The female reproductive tract microbiotas, inflammation, and gynecological conditions.

The intricate interactions between the host cells, bacteria, and immune components that reside in the female reproductive tract (FRT) are essential in maintaining reproductive tract homeostasis. Much of our current knowledge surrounding the FRT microbiota relates to the vaginal microbiota, where 'health' has long been associated with low bacterial diversity and Lactobacillus dominance. This concept has recently been challenged as women can have a diverse vaginal microbial composition in the absence of symptomatic disease. The structures of the upper FRT (the endocervix, uterus, Fallopian tubes, and ovaries) have distinct, lower biomass microbiotas than the vagina; however, the existence of permanent microbiotas at these sites is disputed. During homeostasis, a balance exists between the FRT bacteria and the immune system that maintains immune quiescence. Alterations in the bacteria, immune system, or local environment may result in perturbances to the FRT microbiota, defined as dysbiosis. The inflammatory signature of a perturbed or "dysbiotic" FRT microbiota is characterized by elevated concentrations of pro-inflammatory cytokines in cervical and vaginal fluid. It appears that vaginal homeostasis can be disrupted by two different mechanisms: first, a shift toward increased bacterial diversity can trigger vaginal inflammation, and second, local immunity is altered in some manner, which disrupts the microbiota in response to an environmental change. FRT dysbiosis can have negative effects on reproductive health. This review will examine the increasing evidence for the involvement of the FRT microbiotas and inflammation in gynecologic conditions such as endometriosis, infertility, and endometrial and ovarian cancer; however, the precise mechanisms by which bacteria are involved in these conditions remains speculative at present. While only in their infancy, the use of antibiotics and probiotics to therapeutically alter the FRT microbiota is being studied and is discussed herein. Our current understanding of the intimate relationship between immunity and the FRT microbiota is in its early days, and more research is needed to deepen our mechanistic understanding of this relationship and to assess how our present knowledge can be harnessed to assist in diagnosis and treatment of gynecologic conditions.

Endometrial microbiota is more diverse in people with endometriosis than symptomatic controls.

Endometriosis is a chronic, estrogen-dependent gynecological condition affecting approximately 10% of reproductive age women. The most widely accepted theory of its etiology includes retrograde menstruation. Recent reports suggest the uterus is not sterile. Thus, the refluxed menstrual effluent may carry bacteria, and contribute to inflammation, the establishment and growth of endometriotic lesions. Here, we compared and contrasted uterine bacteria (endometrial microbiota) in people with surgically confirmed presence (N = 12) or absence of endometriosis (N = 9) using next-generation 16S rRNA gene sequencing. We obtained an average of > 9000 sequence reads per endometrial biopsy, and found the endometrial microbiota of people with endometriosis was more diverse (greater Shannon Diversity Index and proportion of 'Other' taxa) than symptomatic controls (with pelvic pain, surgically confirmed absence of endometriosis; diagnosed with other benign gynecological conditions). The relative abundance of bacterial taxa enriched in the endometrial microbiota of people with endometriosis belonged to the Actinobacteria phylum (Gram-positive), Oxalobacteraceae (Gram-negative) and Streptococcaceae (Gram-positive) families, and Tepidimonas (Gram-negative) genus, while those enriched in the symptomatic controls belonged to the Burkholderiaceae (Gram-negative) family, and Ralstonia (Gram-negative) genus. Taken together, results suggest the endometrial microbiota is perturbed in people with endometriosis.

Depot medroxyprogesterone acetate (DMPA) enhances susceptibility and increases the window of vulnerability to HIV-1 in humanized mice.

The progestin-based hormonal contraceptive Depot Medroxyprogesterone Acetate (DMPA) is widely used in sub-Saharan Africa, where HIV-1 is endemic. Meta-analyses have shown that women using DMPA are 40% more likely than women not using hormonal contraceptives to acquire Human Immunodeficiency Virus (HIV-1). Therefore understanding how DMPA increases susceptibility to HIV-1 is an important public health issue. Using C57BL/6 mice and our previously optimized humanized mouse model (NOD-Rag1tm1Mom Il2rgtm1Wjl transplanted with hCD34-enriched hematopoietic stem cells; Hu-mice) where peripheral blood and tissues are reconstituted by human immune cells, we assessed how DMPA affected mucosal barrier function, HIV-1 susceptibility, viral titres, and target cells compared to mice in the diestrus phase of the estrous cycle, when endogenous progesterone is highest. We found that DMPA enhanced FITC-dextran dye leakage from the vaginal tract into the systemic circulation, enhanced target cells (hCD68+ macrophages, hCD4+ T cells) in the vaginal tract and peripheral blood (hCD45+hCD3+hCD4+hCCR5+ T cells), increased the rate of intravaginal HIV-1 infection, extended the window of vulnerability, and lowered vaginal viral titres following infection. These findings suggest DMPA may enhance susceptibility to HIV-1 in Hu-mice by impairing the vaginal epithelial barrier, increasing vaginal target cells (including macrophages), and extending the period of time during which Hu-mice are susceptible to infection; mechanisms that might also affect HIV-1 susceptibility in women.

Factors affecting stability of plasma brain-derived neurotrophic factor.

Circulating concentrations of brain-derived neurotrophic factor (BDNF) have been linked to cancer, neuropsychiatric, diabetes, and gynecological disorders. However, factors influencing plasma storage and subsequent BDNF quantification are incompletely understood. Therefore, the anticoagulant used in plasma separator tubes, storage-time, storage-temperature, and repeated freeze-thaw cycles on circulating BDNF concentrations was evaluated. Peripheral blood samples were collected from healthy women (n = 14) and men (n = 10) recruited prospectively from McMaster University (August 2014). Blood was collected from the cubital vein into plasma separator tubes containing five different anticoagulant systems [K2EDTA, Li-Hep, Li-Hep (gel), Na-Hep, Na-Hep (glass)], and placed on ice for transport to the lab for centrifugation. Plasma samples (n = 16) collected in K2EDTA tubes from women recruited to a previous study (April 2011 to December 2012) were used to determine the effect of multiple freeze-thaw cycles. Plasma BDNF was quantified using a commercially available ELISA kit. Plasma concentrations of BDNF were significantly affected by the type of plasma separator tube, storage-time, and number of freeze-thaw cycles. Storage temperature (- 20 vs. - 80 °C) did not significantly affect the quantity of BDNF measured as mean BDNF concentrations generally fell within our calculated acceptable change limit up to 6 months in the freezer. Our results suggest that for quantification of circulating BDNF blood collected in K2EDTA tubes and plasma stored up to 6 months at either - 20 or - 80 °C produces reproducible results that fall within an acceptable range. However, plasma samples stored beyond 6 months and repeated freeze-thaw cycles should be avoided.

Curcumin Can Decrease Tissue Inflammation and the Severity of HSV-2 Infection in the Female Reproductive Mucosa.

Herpes Simplex Virus Type 2 (HSV-2) is one of the most prevalent sexually transmitted viruses and is a known risk factor for HIV acquisition in the Female Genital Tract (FGT). Previously, we found that curcumin can block HSV-2 infection and abrogate the production of inflammatory cytokines and chemokines by genital epithelial cells in vitro. In this study, we investigated whether curcumin, encapsulated in nanoparticles and delivered by various in vivo routes, could minimize inflammation and prevent or reduce HSV-2 infection in the FGT. Female mice were pre-treated with curcumin nanoparticles through oral, intraperitoneal and intravaginal routes, and then exposed intravaginally to the tissue inflammation stimulant CpG-oligodeoxynucleotide (ODN). Local intravaginal delivery of curcumin nanoparticles, but not intraperitoneal or oral delivery, reduced CpG-mediated inflammatory histopathology and decreased production of pro-inflammatory cytokines Interleukin (IL)-6, Tumor Necrosis Factor Alpha (TNF-α) and Monocyte Chemoattractant Protein-1 (MCP-1) in the FGT. However, curcumin nanoparticles did not demonstrate anti-viral activity nor reduce tissue pathology when administered prior to intravaginal HSV-2 infection. In an alternative approach, intravaginal pre-treatment with crude curcumin or solid dispersion formulations of curcumin demonstrated increased survival and delayed pathology following HSV-2 infection. Our results suggest that curcumin nanoparticle delivery in the vaginal tract could reduce local tissue inflammation. The anti-inflammatory properties of curcumin delivered to the vaginal tract could potentially reduce the severity of HSV-2 infection and decrease the risk of HIV acquisition in the FGT of women.

Medroxyprogesterone acetate alters the vaginal microbiota and microenvironment in women and increases susceptibility to HIV-1 in humanized mice.

The hormonal contraceptive medroxyprogesterone acetate (MPA) is associated with increased risk of human immunodeficiency virus (HIV), via incompletely understood mechanisms. Increased diversity in the vaginal microbiota modulates genital inflammation and is associated with increased HIV-1 acquisition. However, the effect of MPA on diversity of the vaginal microbiota is relatively unknown. In a cohort of female Kenyan sex workers, negative for sexually transmitted infections (STIs), with Nugent scores <7 (N=58 of 370 screened), MPA correlated with significantly increased diversity of the vaginal microbiota as assessed by 16S rRNA gene sequencing. MPA was also significantly associated with decreased levels of estrogen in the plasma, and low vaginal glycogen and α-amylase, factors implicated in vaginal colonization by lactobacilli, bacteria that are believed to protect against STIs. In a humanized mouse model, MPA treatment was associated with low serum estrogen, low glycogen and enhanced HIV-1 susceptibility. The mechanism by which the MPA-mediated changes in the vaginal microbiota may contribute to HIV-1 susceptibility in humans appears to be independent of inflammatory cytokines and/or activated T cells. Altogether, these results suggest MPA-induced hypo-estrogenism may alter key metabolic components that are necessary for vaginal colonization by certain bacterial species including lactobacilli, and allow for greater bacterial diversity in the vaginal microbiota.This article has an associated First Person interview with the first author of the paper.

MicroRNA expression pattern differs depending on endometriosis lesion type.

MicroRNA (miRNA), noncoding segments of RNA involved in post-transcriptional regulation of protein expression are differentially expressed in eutopic endometrium of women with and without endometriosis compared to endometriotic lesions. However, endometriotic lesion types are known to be biochemically distinct and therefore hypothesized that miRNAs are differentially expressed in endometriomas compared to peritoneal and deep-infiltrating lesions. Therefore, endometrial biopsies and ectopic implants from women (n = 38) undergoing laparoscopic surgery for chronic pelvic pain were collected. Samples of endometriomas, peritoneal or deep-infiltrating lesions were selected from our tissue bank for study participants who exclusively had only one lesion type noted on their surgical report. Quantitative real-time polymerase chain reaction for miR-9, miR-21, miR-424, miR-10a, miR-10b, and miR-204 was performed. miR-204 expression was significantly lower (P = 0.0016) in the eutopic endometrium of women with endometriosis compared to controls. Relative expression of miR-21, miR-424, and miR-10b differed significantly (P < 0.05) across endometriotic lesion types. Finally, all miRNAs isolated from endometriomas, peritoneal and deep-infiltrating lesions studied were differentially expressed compared to matched eutopic endometrium samples. We therefore conclude that miRNA expression in the eutopic endometrium from women with endometriosis differs from symptomatic controls. Moreover, miRNA expression pattern is dependent on the endometriotic lesion type studied. We suggest that identification of different miRNA expression patterns for endometriomas, peritoneal and deep-infiltrating lesions could contribute to individualized patient care for women with endometriosis.

Frequency of Human CD45+ Target Cells is a Key Determinant of Intravaginal HIV-1 Infection in Humanized Mice.

Approximately 40% of HIV-1 infections occur in the female genital tract (FGT), primarily through heterosexual transmission. FGT factors determining outcome of HIV-1 exposure are incompletely understood, limiting prevention strategies. Here, humanized NOD-Rag1-/- γc-/- mice differentially reconstituted with human CD34+ -enriched hematopoietic stem cells (Hu-mice), were used to assess target cell frequency and viral inoculation dose as determinants of HIV-1 infection following intravaginal (IVAG) challenge. Results revealed a significant correlation between HIV-1 susceptibility and hCD45+ target cells in the blood, which correlated with presence of target cells in the FGT, in the absence of local inflammation. HIV-1 plasma load was associated with viral dose at inoculation and frequency of target cells. Events following IVAG HIV-1 infection; viral dissemination and CD4 depletion, were not affected by these parameters. Following IVAG inoculation, HIV-1 titres peaked, then declined in vaginal lavage while plasma showed a reciprocal pattern. The greatest frequency of HIV-1-infected (p24+) cells were found one week post-infection in the FGT versus blood and spleen, suggesting local viral amplification. Five weeks post-infection, HIV-1 disseminated into systemic tissues, in a dose-dependent manner, followed by depletion of hCD45+ CD3+ CD4+ cells. Results indicate target cell frequency in the Hu-mouse FGT is a key determinant of HIV-1 infection, which might provide a useful target for prophylaxis in women.

Role of sex hormones and the vaginal microbiome in susceptibility and mucosal immunity to HIV-1 in the female genital tract.

While the prevalence of Human immunodeficiency virus-1 (HIV-1) infection has stabilized globally, it continues to be the leading cause of death among women of reproductive age. The majority of new infections are transmitted heterosexually, and women have consistently been found to be more susceptible to HIV-1 infection during heterosexual intercourse compared to men. This emphasizes the need for a deeper understanding of how the microenvironment in the female genital tract (FGT) could influence HIV-1 acquisition. This short review focuses on our current understanding of the interplay between estrogen, progesterone, and the cervicovaginal microbiome and their immunomodulatory effects on the FGT. The role of hormonal contraceptives and bacterial vaginosis on tissue inflammation, T cell immunity and HIV-1 susceptibility is discussed. Taken together, this review provides valuable information for the future development of multi-purpose interventions to prevent HIV-1 infection in women.

Altered expression of chemokines and their receptors at porcine maternal-fetal interface during early and mid-gestational fetal loss.

Chemokines play a significant role in pregnancy, especially during embryonic attachment and placental development. During early pregnancy, immune cells are recruited extensively to the endometrium in several species including pigs. However, this recruitment is solely mediated by the presence of the conceptus in pigs making it a unique feature compared with other species (humans, primates and mice). To understand the biological significance of chemokine expression and immune cell recruitment in the context of fetal loss, we investigate a well-characterized porcine fetal loss model during the window of early pregnancy at gestational day (gd) 20 and mid-pregnancy (gd50). These periods coincide with 25-40 % of conceptus loss. Using targeted quantitative polymerase chain reaction and Western blot approaches, we screened a specific set of chemokines. Comparisons were made with endometrial lymphocytes (ENDO LY), endometrium and chorioallantoic membranes (CAM) associated with spontaneously arresting and healthy conceptus attachment sites (CAS). mRNA expression studies revealed an increased expression of CXCR3 and CCR5 in ENDO LY and of CXCL10, CXCR3, CCL5 and CCR5 in the endometrium associated with arresting CAS at gd20. DARC was decreased in the endometrium at gd50. CCL1 was increased in CAM associated with arresting CAS at gd50. Some of these differences were also noted at the protein level (CXCL10, CXCR3, CCL5 and CCR5) in the endometrium and CAM. CD45+ immunohistochemistry demonstrated a significantly higher localization in ENDO LY in the endometrium associated with healthy versus arresting counterparts. Most of these differences were observed in early pregnancy and might contribute towards a shift in immune cell functions.

Assessing brain-derived neurotrophic factor as a novel clinical marker of endometriosis.

OBJECTIVE: To evaluate novel clinical markers of endometriosis including the neurotrophins brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), and neurotrophin 4/5 (NT4/5) and compare them to others previously reported in the literature including cancer antigen 125 (CA-125) and C-reactive protein (CRP). DESIGN: Prospective study. SETTING: University hospital. PATIENT(S): One hundred thirty-eight women were prospectively and consecutively recruited (April 2011-April 2015; cases: undergoing endometriosis surgery, n = 96; controls: benign gynecological surgery, n = 24 combined with healthy women, no history of pelvic pain, not undergoing surgery, n = 18). INTERVENTION(S): Collection of peripheral blood, gynecological and demographic information, eutopic biopsy in women undergoing laparoscopy. MAIN OUTCOME MEASURE(S): Circulating BDNF, NGF, NT4/5, CA-125, and CRP were quantified by ELISA. RESULT(S): Plasma concentrations of BDNF were significantly greater in women with endometriosis (1,091.9 pg/mL [640.4-1,683.1]; n = 68, untreated) than in controls (731.4 pg/mL [352.1-1,176.2]; n = 36), whereas circulating NGF, NT4/5, CA-125, and CRP were not different. When assessed for their ability to differentiate between women with revised Classification of the American Society of Reproductive Medicine stage 1 and 2 or stage 3 and 4 disease and controls, BDNF was the only putative marker able to identify stage 1 and 2 disease, with a sensitivity and specificity of 91.7% and 69.4%, respectively, using an arbitrary cutoff value of 1,000 pg/mL. We also demonstrated that circulating BDNF in women with endometriosis who were receiving ovarian suppression for disease was equivalent to that in the control group. This suggests that BDNF may also offer the opportunity to monitor patient response to treatment. CONCLUSION(S): Plasma BDNF is a potentially useful clinical marker of endometriosis that is superior to NGF, NT4/5, CA-125, and CRP.

Estrogen induced changes in uterine brain-derived neurotrophic factor and its receptors.

STUDY QUESTION: Are brain-derived neurotrophic factor (BDNF) and its receptors, NTRK2, NGFR and SORT1, regulated by ovarian steroids in the uterus? SUMMARY ANSWER: BDNF and its low affinity receptor, nerve growth factor receptor (NGFR), are regulated by estradiol in the uterus. WHAT IS KNOWN ALREADY: Recent studies have revealed a central role for neurotrophins in placental development, endometrial stem cell neurogenesis, endometrial carcinoma and endometriosis. Complex signaling pathways involving BDNF and its receptors are regulated by ovarian hormones in the brain, however their expression and regulation in the uterus is poorly defined. STUDY DESIGN, SIZE, DURATION: This experimental animal study involved a total of 80 mice. PARTICIPANTS/MATERIALS, SETTING, METHODS: Female C57BL/6 mice (n = 50) were monitored daily for estrous cycle stage, and uterine horns were collected. A second group of mice (n = 30) were ovariectomized and given estradiol, progesterone, estradiol + progesterone, or saline for 4 days. Uterine expression of BDNF and its receptors were quantified by real-time PCR and western blot, and localized using immunohistochemistry. MAIN RESULTS AND THE ROLE OF CHANCE: During the estrous cycle, expression of BDNF, NTRK2 and SORT1 remained constant, while NGFR declined 11-fold from pro-estrus through to diestrus (P = 0.005). In ovariectomized mice, estradiol treatment increased uterine expression of mature BDNF greater than 6-fold (P = 0.013, 25 kDa; P = 0.003, 27 kDa), pro-BDNF 5-fold (P = 0.041, 37 kDa band; P = 0.046, 40 kDa band), and NGFR 5-fold (P < 0.001) when compared with other treatments. NTRK2 and SORT1 were unaffected by ovarian hormones. NGFR was primarily localized in epithelial cells in mice in diestrus or in ovariectomized mice treated with progesterone (P ≤ 0.001; P ≤ 0.001, respectively). In contrast, NGFR switched to a stromal localization in ovariectomized mice administered estradiol (P = 0.002). LIMITATIONS, REASONS FOR CAUTION: This study was performed in one only species. WIDER IMPLICATIONS OF THE FINDINGS: Results of this study demonstrate the uterine regulation of BDNF and NGFR by estradiol, and highlight the striking difference between hormone exposure during the estrous cycle and daily estradiol exposure after ovariectomy on neurotrophin expression in the uterus. The results also show the spatial regulation of NGFR in the uterus in response to ovarian hormones. Sustained estrogen exposure, as seen in estrogen-dependent disease, may alter the delicate neurotrophin balance and inappropriately activate potent BDNF-NTRK2 pathways which are capable of contributing to endometrial pathology. STUDY FUNDING/COMPETING INTERESTS: This study was supported by the Canadian Institutes of Health Research (CIHR) (W.G.F.), a NSERC Discovery Grant (W.G.F.), and a Vanier Canada Graduate Scholarship-CIHR (J.M.W.). J.M.W. is a member of the CIHR sponsored Reproduction and Early Development in Health training program. The authors declare no conflicts of interest.

Distinct microRNA expression in endometrial lymphocytes, endometrium, and trophoblast during spontaneous porcine fetal loss.

Endometrial lymphocytes are recruited to the porcine maternal-fetal interface by conceptus-derived signals. The transiently recruited lymphocytes adopt a specialized phenotype in the endometrium that regulates various placental physiological processes, including angiogenesis. Small non-coding RNAs, microRNAs (miRNAs) are emerging as principal bio-molecules regulating the development of lymphocytes and their angiogenic functions. However, no information is available in the context of endometrial lymphocytes in pregnancy. We hypothesize that miRNAs are involved in the development of endometrial lymphocytes and their angiogenic functions at the porcine maternal-fetal interface. Using a targeted Q-PCR approach for selected miRNAs involved in immune cell development, angiogenesis, and anti-angiogenesis, we conducted a study to screen endometrial lymphocytes associated with healthy and spontaneously arresting conceptus attachment sites (CAS) at two well-defined periods of fetal loss. Comparisons were made with endometrium and trophoblasts associated with healthy and arresting CAS. In addition, levels of putative mRNA targets and subsequent functional clustering of genes were studied in order to predict the biological mechanisms affected. We found several significant differences for miRNAs involved in immune cell development and angiogenesis (miR-296-5P, miR-150, miR-17P-5P, miR-18a, and miR-19a) between endometrial lymphocytes associated with healthy and arresting CAS. Significant differences were also found in endometrium and trophoblasts for some miRNAs (miR-20b, miR-17-5P, miR-18a, miR-15b-5P, and miR-222). Finally, selected mRNA targets showed differential expression in all groups. Our data, although associative, are the first to unravel the selected miRNAs involved in immune cell development and provide insights into their possible regulation in abortive pregnancy.

mRNA destabilizing factors: tristetraprolin expression at the porcine maternal-fetal interface.

PROBLEM: To evaluate the expression of the tristetraprolin family and their selected targets during porcine pregnancy. METHOD OF STUDY: Using qPCR and Western blot, mRNA and protein levels were compared between endometrium and chorioallantoic membrane (CAM) associated with healthy and impaired conceptuses at gestation day (gd) 20 and gd50, respectively. Immunohistochemistry was performed to determine localization of TIS11 family members at gd20 and 50. RESULTS: Multiple significant differences (P < 0.05) in TIS11 family transcripts were observed in the aforementioned comparisons. GM-CSF was significantly higher in healthy endometrium and CAM from impaired conceptus attachment sites. TNF-α was elevated in CAM as compared to endometrium at gd50, regardless of conceptus health status. Immunohistochemical staining shows TIS11 family expressed in the glandular and luminal epithelium, as well as stromal cells in the uterus. CONCLUSIONS: The shift in the expression of tristetraprolin (TTP) and TIS11D points to a potential role of these genes in regulating spontaneous fetal loss.

Expression of leptin and its long form receptor at the porcine maternal-fetal interface: contrasting healthy and arresting conceptus attachment sites during early and mid-pregnancy.

BACKGROUND: It is well established that spontaneous conceptus loss in swine is associated with an imbalance of both angiogenic and immunological factors. Leptin (LEP), a metabolic hormone, has also been implicated in the promotion of angiogenesis. In this study, LEP and its long form receptor (OB-Rb) were evaluated during porcine pregnancy to assess their basal level of expression and their potential role in conceptus development. METHODS: Expression and secretion of LEP and OB-Rb were quantified in the endometrium of non-pregnant (n = 5), and in the endometrium and chorioallantoic membrane (CAM) of pregnant sows (parity 2 to 5) at gestational days (gd) 20 (n = 8) and 50 (n = 8). Data were analyzed by a 3-way ANOVA testing the effects of conceptus health, tissue type and gestational day. RESULTS: Leptin and OB-Rb transcripts were significantly higher (P < 0.05) in pregnant than in non-pregnant sows. Significantly greater LEP (P < 0.001) was detected in the endometrial tissue at gd20 compared with gd50. At the protein level, the lowest LEP expression (P < 0.01) was detected in the CAM at gd50, while OB-Rb protein was significantly lower (P < 0.01) at gd50 in the CAM than in the endometrium collected from gd20 and gd50 conceptus attachment sites. Immunofluorescence staining confirmed the expression of these proteins at both gestational days and in both tissue types. CONCLUSIONS: Changes in the expression patterns of LEP and OB-Rb between gd20 and gd50 suggest a role for the LEP/OB-R complex at the early stages of porcine pregnancy, possibly affecting the attachment process. Further mechanistic studies are warranted to understand the specific role of leptin in porcine pregnancy.

MicroRNAs, immune cells and pregnancy.

MicroRNAs (miRNAs) are a recently discovered class of non-coding RNAs that are expressed in many cell types, where they regulate the expression of complementary RNAs, thus modulating the stability and translation of mRNAs. miRNAs are predicted to regulate the expression of ∼50% of all protein coding genes in mammals. Therefore, they participate in virtually all cellular processes investigated so far. Altered miRNAs expressions are associated with both physiological (pregnancy) and pathological processes (cancer). As the dynamic maternal-fetal interface plays a critical role in the maintenance of successful pregnancy, it is not surprising that the miRNAs that are unique to reproductive tissues are abundantly expressed. Research in this field has demonstrated the presence and dysregulation of a distinct set of pregnancy-associated miRNAs; however, most studies have centered on localizing various miRNAs in reproductive microdomains associated with normal or complicated pregnancies. Although several independent miRNA regulatory mechanisms associated with endometrial receptivity, immune cells, angiogenesis and placental development have been studied, miRNA-mediated regulation of pregnancy remains poorly understood. This review provides a summary of the current data on miRNA regulation as well as functional profiles of miRNAs that are found in the uterus, in immune cells associated with maternal tolerance to the fetus, and those involved in angiogenesis and placental development.

The brain-uterus connection: brain derived neurotrophic factor (BDNF) and its receptor (Ntrk2) are conserved in the mammalian uterus.

The neurotrophins are neuropeptides that are potent regulators of neurite growth and survival. Although mainly studied in the brain and nervous system, recent reports have shown that neurotrophins are expressed in multiple target tissues and cell types throughout the body. Additionally, dysregulation of neurotrophins has been linked to several disease conditions including Alzheimer's, Parkinson's, Huntington's, psychiatric disorders, and cancer. Brain derived neurotrophic factor (BDNF) is a member of the neurotrophin family that elicits its actions through the neurotrophic tyrosine receptor kinase type 2 (Ntrk2). Together BDNF and Ntrk2 are capable of activating the adhesion, angiogenesis, apoptosis, and proliferation pathways. These pathways are prominently involved in reproductive physiology, yet a cross-species examination of BDNF and Ntrk2 expression in the mammalian uterus is lacking. Herein we demonstrated the conserved nature of BDNF and Ntrk2 across several mammalian species by mRNA and protein sequence alignment, isolated BDNF and Ntrk2 transcripts in the uterus by Real-Time PCR, localized both proteins to the glandular and luminal epithelium, vascular smooth muscle, and myometrium of the uterus, determined that the major isoforms expressed in the human endometrium were pro-BDNF, and truncated Ntrk2, and finally demonstrated antibody specificity. Our findings suggest that BDNF and Ntrk2 are transcribed, translated, and conserved across mammalian species including human, mouse, rat, pig, horse, and the bat.

Animal models for anti-angiogenic therapy in endometriosis.

Endometriosis is a gynecological disease characterized by the growth of endometrium outside of the uterine cavity. It is often associated with dysmenorrhea, dyspareunia, pelvic pain and infertility. One of the key requirements for endometriotic lesions to survive is development of a blood supply to support their growth. Indeed, dense vascularization is characteristic feature of endometriotic lesions. This has led to the idea that suppression of blood vessel growth (anti-angiogenic therapy) may be a successful therapeutic approach for endometriosis. Potential effectiveness of anti-angiogenic therapies has been assessed in some animal models but there are no reports of human clinical trials. Without understanding the specific mechanism by which endometriosis lesions establish a new blood supply, short-term animal experiments will have limited value for translation into human medicine. Further, it is crucial to use appropriate animal models to assess efficacy of anti-angiogenic compounds. Syngeneic and autologous rodent models, where endometrial fragments are auto-transplanted into the peritoneal cavity are commonly used in anti-angiogenic therapy studies. Another approach is xenograft models where human endometrium is engrafted into immunodeficient mice. Here we review the animal models and experimental techniques used to evaluate anti-angiogenic therapies for endometriosis. We also review our own work on the role of stromal cell derived factor-1 in the recruitment of endothelial progenitor cells in endometriotic lesion angiogenesis, and the effects of the anti-angiogenic peptide ABT-898, a thrombospondin-1 mimetic, on endometriotic lesion growth and vascular development.

Expression of angiogenic basic fibroblast growth factor, platelet derived growth factor, thrombospondin-1 and their receptors at the porcine maternal-fetal interface.

BACKGROUND: Commercial swine breeds in North America undergo two waves of spontaneous fetal loss; one during peri-attachment and another during mid-gestation. Although an exact mechanism for this loss is not known, deficits in vasculature at the attachment sites appear to be a major cause. We hypothesized that a balance between pro-angiogenic and anti-angiogenic factors is needed at the maternal-fetal interface for successful conceptus development. Six selected members of the pro-angiogenic fibroblast growth factor (FGF) and platelet derived growth factor (PDGF) families and anti-angiogenic factor thrombospondin-1 (TSP-1) and its receptor CD36 were quantified and localized at the porcine maternal-fetal interface at early and midgestation time points. METHODS: Mesometrial endometrium was collected from non-pregnant gilts (n = 8). Endometrial and chorioallantoic membrane samples were collected from healthy and arresting conceptus attachment sites at gestation day (gd) 20 (n = 8) and gd 50 (n = 8). At gd20 arresting conceptus attachment sites were distinguished by decreased vasculature of the placental membranes and decreased conceptus size. At gd50 arresting conceptuses attachment sites were identified by smaller conceptus length and weight measurements. Quantitative real time PCR was used to determine relative transcript levels of genes of interest, and cellular localization was determined by immunohistochemistry in paraffin embedded endometrial sections. RESULTS: At gd20, endometrial samples from arresting conceptuses had elevated transcripts for bFGF, and PDGF-bb than healthy sites (p < 0.05). At gd50, bFGF, FGFR2, and CD36 were more abundant at arresting than at healthy conceptus attachment sites (p < 0.05). Chorioallantoic membrane from arresting conceptus attachment sites at gd20 had elevated transcripts for bFGF, FGFR1, FGFR2 and CD36 compared with healthy sites (p < 0.05). FGFR2 transcripts were more abundant in chorioallantoic membrane from arresting conceptuses at gd 50 (p < 0.05). Immunohistochemical localization of selected pro- and anti-angiogenic factors and receptors revealed their abundance in the luminal epithelium, uterine glands and perivascular areas of endometrium at gd20 and gd50. CONCLUSIONS: We provide comprehensive analysis of pro and anti-angiogenic factors at the porcine maternal fetal interface during early and mid-pregnancy. At mRNA levels, the majority of pro-angiogenic factors investigated were elevated at the sites of fetal arrest. These observations contrast with our previous findings of decreased Vascular Endothelial Growth Factor (VEGF) family members at arresting sites, and suggest that the bFGF family functions as a compensatory survival mechanism when major angiogenic proteins are decreasing at the sites of fetal arrest.

Expression of chemokine decoy receptors and their ligands at the porcine maternal-fetal interface.

Successful pregnancy requires coordinated maternal-fetal cross-talk to establish vascular connections that support conceptus growth. In pigs, two waves of spontaneous fetal loss occur and 30-40% of conceptuses are lost before parturition. Previous studies associated these losses with decreased angiogenic and increased inflammatory cytokines. Chemokines, a sub-category of cytokines, and decoy receptors control leukocyte trafficking, angiogenesis and development. The availability of chemokines is regulated by three non-signalling decoy receptors: chemokine decoy receptor (D6), Duffy antigen receptor for chemokines (DARC) and Chemocentryx decoy receptor (CCX CKR). We hypothesized that the expression of these receptors and their chemokine ligands regulate the porcine pregnancy success or failure. Here, we describe for the first time the transcription and translation of all three decoy receptors and several chemokine ligands in endometrium and trophoblast associated with healthy and arresting conceptuses at gestation day (gd) 20 and gd50. Among decoy receptors, transcripts for DARC were significantly reduced in endometrium, whereas that for CCX CKR were significantly increased in endometrium and trophoblast at gd50 arresting compared with healthy sites. However, western blot analysis revealed no differences in decoy receptor expression between healthy and arresting tissues. Transcripts for decoy receptor ligands CCL2, CCL3, CCL4, CCL5, CCL11, CCL19, CCL21, CXCL2 and CXCL8 were stable between healthy and arresting littermates. Quantification by SearchLight chemiluminescent protein array confirmed ligand expression at the protein level. These data indicate that decoy receptors and ligands are expressed at the porcine maternal-fetal interface and dysregulation of decoy receptor (DARC and CCX CKR) transcripts occurs at sites of fetal arrest.