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Problem: Endometrium, the innermost mucosal layer of the uterus, serves as a lodge for the embryo in eutherian mammals. The endometrium is constituted of various cell types, and each cell type executes specific functions to facilitate embryo implantation and development. It is well established that the endometrium, despite being non-permissive to the embryo for the major period of a menstrual cycle, is irreplaceable in the scheme of events essential for procreation. However, the embryo, before initiating physical contact with the endometrium, encounters the uterine cavity that remains bathed in uterine fluid. Uterine fluid is an admixture of endometrial secretions, plasma transudates, and oviductal fluid. Uterine fluid components are believed to play important roles in immunosuppression and embryo development during peri-implantation period. Uterine fluid is also involved in defense against pathogens, sperm migration, and lubrication of endometrium. The advent of high-throughput functional genomics tools has created enormous opportunities to investigate the uterine fluid for its protein repertoire and modulation during the receptive phase of an endometrial cycle in animals and humans. Towards this, few investigations have been conducted in recent years. The data obtained using non-targetted functional genomics approaches need to be assimilated with the existing information on specific components of uterine fluid., Method: This review compiles existing information on the composition of uterine fluid and its significance in endometrial functions and dysfunctions., Result: Collectively, investigations based on targetted and non-targetted approaches have revealed the presence of several cytokines, growth factors, ions, carbohydrates, and steroids, in human uterine fluid., Conclusion: Detailed investigations of human uterine fluid, especially directed towards the elucidation of functional relevance of different proteins in uterine fluid, will help identify novel markers of endometrial receptivity and also gain significant insights into the mechanisms underlying unexplained infertility, recurrent pregnancy losses, and other endometrial pathologies., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Chemokines are a group of small cytokines that have an ability to induce leukocyte migration. Chemokines exert their functions by binding and activating specific G protein-coupled receptors. Studies have unveiled pleiotropic bioactivities of chemokines in various phenomena ranging from immunomodulation, embryogenesis, and homeostasis to pathogenesis. In the mammalian reproductive system, chemokines unexceptionally serve in multimodal events that are closely associated with establishment, maintenance, and deterioration of fecundity. The aim of this review is to update the knowledge on chemokines in male and female genital organs, with a focus on their potential pathophysiological roles in human reproduction., (© 2010 John Wiley & Sons A/S.)

Problem: CCL20/MIP3alpha is a chemokine for immature dendritic cells as well as an antibacterial against gram-positive and gram-negative bacteria. The role of CCL20/MIP3alpha as an antiviral is unknown. In this study, we have examined the production of CCL20/MIP3alpha by epithelial cells from the upper female reproductive tract as well as its activity as an antiviral molecule., Method of Study: Primary uterine and Fallopian tube epithelial cells were treated with Poly(I:C) and CCL20/MIP3alpha mRNA and protein was measured by Realtime RT-PCR and ELISA assays. Anti-HIV activity was determined using an indicator cell line TZM-bl and quantified by using a luminometer., Results: Primary uterine and Fallopian tube epithelial cells produce CCL20/MIP3alpha constitutively and the production is enhanced following stimulation with viral double-stranded RNA mimic Poly(I:C). Recombinant CCL20/MIP3alpha was able to inhibit both T-cell-tropic X4/IIIB and macrophage-tropic R5/BaL HIV-1 when virus was directly incubated with CCL20/MIP3alpha but not when CCL20/MIP3alpha was added to cells either prior to infection or post-infection. This suggests that the mechanism of inhibition is likely to be a direct interaction between HIV-1 and CCL20/MIP3alpha., Conclusion: This study demonstrates that CCL20/MIP3alpha is an important endogenous anti-HIV-1 microbicide of the female reproductive tract.

Problem: The role of neutrophils in the human Fallopian tube (FT) is unknown. In order to provide insights into their functions in the FT, we systematically compared neutrophils from normal FT and peripheral blood (PB)., Method of Study: Flow cytometric analysis of surface receptors, granule proteins, and intracellular cytokines expressed by neutrophils from enzymatically dispersed FT and PB was performed., Results: Fallopian tube neutrophils expressed significantly higher levels of CD64, human class II histocompatibility antigen DR (HLA-DR), gamma-interferon, and vascular endothelial growth factor than those from PB. Fewer FT neutrophils expressed IL-8 receptors compared to PB, while more expressed the receptor for the bacterial-derived chemoattractant formyl-Met-Leu-Phe (fMLP). The number of FT neutrophils containing the granule proteins matrix metalloproteinase-9, lactoferrin, and myeloperoxidase was decreased versus PB., Conclusion: Fallopian tube neutrophils exhibit a phenotype distinct from PB neutrophils, suggesting functional activation of innate immune defense in the female reproductive tract as well as a potential role in maintaining normal FT physiology.

Problem: Mechanisms of protective immunity in the female reproductive tract are poorly understood. For sexually transmitted diseases, bovine trichomoniasis is a useful model because it resembles human trichomoniasis to some extent, and antibodies play an important role in protection against these extracellular parasites. Protective efficacy was compared in animals with genital responses of predominantly immunoglobulin G (IgG) or predominantly IgA antibodies to a purified surface antigen of Tritrichomonas foetus., Method of Study: Immunization of mice by various routes with immunoaffinity-purified T. foetus surface antigen (TF1.17) or killed cells was used to define the best routes and antigen combinations to give predominantly IgG or IgA antibodies to TF1.17 antigens in genital secretions. Cattle were then immunized either subcutaneously (SC) two times with TF1.17 antigen and once SC with killed T. foetus or twice SC with TF1.17 antigen and once intravaginally with killed T. foetus. All immunizations were in Quil A adjuvant. Controls were not immunized. Animals were challenged intravaginally with 10(6) T. foetus 3 weeks after the third immunization. Vaginal mucus was collected weekly for culture and antibody assays. Serum was collected weekly, and uterine secretions were collected at 10 weeks post-challenge. Tissues were fixed at 10 weeks also., Results: Murine studies showed systemic priming with vaginal boosting gave the highest genital IgA responses. In cattle, systemic immunization (group S) induced high IgG1 antibody levels in vaginal secretions. Systemic priming with vaginal boosting (group S/V) primed for an anamnestic vaginal IgA response after challenge with T. foetus. Cattle with predominantly IgG or predominantly IgA responses in vaginal secretions either did not become infected or cleared infection faster than controls. Uterine IgA responses at 10 weeks were highest in the vaginally boosted group, but other responses were not different from the controls at this time point. Microscopic examination of genital tissues showed subepithelial infiltration of mononuclear cells in all groups. Lymphoid aggregates or nodules were detected in vaginal sections in cattle of groups S/V and C as well as in uterine sections of all animals in all three groups., Conclusions: Both IgG and IgA antibodies to T. foetus superficial antigen were associated with protection. The timing of the response was related to the time of clearance. Lymphoid organization in the vagina and uterine tissues suggested development of mucosal inductive sites.

Problem: Leukemia inhibitory factor (LIF) is a pleiotropic cytokine of the interleukin-6 family and has different biological actions in various tissue systems. Although named for its ability to inhibit proliferation of a myeloid leukemic cell line by inducing differentiation, it also regulates the growth and differentiation of embryonic stem cells, primordial germ cells, peripheral neurons, osteoblasts, adipocytes, and endothelial cells. LIF is crucial for successful implantation of the embryo in mice. Currently, there is an accumulation of data about the role of LIF in human reproduction., Method of Study: This review of the literature and of our studies focuses on the expression, regulation, and effects of LIF in the human endometrium, fallopian tube, and ovarian follicle., Results: Human endometrium expresses LIF in a menstrual cycle-dependent manner. Maximal expression is observed between days 19 and 25 of the menstrual cycle, coinciding with the time of implantation. Various cytokines and growth factors induce endometrial LIF expression in vitro. LIF receptor is expressed in endometrial tissue throughout the menstrual cycle and on human blastocysts in a stage-dependent manner. Affecting the trophoblast differentiation pathway toward the adhesive phenotype, LIF plays a role in implantation. LIF is also expressed and secreted by the epithelial cells of the fallopian tube. Its increased expression in the tubal stromal cell cultures by the inflammatory cytokines suggests a link between salpingitis and ectopic implantation in the tube. The rising follicular fluid LIF level around the time of ovulation indicates that LIF may play a role in ovulatory events, early embryonic development, and implantation., Conclusions: There is growing evidence that LIF may be one of the entities that plays a role in human reproduction.

Problem: The tissues of the human female reproductive tract (Fallopian tube, uterus, cervix, and vagina) may play different roles in the provision of mucosal immunity. The purpose of this study was to develop a uniform method suitable for quantitative comparison of the leukocytes from all these tissues., Method of Study: Tissues, typically 0.5-1.0 g, were dispersed by enzyme treatment. A flow cytometric gating procedure based on CD45-positivity and low far-red autofluorescence permitted unfractionated, freshly dispersed cells to be phenotyped with respect to T lymphocytes, B lymphocytes, macrophages, and granulocytes., Results: Reproductive tract tissues contain leukocytes that represent approximately 6-20% of the total number of cells, with the Fallopian tubes and uterus containing a higher proportion of leukocytes than the cervix and vagina. The uterine endometrium from post-menopausal women has fewer leukocytes than does uterine endometrium from pre-menopausal women. T lymphocytes are a major constituent (30-60%) of leukocytes from all tissues. The Fallopian tube contains granulocytes as another major constituent; granulocytes are significantly less numerous in the other tissues. All tissues contain B lymphocytes and macrophages as clearly detectable but minor components., Conclusions: Three-color flow cytometry is an appropriate method for quantitative comparison of leukocytes from the different tissues of the female reproductive tract, during all phases of the menstrual cycle and within post-menopausal samples. Results indicate that the tissues differ from each other, particularly with respect to the large number of granulocytes in the Fallopian tubes.

Problem: How is it possible that the female genital tract immunologically does not reject spermatooa not the preimplantation and nidating embryo?, Methods: Four fluids of the human reproductive tract, i.e., human oviductal fluid (hOF), follicular fluid (FF), amniotic fluid (AF), and seminal plasma (SP) were investigated by specific ELISA for 18 cytokines. The concentrations, presence or absence of these compounds were evaluated for their possible role in the immunology of the reproductive process., Results: Stem cell factor and IL-11 were detected in all reproductive tract fluids examined whereas large amounts of IL-1 beta and IL-1RA was found in AF and hOF. Follicular fluid revealed IL-2. HOF contained IL 2, IL-6, IL-8, TNF-alpha, MIP-1 alpha, IFN-gamma, and high levels of IL-1 beta, IL-10, IL-1RA, and sIL-2R. Amniotic fluid contained sIL-2R, IL-8, IL-1 beta, IL-1RA, IL-6, TNF-alpha, and MIP-1 alpha. No IL-12 or IL-13 was detected in hOF follicular fluid or amniotic fluid. Almost no free TGF-beta 1 or TGF-beta 2 was found in any reproductive tract fluid except seminal plasma. Seminal plasma contained large quantities of free TGF-beta 1 (9,220 +/- 3,635 pg/mL) in addition to large quantities of latent TGF-beta 2 (2,933 +/- 2,169 pg/mL) and TGF-beta 1 (71,000 +/- 3,240 pg/mL). Furthermore, considerable concentrations of IL-8 (1900 +/- 374 pg/mL) and sIL-2R (350 mu/mL) exist in seminal plasma., Conclusions: HOF contains a high level of IL-10 (588 +/- 304 pg/mL), a powerful immune suppressor which probably plays a role in regulating immune responses in the fallopian tube and possibly in the endometrial cavity. Our observations suggest that seminal plasma with its huge content of TGF beta provides immune protection for sperm. Unfortunately, such high concentrations of TGF beta may also inhibit an immune defense in any organ in which semen is deposited.

Problem: Systematically to investigate the expression of human leucocyte antigen (HLA) DR on the columnar epithelium and the content of T-lymphocytes in the fimbriae specimens of the fallopian tube from healthy women and from women with infertility and pelvic adhesions., Method: The staining of HLA-DR expression on the columnar epithelium was judged on a four-graded scale according to the distribution of HLA-DR expression. The amount and distribution of T-lymphocytes were registered., Results: The biopsies from normal fimbriae contained sparsely infiltrating T-lymphocytes. On the columnar epithelial cells a patchy HLA-DR expression was observed that was more widespread in preovulatory cases. In some biopsies from sactosalpinx, the HLA-DR expression was found completely to cover the columnar epithelial cells and was associated with a heavy infiltration of T-lymphocytes. CD4+ T-lymphocytes dominated in all but one adhesion biopsies., Conclusion: The findings indicate an ongoing immunological activity in pelvic adhesions that might immunologically influence fertility.

Problem: It is well known that sperm migration in cervical mucus is impaired by sperm immobilizing antibodies secreted in the mucus. However, it is not clear yet whether sperm migration from the uterine cavity through the fallopian tubes to the peritoneal cavity is impaired by sperm immobilizing antibodies. To test the possible impairment of sperm migration in the tubes, laparoscopic examinations were carried out and the presence of motile sperm in the peritoneal fluid after intra-uterine insemination was investigated., Method: Peritoneal sperm recovery tests were performed in 28 infertile women with sperm immobilizing antibodies in their sera, and the results were compared with those in 322 infertile women without the antibodies. Both the sperm immobilizing antibody titers (SI50) and complement activities (C'H50) in peritoneal fluid were compared with those in patients' sera. In some experiments, the supernatant of the peritoneal fluid was used as a source of complement for the sperm immobilization tests instead of guinea pig serum., Results: Among couples with normal semen characteristics by the criteria of WHO, sperm recovery in the peritoneal fluid was observed in only 3 (11.1%) of 27 patients with sperm immobilizing antibodies, compared with 72 (34.0%) of 212 patients without the antibodies (P < 0.025). The antibody titers of the patients with the sperm recovery were very low by the quantitative sperm immobilization test. In most patients, a similar amount of sperm immobilizing antibodies was present in the peritoneal fluid and the sera. Though the complement activities in the peritoneal fluid were less than those in sera, the former were still found to be sufficient to immobilize sperm in vivo., Conclusions: These results suggest that the complement-dependent sperm immobilizing antibodies could interfere with sperm migration in the female genital tract at the level of the fallopian tubes.