E-&ndashcadherin (CDH1) is a cell adhesion molecule that coordinates key morphogenetic processes regulating cell growth, cell proliferation and apoptosis. Loss of CDH1 is a trademark of the cellular event epithelial to mesenchymal transition (EMT), which increases the metastatic potential of malignant cells. PTEN is a tumor suppressor gene commonly mutated in many human cancers, including endometrial cancer. In the mouse uterus, ablation of Pten induces epithelial hyperplasia, leading to endometrial carcinomas. However, loss of Pten alone does not affect longevity until around 5 months. Similarly, conditional ablation of Cdh1 alone does not predispose mice to cancer. We characterized the impact of dual Cdh1 and Pten ablation using Pgr-Cre (Cdh1d/d Ptend/d) in the mouse uterus. We observed that Cdh1d/d Ptend/d mice died at postnatal day 15-&ndash19 with massive blood loss from their reproductive tract (abnormal metrorrhagia) with prevalent vascularization in both the endometrium and myometrium. Their uteri were abnormally structured with curly horns, disorganized epithelial structure, and increased cell proliferation. Co-&ndashimmunostaining of KRT8 and ACTA2 showed invasion of epithelial cells into the myometrium. Further, the uteri of Cdh1d/d Ptend/d mice had prevalent vascularization in both the endometrium and myometrium. We also observed reduced expression of estrogen and progesterone receptors, loss of cell adherens and tight junction molecules (CTNNB1 and claudin), as well as activation of AKT in the uteri of Cdh1d/d Ptend/d mice. However, complex hyperplasia was not found in the uteri of Cdh1d/d Ptend/d. Collectively, these findings suggest that ablation of Pten with Cdh1 in the uterus accelerates cellular invasiveness and angiogenesis, and causes early death. Thus, this model does not allow sufficient time for the emergence of advanced, clinically over aggressive endometrial tumorigenesis and metastasis. Additionally, we looked at a new Cre system to ablate Pten and Cdh1 only in the epithelial cells of the uterus. Sprr2f, an estrogen dependent gene that is found highly expressed in the uterus, helps with structure and barrier function of epithelial cells. Prg-Cre turns on at postnatal day 3-5 before development of the uterus; whereas, Sprr2f-Cre is active around 3 weeks which is after uterine development. We have driven the ablation of Cdh1d/d Ptend/d using the Sprr2f-Cre. The Sprr2f-Cre Cdh1d/d Ptend/d mice successfully lived to 2 months. The Sprr2f-Cre Ptend/d mice displayed hyperplastic epithelial cells, most prominently in the glandular like structures of the uterus. Lack of cellular structure was observed in the Sprr2f-Cre Cdh1d/d Ptend/d mice. We also developed a model of orthotopic tumor transplantation to study further tumor development including cell invasion, dissemination and metastasis. The uteri of control, Cdhd/d, Ptend/d and Cdhd/d Ptend/d mice were collected and dissected to approximately ~1 mm in diameter. Then, the tissue fragments were orthotopically implanted into the uterine wall (endometrium) of wild-type syngeneic host mice. We have observed successful implantation and sustainability of the tissue through this technique. The tissue viability was successfully verified by implanting donor uterine pieces under the kidney capsule of recipient wild type mice. This study has shown that the ablation of Cdh1 and Pten in the mouse uterus initiates a more aggressive form of type I endometrial carcinoma when using Pgr-Cre as well as Sprr2f-Cre. However, neither conditional ablation approaches allowed us to fully observe the progression of the carcinoma to a metastatic disease. Our intrauterine endometrial/myometrial implantation technique proved to be an incomplete method to further study the metastatic potential of the PgrCre/+ Cdh1f/f Ptenf/f mice.

Worldwide, more than half of all HIV-infected individuals are women. Since mucosal surfaces are the primary gateway for HIV entry, maintaining the integrity of the female reproductive tract (FRT) is essential for preventing infection. The FRT employs many immune mechanisms that serve as the first line of defense against HIV transmission. Among these are vaginal fluid secretions rich in antimicrobial peptides, and commensal bacteria that colonize the vagina and prevent infections. We sought to study vaginal fluid as an innate immune component of the FRT in the prevention of HIV infection. Additionally, we investigated the anti-HIV microbicide candidate RC-101 as a possible treatment against pathogenic bacteria that disrupt the healthy microbiota of the FRT and create a suboptimal immune state that increases host susceptibility to viruses, such as HIV. Here we report that vaginal fluid collected from healthy females inhibits HIV infection. Moreover, our studies reveal that vaginal fluid collected from Black and White women exhibit disparate anti-HIV activity, possibly rendering Black women more susceptible to HIV infection. In addition, we show that RC-101, which is active against HIV, can also inhibit pathogenic bacteria that compromise FRT innate immunity, providing a dual mechanism of protection against HIV acquisition. Overall, these findings show that vaginal fluid is an important part of female innate immunity that protects the host from heterosexual HIV acquisition. Furthermore, the microbicide RC-101 may prevent HIV infection by both directly preventing viral entry, and by restricting the growth of pathogenic bacteria that disrupt the protective commensal vaginal flora. Together, innate mechanisms and bolstered protection present a multifaceted approach to maintaining effective host immunity.

Human immunodeficiency virus (HIV) infects 30 million people worldwide. In sub-Saharan Africa, the region most affected by HIV, women comprise 60% of the infected population. Heterosexual transmission is a major mode of viral acquisition, mandating further research of the process and prevention of HIV acquisition via the female reproductive tract (FRT). The FRT is a dynamic environment, protected by host immune mechanisms and commensal microbes. The disruption of either of these elements can increase susceptibility to HIV. Accordingly, one common risk factor for HIV acquisition is the microbial shift condition known as bacterial vaginosis (BV), which is characterized by the displacement of healthy lactobacilli by an overgrowth of pathogenic bacteria. As the bacteria responsible for BV pathogenicity and their interactions with host immunity are not understood, we sought to evaluate the effects of BV-associated bacteria on reproductive epithelia. Here we have characterized the interaction between BV-associated bacteria and the female reproductive tract by measuring cytokine and defensin induction in FRT epithelial cells following bacterial inoculation. Four BV-associated bacteria were evaluated alongside six lactobacilli for a comparative assessment. Our model showed good agreement with clinical BV trends; we observed a distinct cytokine and human β- defensin-2 response to BV-associated bacteria, especially Atopobium vaginae, compared to most lactobacilli. One lactobacillus species, Lactobacillus vaginalis, induced an immune response similar to that elicited by BV-associated bacteria. These iii data provide an important prioritization of BV-associated bacteria and support further characterization of reproductive bacteria and their interactions with host epithelia. We next evaluated the effect of this interaction on HIV infection by investigating the soluble effectors secreted when FRT epithelial cells were cocultured with A. vaginae. We observed increased proviral activity mediated by secreted low molecular weight effectors, and determined that this activity was not likely mediated by cytokine responses. Instead, we identified a complex mixture containing several upregulated host proteins. Selected individual proteins from the mixture exhibited HIV-enhancing activity only when applied with the complex mixture of proviral factors, suggesting that HIV enhancement might be mediated by synergistic effects. In addition to characterizing the immune interactions that mediate the enhanced HIV acquisition associated with BV, we also evaluated the safety and efficacy of RC- 101, a candidate vaginal microbicide being developed for the prevention of HIV transmission. RC-101 has been effective and well tolerated in preliminary cell culture and macaque models. However, the effect of RC-101 on primary vaginal tissues and resident vaginal microflora requires further evaluation. Here, we treated primary vaginal tissues and vaginal bacteria, both pathogenic and commensal, with RC-101 to investigate compatibility of this microbicide with FRT tissue and microflora. RC-101 was well tolerated by host tissues and commensal vaginal bacteria, while BV-associated bacteria were inhibited by RC-101. By establishing vaginal microflora, the specific antibacterial activity of RC-101 may provide a dual mechanism of HIV protection.

Antimicrobial peptides (AMPs) are small proteins produced by epithelial surfaces, which have broad-spectrum antimicrobial and immunomodulatory activities. In the lung, skin and alimentary tract AMPs are known to be important in infectious and inflammatory conditions. Far less is known regarding the role of AMPs within the female reproductive tract, but as infection and inflammation are causes of preterm labour, AMPs may have a key function in maintain and protecting pregnancy. The major groups of human AMPs include the human beta defensins (HBDs), two antileukoproteinases (secretory leukocyte protease inhibitor (SLPI) and Trappin-2/Elafin), and the human cathelicidin hCAP18/LL-37, with several studies identifying their presence at sites throughout the reproductive tract. The cervix in pregnancy is positioned between the upper genital tract containing the developing fetus and the lower tract where infections usually arise. I hypothesise that AMPs are fundamental to mucosal immune defence of the cervix in pregnancy, preventing ascending infection and excessive inflammation that can cause preterm labour. This thesis focused on the human cathelicidin hCAP18/LL-37 and its role within the cervix and vagina. The aims of this thesis were to; investigate the inflammatory effects of LL-37 from cervical and vaginal derived epithelial cells and determine the pathways and receptors in which LL-37 may elicit its effects and how production may be regulated; investigate the role of CRAMP in a mouse model of preterm birth; and determine the production of AMPs by the pregnant cervix whilst investigating the relationship between AMP concentrations in cervicovaginal secretions and preterm labour. The inflammatory effect of LL-37 was investigated using cell lines derived from endocervical, ectocervical and vaginal epithelium. The study of these cell lines suggests divergent responses of cervical and vaginal epithelial cells. LL-37 mediated induction of IL-8 and IL-6 production from endocervical epithelial cells was observed in a dose-dependent and time-dependent manner, whilst ectocervical and vaginal cells also respond to treatment with LL-37 through IL-8 and IL-6 production. To determine a possible mechanism of action of LL-37 on IL-8 and IL-6 in the three cell lines, inhibitors against MAPK cascades, ERK, p38 MAPK and JNK, and known LL-37 receptors were investigated. In endocervical cells LL-37 mediated IL-8 occurs via activation of unidentified GPCRs, whilst in ectocervical cells this effect on IL‐8 and IL-6 is via the activation of ERK and p38 MAPK cascades. The mechanism by which LL-37 induces IL-8 secretion in vaginal epithelial cells remains unknown. Expression of LL-37 was shown to be mediated by vitamin D3 in vitro in cervical and vaginal epithelial cells. However when this relationship was investigated in vivo, using matched serum and cervicovaginal secretions from woman at early pregnancy, no correlation was observed between circulating vitamin D and cervicovaginal or circulating hCAP18/LL-37. However, the majority of women in this study reported with insufficient levels of vitamin D, which may effect the relationship observed with hCAP18/LL-37. Using a mouse model of LPS-induced preterm labour, to mimic the presence of intrauterine infection bacterial infection, I aimed to characterise the role of CRAMP, the mouse orthologue of hCAP18/LL-37, in the lower inflammatory and immune response that results in preterm labour. Wild type C57Bl/6J mice receiving an intrauterine injection of LPS deliver prematurely, within 24 hours of injection. However mice deficient in CRAMP (Camp -/-) receiving an intrauterine injection of LPS deliver significantly later and have a non-significant increase in pup survival compared to wild type C57Bl/6J mice. Cervical tissue collected post partum showed no difference in inflammatory markers between wild type C57Bl/6J and Camp -/- mice, however there was increased expression of the neutrophil chemoattractant marker, Cxcl5, and the neutrophil marker, Ngp in Camp -/- mice. In the lower genital tract, levels of antimicrobial peptides were determined in samples of cervicovaginal secretions collected from pregnant women. AMPs, hCAP18/LL-37, HBD-2 and SLPI were found in cervicovaginal secretions, and levels of hCAP18/LL-37 were increased in women with the common vaginal infection bacterial vaginosis. However no relationship was identified between the concentration of AMPs and preterm birth in this study. This work has shown that the lower genital tract, where infections that are associated with preterm labour originate, expresses the human cathelicidin hCAP18/LL-37. It may play an important role in modulating the immune response to invading infection associated with preterm labour. Further investigation of these responses may increase understanding of the physiology and pathophysiology of labour, and lead to strategies for the prevention of premature delivery.

Infertility affects 15 % of couples, which corresponds to 60 - 80 million worldwide. The microenvironments in which the oocyte, embryo and fetus mature are vital to the establishment and development of a healthy pregnancy. Different biological systems, such as angiogenesis, the immune system and apoptosis need to be adequately regulated for pregnancy to occur and progress normally. The overall aim of this thesis was to investigate the impact of Histidine-rich glycoprotein (HRG) and Src homology 2 domain-containing adapter protein B (SHB) on human female fertility. HRG is a plasma protein that regulates angiogenesis, the immune system, coagulation/fibrinolysis and apoptosis, by building complexes with various ligands. The impact of HRG on fertility is studied here for the first time. HRG is present in follicular fluid, the Fallopian tube, endometrium, myometrium and placenta. HRG distribution within embryo nuclei depends on developmental stage. Blastocysts express and secrete HRG. The HRG C633T single nucleotide polymorphism (SNP) appears to affect the chance of pregnancy and, correspondingly, parameters associated with pregnancy in IVF. Additionally, this HRG genotype may increase the risk in IVF of only developing embryos unfit for transfer. SHB is an adaptor protein involved in intracellular signaling complexes that regulate angiogenesis, the immune system and cell proliferation/apoptosis. Shb knockout mice have altered oocyte/follicle maturation and impaired embryogenesis. The impact of three SHB polymorphisms (rs2025439, rs13298451 and rs7873102) on human fertility is studied for the first time. The SNP prevalences did not differ between infertile and fertile women. BMI, gonadotropin dosages, the percentage of immature oocytes, the number of fertilized oocytes, the percentage of good-quality embryos and the day of embryo transfer seems to be affected by SHB genotype. In conclusion, HRG and SHB appear to influence female fertility. They are potential biomarkers that might be used for predicting pregnancy chance in infertile women. Knowledge of these genotypes may improve patient counseling and individualization of treatment.