglobulin (Ig) G in cervico-vaginal secretions. We show here that in the mouse, IgG accumulation was enhanced approximately 5-fold post-ovulation, and was accompanied by an influx of neutrophils into the FRT. To determine whether these two events were causally related, we performed short-term neutrophil depletion experiments at individual stages throughout the estrous cycle. Our results demonstrate that neutrophils were not necessary for cycle-dependent tissue remodeling and cycle progression and that cycle-dependent IgG accumulation occurred independent of neutrophils. We thus conclude that neutrophil influx and IgG accumulation are independent events that occur in the FRT during the reproductive cycle.     

Organoid systems to study the human female reproductive tract and pregnancy

Both the proper functioning of the female reproductive tract (FRT) and normal placental development are essential for women’s health, wellbeing, and pregnancy outcome. The study of the FRT in humans has been challenging due to limitations in the in vitro and in vivo tools available. Recent developments in 3D organoid technology that model the different regions of the FRT include organoids of the ovaries, fallopian tubes, endometrium and cervix, as well as placental trophoblast. These models are opening up new avenues to investigate the normal biology and pathology of the FRT. In this review, we discuss the advances, potential, and limitations of organoid cultures of the human FRT.Subject terms: Cell biology, Physiology, Diseases

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Differential Binding of IgG and IgA to Mucus of the Female Reproductive Tract

Cells of the endocervix are responsible for the secretion of mucins, which provide an additional layer of protection to the female reproductive tract (FRT). This barrier is likely fortified with IgA as has previously been shown in the gastrointestinal tract and lungs of mice. Mucus associated IgA can facilitate clearance of bacteria. While a similar function for IgG has been proposed, an association with mucus has not yet been demonstrated. Here we find that IgA and IgG are differentially associated with the different types of mucus of the FRT. We observed that while both IgA and IgG are stably associated with cervical mucus, only IgG is associated with cervicovaginal mucus. These findings reveal that antibodies can bind tightly to mucus, where they can play a significant role in the fortification of the mucus barriers of the FRT. It may be possible to harness this interaction in the development of vaccines designed to protect the FRT mucosal barriers from sexually transmitted diseases such as HIV.     

The impact of aging on innate and adaptive immunity in the human female genital tract

Mucosal tissues in the human female reproductive tract (FRT) are primary sites for both gynecological cancers and infections by a spectrum of sexually transmitted pathogens, including human immunodeficiency virus (HIV), that compromise women''s health. While the regulation of innate and adaptive immune protection in the FRT by hormonal cyclic changes across the menstrual cycle and pregnancy are being intensely studied, little to nothing is known about the alterations in mucosal immune protection that occur throughout the FRT as women age following menopause. The immune system in the FRT has two key functions: defense against pathogens and reproduction. After menopause, natural reproductive function ends, and therefore, two overlapping processes contribute to alterations in immune protection in aging women: menopause and immunosenescence. The goal of this review is to summarize the multiple immune changes that occur in the FRT with aging, including the impact on the function of epithelial cells, immune cells, and stromal fibroblasts. These studies indicate that major aspects of innate and adaptive immunity in the FRT are compromised in a site‐specific manner in the FRT as women age. Further, at some FRT sites, immunological compensation occurs. Overall, alterations in mucosal immune protection contribute to the increased risk of sexually transmitted infections (STI), urogenital infections, and gynecological cancers. Further studies are essential to provide a foundation for the development of novel therapeutic interventions to restore immune protection and reverse conditions that threaten women''s lives as they age.     

Wnt5a is required for proper epithelial-mesenchymal interactions in the uterus

Epithelial-mesenchymal interactions play a crucial role in the correct patterning of the mammalian female reproductive tract (FRT). Three members of the Wnt family of growth factors are expressed at high levels in the developing FRT in the mouse embryo. The expression of Wnt genes is maintained in the adult FRT, although levels fluctuate during estrous. Wnt4 is required for Müllerian duct initiation, whereas Wnt7a is required for subsequent differentiation. In this study, we show that Wnt5a is required for posterior growth of the FRT. We further demonstrate that the mutant FRT has the potential to form the posterior compartments of the FRT using grafting techniques. Postnatally, Wnt5a plays a crucial role in the generation of uterine glands and is required for cellular and molecular responses to exogenous estrogens. Finally, we show that Wnt5a participates in a regulatory loop with other FRT patterning genes including Wnt7a, Hoxa10 and Hoxa11. Data presented provide a mechanistic basis for how uterine stroma mediates both developmental and estrogen-mediated changes in the epithelium and demonstrates that Wnt5a is a key component in this process. The similarities of the Wnt5a and Wnt7a mutant FRT phenotypes to those described for the Hoxa11 and Hoxa13 mutant FRT phenotypes reveal a mechanism whereby Wnt and Hox genes cooperate to pattern the FRT along the anteroposterior axis.     

MSX2 promotes vaginal epithelial differentiation and wolffian duct regression and dampens the vaginal response to diethylstilbestrol

In utero exposure to diethylstilbestrol (DES) leads to patterning defects in the female reproductive tract (FRT) and a propensity to the development of vaginal adenocarcinomas in humans. In the mouse, DES treatment similarly induces a plethora of FRT developmental defects, including stratification of uterine epithelium and presence of glandular tissue in cervix and vagina. Uterine abnormalities are associated with repression of the homeobox gene Msx2, and DES leads to an altered uterine response in Msx2 mutants including a dilated uterine lumen. Here we investigate the role of Msx2 in normal vaginal development and in FRT response to DES. During vaginal development, Msx2 is required for Tgfbeta2 and Tgfbeta3 expression and for proper vaginal epithelial differentiation. Moreover, Msx2 is involved in caudal Wolffian duct regression by promoting apoptosis. Consistently, neonatal DES exposure represses Msx2 expression in the Wolffian duct epithelium and inhibits its apoptosis and subsequent regression. Intriguingly, although DES treatment also represses Msx2 expression in the vaginal epithelium, a much more severe DES-induced vaginal phenotype was observed in Msx2 mutant mice, including a complete failure of Müllerian vaginal epithelial stratification and a severely dilated vaginal lumen, accompanied by loss of p63 and water channel protein expression. These results demonstrate a critical role for Msx2 in counteracting the effect of DES on FRT patterning and suggest that the response to DES may be highly variable depending on the genotype of an individual.     

Neonatal phytoestrogen exposure alters oviduct mucosal immune response to pregnancy and affects preimplantation embryo development in the mouse

Treatment of neonatal mice with the phytoestrogen genistein (50 mg/kg/day) results in complete female infertility caused in part by preimplantation embryo loss in the oviduct between Days 2 and 3 of pregnancy. We previously demonstrated that oviducts of genistein-treated mice are "posteriorized" as compared to control mouse oviducts because they express numerous genes normally restricted to posterior regions of the female reproductive tract (FRT), the cervix and vagina. We report here that neonatal genistein treatment resulted in substantial changes in oviduct expression of genes important for the FRT mucosal immune response, including immunoglobulins, antimicrobials, and chemokines. Some of the altered immune response genes were chronically altered beginning at the time of neonatal genistein treatment, indicating that these alterations were a result of the posteriorization phenotype. Other alterations in oviduct gene expression were observed only in early pregnancy, immediately after the FRT was exposed to inflammatory or antigenic stimuli from ovulation and mating. The oviduct changes affected development of the surviving embryos by increasing the rate of cleavage and decreasing the trophectoderm-to-inner cell mass cell ratio at the blastocyst stage. We conclude that both altered immune responses to pregnancy and deficits in oviduct support for preimplantation embryo development in the neonatal genistein model are likely to contribute to infertility phenotype.     

Generation of human female reproductive tract epithelium from human embryonic stem cells

Background

Recent studies have identified stem/progenitor cells in human and mouse uterine epithelium, which are postulated to be responsible for tissue regeneration and proliferative disorders of human endometrium. These progenitor cells are thought to be derived from Müllerian duct (MD), the primordial female reproductive tract (FRT).

Methodology/Principal Findings

We have developed a model of human reproductive tract development in which inductive neonatal mouse uterine mesenchyme (nMUM) is recombined with green fluorescent protein (GFP)-tagged human embryonic stem cells (hESCs); GFP-hESC (ENVY). We demonstrate for the first time that hESCs can be differentiated into cells with a human FRT epithelial cell phenotype. hESC derived FRT epithelial cells emerged from cultures containing MIXL1⁺ mesendodermal precursors, paralleling events occurring during normal organogenesis. Following transplantation, nMUM treated embryoid bodies (EBs) generated epithelial structures with a typical MD phenotype that expressed the MD markers PAX2, HOXA10. Functionally, the hESCs derived FRT epithelium responded to exogenous estrogen by proliferating and secreting uterine-specific glycodelin A (GdA).

Conclusions/Significance

These data show nMUM can induce differentiation of hESC to form the FRT epithelium. This may provide a model to study early developmental events of the human FRT.     

Normal and abnormal epithelial differentiation in the female reproductive tract

In mammals, the female reproductive tract (FRT) develops from a pair of paramesonephric or Müllerian ducts (MDs), which arise from coelomic epithelial cells of mesodermal origin. During development, the MDs undergo a dynamic morphogenetic transformation from simple tubes consisting of homogeneous epithelium and surrounding mesenchyme into several distinct organs namely the oviduct, uterus, cervix and vagina. Following the formation of anatomically distinctive organs, the uniform MD epithelium (MDE) differentiates into diverse epithelial cell types with unique morphology and functions in each organ. Classic tissue recombination studies, in which the epithelium and mesenchyme isolated from the newborn mouse FRT were recombined, have established that the organ specific epithelial cell fate of MDE is dictated by the underlying mesenchyme. The tissue recombination studies have also demonstrated that there is a narrow developmental window for the epithelial cell fate determination in MD-derived organs. Accordingly, the developmental plasticity of epithelial cells is mostly lost in mature FRT. If the signaling that controls epithelial differentiation is disrupted at the critical developmental stage, the cell fate of MD-derived epithelial tissues will be permanently altered and can result in epithelial lesions in adult life. A disruption of signaling that maintains epithelial cell fate can also cause epithelial lesions in the FRT. In this review, the pathogenesis of cervical/vaginal adenoses and uterine squamous metaplasia is discussed as examples of such incidences.     

Pacific and Atlantic herring produce burst pulse sounds

The commercial importance of Pacific and Atlantic herring (Clupea pallasii and Clupea harengus) has ensured that much of their biology has received attention. However, their sound production remains poorly studied. We describe the sounds made by captive wild-caught herring. Pacific herring produce distinctive bursts of pulses, termed Fast Repetitive Tick (FRT) sounds. These trains of broadband pulses (1.7-22 kHz) lasted between 0.6 s and 7.6 s. Most were produced at night; feeding regime did not affect their frequency, and fish produced FRT sounds without direct access to the air. Digestive gas or gulped air transfer to the swim bladder, therefore, do not appear to be responsible for FRT sound generation. Atlantic herring also produce FRT sounds, and video analysis showed an association with bubble expulsion from the anal duct region (i.e. from the gut or swim bladder). To the best of the authors' knowledge, sound production by such means has not previously been described. The function(s) of these sounds are unknown, but as the per capita rates of sound production by fish at higher densities were greater, social mediation appears likely. These sounds may have consequences for our understanding of herring behaviour and the effects of noise pollution.     

Vaginal Challenge with an SIV-Based Dual Reporter System Reveals That Infection Can Occur throughout the Upper and Lower Female Reproductive Tract

The majority of new HIV infections occur in women as a result of heterosexual intercourse, overcoming multiple innate barriers to infection within the mucosa. However, the avenues through which infection is established, and the nature of bottlenecks to transmission, have been the source of considerable investigation and contention. Using a high dose of a single round non-replicating SIV-based vector containing a novel dual reporter system, we determined the sites of infection by the inoculum using the rhesus macaque vaginal transmission model. Here we show that the entire female reproductive tract (FRT), including the vagina, ecto- and endocervix, along with ovaries and local draining lymph nodes can contain transduced cells only 48 hours after inoculation. The distribution of infection shows that virions quickly disseminate after exposure and can access target cells throughout the FRT, with an apparent preference for infection in squamous vaginal and ectocervical mucosa. JRFL enveloped virions infect diverse CD4 expressing cell types, with T cells resident throughout the FRT representing the primary target. These findings establish a new perspective that the entire FRT is susceptible and virus can reach as far as the ovary and local draining lymph nodes. Based on these findings, it is essential that protective mechanisms for prevention of HIV acquisition must be present at protective levels throughout the entire FRT to provide complete protection.     

Th1 cells are dispensable for primary clearance of Chlamydia from the female reproductive tract of mice

Protective immune responses to Chlamydia infection within the female reproductive tract (FRT) are incompletely understood. MHC class II-restricted CD4 Th1 responses are believed to be vital for bacterial clearance due to their capacity to secrete IFN-γ, but an essential requirement for T-bet-expressing Th1 cells has yet to be demonstrated in the mouse model of Chlamydia infection. Here, we investigated the role of T-bet and IFN-γ in primary clearance of Chlamydia after FRT infection. Surprisingly, IFN-γ producing CD4 T cells from the FRT expressed low levels of T-bet throughout infection, suggesting that classical T-bet-expressing Th1 cells are inefficiently generated and therefore unlikely to participate in bacteria clearance. Furthermore, mice deficient in T-bet expression or with a CD4-specific T-bet deficiency cleared FRT infection similarly to wild-type controls. T-bet-deficient mice displayed significant skewing of FRT CD4 T cells towards Th17 responses, demonstrating that compensatory effector pathways are generated in the absence of Th1 cells. In marked contrast, IFN-γ-, and IFN-γR-deficient mice were able to reduce FRT bacterial burdens, but suffered systemic bacterial dissemination and 100% mortality. Together, these data demonstrate that IFN-γ signaling is essential to protect mice from fatal systemic disease, but that classical T-bet-expressing Th1 cells are non-essential for primary clearance within the FRT. Exploring the protective contribution of Th1 cells versus other CD4 effector lineages could provide important information for the generation of new Chlamydia vaccines.     

A Mushroom-Inducing DNA Sequence Isolated from the Basidiomycete, Schizophyllum Commune

A DNA sequence capable of inducing the de novo development of fruiting bodies (mushrooms) when integrated into the genome of unmated, nonfruiting strains of the Basidiomycete Schizophyllum commune has been isolated and partially characterized. This sequence, designated FRT1, overrides the normal requirement of a mating interaction for fruiting in this organism. It has been shown to integrate stably in different chromosome locations and appears to be trans-acting. It also enhances the normal process of fruiting that occurs after mating. Additional DNA sequences with similarity to FRT1 were detected within the genome of the strain of origin by hybridization of labeled FRT1 DNA to blots of digested genomic DNAs. FRT1 and the genomic sequences similar to it were shown to be genetically linked. Southern hybridization experiments suggested sequence divergence at the FRT1 locus between different strains of S. commune. A testable model for how FRT1 may act as a key element in the pathway for the differentiation of fruiting bodies is presented as a working hypothesis for further investigation.     

FLP recombinase-mediated site-specific recombination in silkworm, Bombyx mori

A comprehensive understanding of gene function and the production of site-specific genetically modified mutants are two major goals of genetic engineering in the post-genomic era. Although site-specific recombination systems have been powerful tools for genome manipulation of many organisms, they have not yet been established for use in the manipulation of the silkworm Bombyx mori genome. In this study, we achieved site-specific excision of a target gene at predefined chromosomal sites in the silkworm using a FLP/FRT site-specific recombination system. We first constructed two stable transgenic target silkworm strains that both contain a single copy of the transgene construct comprising a target gene expression cassette flanked by FRT sites. Using pre-blastoderm microinjection of a FLP recombinase helper expression vector, 32 G3 site-specific recombinant transgenic individuals were isolated from five of 143 broods. The average frequency of FLP recombinase-mediated site-specific excision in the two target strains genome was approximately 3.5%. This study shows that it is feasible to achieve site-specific recombination in silkworms using the FLP/FRT system. We conclude that the FLP/FRT system is a useful tool for genome manipulation in the silkworm. Furthermore, this is the first reported use of the FLP/FRT system for the genetic manipulation of a lepidopteran genome and thus provides a useful reference for the establishment of genome manipulation technologies in other lepidopteran species.     

Protein-Coated Nanoparticles Are Internalized by the Epithelial Cells of the Female Reproductive Tract and Induce Systemic and Mucosal Immune Responses

The female reproductive tract (FRT) includes the oviducts (fallopian tubes), uterus, cervix and vagina. A layer of columnar epithelium separates the endocervix and uterus from the outside environment, while the vagina is lined with stratified squamous epithelium. The mucosa of the FRT is exposed to antigens originating from microflora, and occasionally from infectious microorganisms. Whether epithelial cells (ECs) of the FRT take up (sample) the lumen antigens is not known. To address this question, we examined the uptake of 20–40 nm nanoparticles (NPs) applied vaginally to mice which were not treated with hormones, epithelial disruptors, or adjuvants. We found that 20 and 40 nm NPs are quickly internalized by ECs of the upper FRT and within one hour could be observed in the lymphatic ducts that drain the FRT, as well as in the ileac lymph nodes (ILNs) and the mesenteric lymph nodes (MLNs). Chicken ovalbumin (Ova) conjugated to 20 nm NPs (NP-Ova) when administered vaginally reaches the internal milieu in an immunologically relevant form; thus vaginal immunization of mice with NP-Ova induces systemic IgG to Ova antigen. Most importantly, vaginal immunization primes the intestinal mucosa for secretion of sIgA. Sub-cutaneous (s.c) boosting immunization with Ova in complete Freund''s adjuvant (CFA) further elevates the systemic (IgG1 and IgG2c) as well as mucosal (IgG1 and sIgA) antibody titers. These findings suggest that the modes of antigen uptake at mucosal surfaces and pathways of antigen transport are more complex than previously appreciated.     

Potential innate immunity-related markers of endometrial receptivity and recurrent implantation failure (RIF)

The successful implantation of the embryo into a receptive endometrium is essential for the establishment of a viable pregnancy while recurrent implantation failure (RIF) is a real challenge in assisted reproduction. The maternal innate immune system, specifically the Toll-like receptors (TLRs), are involved in maintaining immunity in the female reproductive tract (FRT) required for fertility. In this study, we aimed to investigate the importance of innate immunity-related gene expression in the regulation of human fertility and as a prediction of potential outcome of in vitro fertilization - embryo transfer (IVF-ET), thus, we assessed the gene expression levels of TLR signalling molecules using quantitative real-time PCR between endometrial biopsies of healthy fertile women, and the patients experiencing RIF. Interestingly, our results showed that, TRIB2 and TLR9 genes were differentially expressed between the endometrial biopsies of healthy women and those with RIF. However, comparing expression levels of same genes between pre-receptive and receptive healthy endometrial biopsies showed different genes (ICAM1, NFKBIA, VCAM1, LIF, VEGFB, TLR5) had significantly altered expression, suggesting their involvement in endometrial receptivity. Thus, further investigations will enable us to better understand the role of these genes in the biology of FRT and as a possible target for the improvement of infertility treatments and/or development of non-hormonal contraception.     

Synergy between IL-8 and GM-CSF in reproductive tract epithelial cell secretions promotes enhanced neutrophil chemotaxis

Neutrophils occur in tissues of the female reproductive tract (FRT) under non-infected conditions. These cells generally enter tissues under the influence of chemoattractants called chemokines. Primary epithelial cells (EC) from FRT were a potent source of chemokines, IL-8 being the chief neutrophil chemoattractant secreted. Blocking with neutralizing anti-IL-8 showed that IL-8 did not account for all of the chemoattraction observed. A mixture of 25 ng/mL rIL-8 and 1 ng/mL rGM-CSF mediated 2.7-fold more chemotaxis than that expected if the two agents were additive. We then found that GM-CSF was produced by EC in amounts that synergised strongly with IL-8 to enhance chemotaxis. Treatment of uterine EC conditioned medium with saturating doses of anti-IL-8 plus anti-GM-CSF antibodies produced an 84% inhibition of chemotaxis. These findings demonstrate that the majority of neutrophil chemoattractant activity produced by FRT EC results from the synergistic effects of IL-8 and GM-CSF.     

Aging impacts CD103+CD8+ T cell presence and induction by dendritic cells in the genital tract

As women age, susceptibility to systemic and genital infections increases. Tissue‐resident memory T cells (TRMs) are CD103⁺CD8⁺ long‐lived lymphocytes that provide critical mucosal immune protection. Mucosal dendritic cells (DCs) are known to induce CD103 expression on CD8⁺ T cells. While CD103⁺CD8⁺ T cells are found throughout the female reproductive tract (FRT), the extent to which aging impacts their presence and induction by DCs remains unknown. Using hysterectomy tissues, we found that endometrial CD103⁺CD8⁺ T cells were increased in postmenopausal compared to premenopausal women. Endometrial DCs from postmenopausal women were significantly more effective at inducing CD103 expression on allogeneic naïve CD8⁺ T cells than DCs from premenopausal women; CD103 upregulation was mediated through membrane‐bound TGFβ signaling. In contrast, cervical CD103⁺ T cells and DC numbers declined in postmenopausal women with age. Decreases in DCs correlated with decreased CD103⁺ T cells in endocervix, but not ectocervix. Our findings demonstrate a previously unrecognized compartmentalization of TRMs in the FRT of postmenopausal women, with loss of TRMs and DCs in the cervix with aging, and increased TRMs and DC induction capacity in the endometrium. These findings are relevant to understanding immune protection in the FRT and to the design of vaccines for women of all ages.