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Triggering receptor expressed on myeloid cells-2 (TREM-2) is an innate immune receptor that initiates cellular activation upon ligation. In this study, we examined the interaction of TREM-2 with Neisseria gonorrhoeae using murine TREM-2A, as it has been reported to recognize bacterial ligands. Using a whole-bacteria enzyme-linked immunosorbent assay (ELISA), TREM-2A bound to all six strains in variable degrees. Far-western blots of gonococcal outer membranes revealed TREM-2A binding to lipooligosaccharide (LOS) and opacity (Opa) protein, with predominant binding to LOS. Binding of TREM-2A to LOS was confirmed by ELISA and surface plasmon resonance. O-deacylation of the lipid A significantly reduced binding. Flow cytometry and reporter cell assays showed that gonococci bound to TREM-2A-transfected cells and induced transmembrane signaling. In humans, TREM-2 was constitutively expressed by genitourinary and fallopian tube epithelial cells, both of which are primary targets of gonococcal invasion. Ligation of TREM-2 by LOS induced interleukin-6 production in HeLa cervical carcinoma cells. To our knowledge, this is the first report of the expression of human TREM-2 by cells deriving from a non-myeloid lineage. We conclude that gonococci can interact with TREM-2 receptors through binding to LOS and Opa protein and initiate cell signaling and cytokine production.

Objective: To examine the expression of toll-like receptors (TLR) by primary human Fallopian tube epithelial cells (FTEC) and to determine whether exposure to the TLR3 agonist poly(I:C) induces an antiviral response., Design: Tissue culture study., Setting: University medical center., Patient(s): Premenopausal women undergoing hysterectomy., Intervention(s): Primary human FTEC were grown to confluence and high transepithelial resistance and treated with TLR agonists. Conditioned media was collected and RNA was extracted and analyzed for the expression of cytokines, chemokines, and antimicrobial genes., Main Outcome Measure(s): The RNA was analyzed by real-time polymerase chain reaction and protein levels were assessed by enzyme-linked immunosorbent assay., Result(s): The FTEC were demonstrated to express TLR1-9 but not 10. Treatment of FTEC with TLR3 agonist poly(I:C) resulted in increased expression of interleukin-8, tumor-necrosis factor alpha, human beta-defensin 2, interferon beta, and interferon stimulated genes myxovirus resistance gene 1, 2',5'-oligoadenylate synthetase, and protein kinase R. Additionally, FTEC exposed to poly(I:C) also resulted in the induction of TLR2, TLR3, and TLR7., Conclusion(s): Our results suggest that FTEC are sensitive to viral infection and/or exposure to viral double-stranded RNA and can respond by secreting proinflammatory cytokines that mediate the initiation of an inflammatory response as well as expressing genes that can directly inhibit viral replication.

The MUC1 mucin is normally expressed in the epithelium of the fallopian tube, and its expression on the epithelium of the tubes with ectopic pregnancies has not been studied. The mean (+/- SEM) histologic score for MUC1 was 1.4 +/- 0.2 in the luminal epithelium of tubes with ectopic pregnancies, and the histologic score for normal tubes was 2.8 +/- 0.2; however, these findings must be confirmed with the identification of the cytoplasmic tail of MUC1.

The fallopian tubes are essential for the normal transport of gametes, fertilisation and early embryonic development and transport. Their locomotive force is mainly due to the contractility of the smooth muscle cells, as well as to the ciliary activity of the tubal epithelium. Steroid hormones such as oestradiol and progesterone mediate changes in tubal morphology, in particular the tubal epithelium. It is well known that macrophages participate in the immune system, but recent studies have shown that they also play other roles under physiological conditions. They are known to be a source of prostaglandins of the E series, which influence the contractility of the uterine tube. Lymphocytes in the tubal mucosa can be involved in the process of immune tolerance, which could enable sperms and blastocysts to be transported through the oviduct under normal conditions without the activation of local immune mechanisms. Most of the evidence for mucosal immune responses in the female reproductive tract is related to the vagina, with less information available for the uterus. The less known segment in this regard is the oviduct, which prompted us to review and summarise the current state of knowledge of the immune system at the level of the human oviduct. The present study was therefore undertaken to examine the distribution and morphological properties of macrophages in the endosalpingeal stroma and smooth muscle layer of the human fallopian tubes. Thirty fresh fallopian tubes were examined, taken at the proliferative (7 cases) and secretory (12 cases) phases of the menstrual cycle, and during the postmenopausal period (11 cases). Sections were stained by immunocytochemistry with a primary antibody (CD 68) and were used for counting the macrophages. Ultrathin sections were stained with lead citrate and uranyl acetate and studied by means of electron microscopy to asses the ultrastructure of the macrophages. A significant difference was observed between reproductive and postmenopausal women in the number of macrophages (p<0.05). This study may help to clarify the possible role of macrophages of the uterine tubes in some cases of infertility in females.

Introduction: Tubal rupture seems to be linked to a disturbance in maternal immune response and trophoblast cell invasion. The immunomodulating activity of endometrial cells is necessary for the coexistence of activated immune cells and endometrial cells. RCAS1 and metallothionein (MT) participate in this process., Material and Methods: Tissue samples derived from fallopian tubes and endometrium were collected during one surgical procedure and divided into three groups: unruptured ectopic pregnancy (EP) without bleeding, unruptured EP with hemorrhage into the peritoneal cavity, and ruptured tubal pregnancy. Immunoreactivity of MT, RCAS1, CD56, CD3, CD69 and CD25 were assessed by immunohistochemical methods., Results: The number of CD3+ and CD56+ cells as well as CD69 antigen immunoreactivity in ruptured tubal mucosa of EP were statistically significantly higher than those measured for unruptured EP without bleeding, while at the same time the number of CD56+ cells in endometrium was statistically significantly lower. The growth of immune cell numbers in tubal mucosa during tubal rupture was not associated with an adequate MT and RCAS1 level., Conclusion: Tubal perforation seems to be linked to a concentration of immune cells and a growth of their activity without an adequate increase of the level of proteins compensating for immune cell response., ((c) 2007 S. Karger AG, Basel)

Chlamydia trachomatis infection is associated with severe Fallopian tube tissue damage leading to tubal infertility and ectopic pregnancy. To explore the molecular mechanisms behind infection an ex vivo model was established from human Fallopian tubes and examined by scanning electron microscopy and immunohistochemistry. Extensive tissue destruction affecting especially ciliated cells was observed in C. trachomatis infected human Fallopian tube organ culture. Interleukin-1 (IL-1) produced by epithelial cells was detected after infection. Addition of IL-1 receptor antagonist (IL-1RA) completely eliminated tissue destruction induced by C. trachomatis. The anti-inflammatory cytokine IL-10 reduced the damaging effect of C. trachomatis infection, however, to a lesser extent than IL-1RA. Furthermore, IL-1 was found to induce IL-8, a neutrophil attractant, using a signal transduction pathway involving p38 MAP kinase. Consequently, IL-1 has the potential to generate a cellular infiltrate at the site of infection in vivo. Blocking the IL-1 receptors by IL-1RA eliminated tissue destruction and cytokine production. Hence, these studies show the importance of IL-1 in initiating the tissue destruction observed in the Fallopian tube following C. trachomatis infection. Because leukocytes are absent in the ex vivo model, this study strongly indicates that IL-1 is the initial proinflammatory cytokine activated by C. trachomatis infection.

Chlamydia trachomatis is the most prevalent sexually transmitted bacterial infection in the world. In women, genital infection can cause endometritis and pelvic inflammatory disease with the severe sequelae of ectopic pregnancy or infertility. Chlamydia sp. do not damage tissues directly, but induce an injurious host inflammatory response at the infected site. In the murine model of genital disease with Chlamydia muridarum, TLR2 plays a role in both early production of inflammatory mediators and development of chronic oviduct pathology. We report the results of studies with plasmid-cured C. muridarum mutants that retain the ability to infect the murine genital tract, but fail to cause disease in the oviduct. These mutants do not stimulate TLR2-dependent cytokine production in mice, nor in innate immune cells or epithelial cells in vitro. They induce an effective Th1 immune response, with no evidence for Th1-immune-mediated collateral tissue damage. Furthermore, mice previously infected with the plasmid-deficient strains are protected against oviduct disease upon challenge with virulent C. muridarum. If plasmid-cured derivatives of human C. trachomatis biovars exhibit similar phenotypic characteristics, they have the potential to serve as vaccines to prevent human disease.

Epithelial inclusion cysts (EICs) are considered a preferential site for ovarian carcinogenesis. Local inflammation, associated to ovulatory wound repair and epithelial inflammatory conditions, facilitates EIC formation and involves activation of macrophages. The aim of this study was to analyse the presence and numbers of macrophages in the ovarian surface epithelium (OSE), in EICs, and in the fallopian tubes, as tubal metaplasia is a common finding in EICs. Immunohistochemical analysis of macrophages was performed in 25 fallopian tubes in different phases of the menstrual cycle, and in 30 ovaries showing EICs from cycling and postmenopausal women. In the fallopian tube, macrophages were abundant and underwent cyclic changes during the menstrual cycle, being particularly abundant within the epithelium at early and mid-luteal phases. Macrophages were not found in the normal OSE. However, OSE areas and EICs showing tubal metaplasia were invariably associated with infiltration by abundant macrophages. Macrophages were present among epithelial cells, infiltrating the cyst wall, as well as free in the cyst lumen. No significant differences existed between follicular and luteal phases of the cycle, or between cycling and postmenopausal women. This study has demonstrated that macrophages are associated with metaplastic EICs, and raises the possibility that these cells contribute to the particular microenvironment of EICs through secretion of cytokines and growth factors that may reach bioactive concentrations in the confined space of the EICs.

Background: The aim of this study was to evaluate the site-specific immunoregulatory mechanisms against viral infection in human Fallopian tubes., Methods: We therefore investigated the effects of double-stranded RNA (dsRNA) on the production of interleukin (IL)-6, IL-8 and granulocyte chemotactic protein-2 (GCP-2) by cultured oviductal epithelial cells (OECs) using enzyme-linked immunosorbent assays. Phosphorylation of inhibitor kappaB-alpha (IkappaB-alpha) protein after dsRNA stimulation and the expression of Toll-like receptor (TLR) 3 in these cells were also evaluated by western blot analysis., Results: Polyriboinosinic:polyribocytidylic acid (poly I:C), a synthetic dsRNA that antagonizes TLR3, stimulated the secretion of IL-6, IL-8 and GCP-2 by OECs. Poly I:C-induced production of these cytokines by OECs was inhibited by the pretreatment of these cells with anti-TLR3 antibody. The phosphorylation of IkappaB-alpha protein was detected in OECs after stimulation by poly I:C. The expression of TLR3 was also detected in OECs., Conclusion: These results suggest that the epithelial cells of the human Fallopian tube have evolved a unique, site-specific mechanism for recognizing viral infection. TLR3-mediated production of proinflammatory cytokines and chemokines in OECs in response to viral dsRNA may be important for antiviral immunity in the human female reproductive tract.

The aim of this study was to investigate the influence of pre- and post-ovulatory insemination and early pregnancy on the distribution of immune cells in the oviduct. Eighteen sows were pre-ovulatory and sixteen sows were post-ovulatory inseminated and slaughtered at different times, 5-6 h after insemination, 20-25 h and approximately 70 h after ovulation, day 11 and day 19. Immediately after slaughter, oviductal samples of three different segments (isthmus, ampulla and infundibulum) were fixed, embedded in plastic resin and stained with toluidine blue or cryofixed and stored in a freezer at -70 degrees C until analysed by immunohistochemistry (pre-ovulatory inseminated sows) with an avidin-biotin peroxidase method. Quantitative and qualitative examinations of oviductal epithelium and subepithelial connective tissue were performed by light microscopy. After pre- or post-ovulatory insemination, neutrophils were not observed in the oviductal epithelium from any of the segments or groups. The numbers of intraepithelial lymphocytes of all sows as well as CD2- and CD3-positive cells of the pre-ovulatory inseminated sows were higher in the infundibulum than in the other segments (p < or = 0.001). In the subepithelial connective tissue of the pre-ovulatory inseminated sows, significantly higher numbers of lymphocytes (p < or = 0.001) and plasma cells (p < or = 0.001) were found in infundibulum than in isthmus. Neutrophils were found mainly in infundibulum, the number approximately 40 h after pre-ovulatory insemination was significantly higher (p < or = 0.05) than in the other groups and segments. Significantly higher numbers of CD2 than CD3-positive cells were found for all groups and segments. In the subepithelial connective tissue of post-ovulatory inseminated sows, the numbers of lymphocytes was higher (p < or = 0.001) at day 19 than up to 50 h after insemination and lower (p < or = 0.001) in isthmus than in ampulla and infundibulum. Neutrophils were found in infundibulum in almost all groups and the number was significantly higher (p < or = 0.05) in the infundibulum up to 50 h after insemination than in other segments. In the oviductal epithelium, no influence of insemination was found on the presence of phagocytes, i.e. neutrophils and macrophages, but on lymphocytes. In the infundibular connective tissue, pre-ovulatory insemination had an effect on neutrophil distribution, indicating an active immune response to insemination in the upper segment. Post-ovulatory insemination changed the oviductal immune cell pattern.

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.

Introduction: In spite of increasing number of immune cells in the endometrium during the decidualization, the integrity of endometrial tissue in each menstrual cycle is maintained by adaptive changes in expression of several factors that regulate activity of immune cells and concomitant hormonal alterations during the menstrual cycle. This regulatory function of endometrium is also related to resistance to apoptosis, in which metallothionein (MT) may play a role., Materials and Methods: Study group included 26 women with spontaneous abortion and 18 patients with tubal ectopic pregnancy. Control group included 17 women whose endometrial tissue samples were taken during the normal secretory cycle phase. Expression of metallothionein (MT), CD56 and CD69 were assessed in tissue samples by immunohistochemistry., Results: The number of CD56-positive cells was significantly higher in women with ruptured than unruptured ectopics. MT expression was higher in tubal mucosa distant from the implantation site in ruptured compared to unruptured ectopics. It was found also to be significantly lower than in decidua taken from women with spontaneous abortion. CD69 expression was similar in women with spontaneous abortion as well as patients with ruptured ectopics compared to the control group. On the other hand, CD69 expression in unruptured ectopics was significantly lower than in women with spontaneous abortion and the control group., Conclusion: The concentration of immune cells and increase of their activity in tubal mucosa, with insufficient protection against immune-mediated apoptosis assessed by MT expression, might result in tubal rupture during ectopic pregnancy.

Chlamydia trachomatis is the most common bacterial sexually transmitted disease in the United States and a major cause of female infertility due to infection-induced Fallopian tube scarring. Epithelial cells are likely central to host defense and pathophysiology as they are the principal cell type productively infected by C. trachomatis. We generated cloned murine oviduct epithelial cell lines without viral or chemical transformation to investigate the role of the TLRs and cytosolic nucleotide binding site/leucine-rich repeat proteins Nod1 and Nod2 in epithelial responses to Chlamydia muridarum infection. RT-PCR assays detected mRNA for TLR2 (TLRs 1 and 6), TLR3, and TLR5. No mRNA was detected for TLRs 4, 7, 8, and 9. Messenger RNAs for Nod1 and Nod2 were present in the epithelial cell lines. Oviduct epithelial cell lines infected with C. muridarum or exposed to the TLR2 agonist peptidoglycan secreted representative acute phase cytokines IL-6 and GM-CSF in a MyD88-dependent fashion. Infected epithelial cell lines secreted the immunomodulatory cytokine IFN-beta, even though C. muridarum does not have a clear pathogen-associated molecular pattern (PAMP) for triggering IFN-beta transcription. The oviduct epithelial lines did not secrete IFN-beta in response to the TLR2 agonist peptidoglycan or to the TLR3 agonist poly(I:C). Our data identify TLR2 as the principal TLR responsible for secretion of acute phase cytokines by C. muridarum-infected oviduct epithelial cell lines. The pattern recognition molecule responsible for infection-induced IFN-beta secretion by oviduct epithelial cells remains to be determined.

The aim of this study was to investigate the morphological changes of the sow endosalpinx and the distribution of leukocytes throughout the oestrous cycle and at anoestrus. Nineteen crossbred sows (Swedish Landrace x Swedish Yorkshire) at late dioestrus (three), prooestrus (three), oestrus (three), early dioestrus (three), dioestrus (three) and anoestrus (four) were used. Oviductal samples from three different parts (isthmus, ampulla and infundibulum), taken immediately after slaughter, were fixed, embedded in plastic resin and stained with toluidine blue or stored in a freezer at -70 degrees C until analysed by immunohistochemistry (prooestrus and anoestrus) with an avidin-biotin peroxidase method. Quantitative and qualitative examinations of oviductal epithelium and subepithelial connective tissue were performed by light microscopy. During all stages, a lower degree of morphological changes (pseudostratification, mitosis and secretory granules) was found in the isthmus compared with ampulla and infundibulum. In ampulla and infundibulum, pseudostratification, mitotic activity and secretory granules of the epithelium were high at prooestrus/oestrus. Cytoplasmic protrusions of epithelial cells with some extruded nuclei were prominent in ampulla and infundibulum at all stages except for oestrus and early dioestrus. Lymphocytes as well as CD2- and CD3-positive cells were the predominant immune cells in the epithelial layer. The numbers of lymphocytes and CD3-positive cells did not differ among segments and stages. Numbers of CD2-positive cells did not differ between prooestrus and anoestrus while the numbers were significantly higher in the infundibulum than in ampulla and isthmus. Neutrophils were only occasionally found and mainly in the infundibulum. In the subepithelial connective tissue layer, the two most commonly observed immune cell types were lymphocytes and plasma cells. The numbers of lymphocytes as well as CD2- and CD3-positive cells was lower in isthmus than in the other segments (p < or = 0.001). Higher numbers of plasma cells (p < or = 0.001) were found in infundibulum than in ampulla and isthmus. The numbers of lymphocytes and plasma cells were not significantly different between stages of the oestrous cycle. However, the number of neutrophils differed and were highest at prooestrus in ampulla and infundibulum. The numbers of CD2-, CD3- and CD79-positive cells did not differ between prooestrus and anoestrus whereas for CD14- and SWC3-positive cells, the numbers were higher at prooestrus (p < or = 0.05) than at anoestrus. In the oviduct, the morphology differed in ampulla and infundibulum with oestrous cycle stages, which indicates an effect by ovarian steroid hormones. The immune cell infiltration was less influenced by cyclic changes. However, the immune cell infiltration (in the connective tissue) in the upper part, especially infundibulum, differed significantly from the one in the lower part, isthmus, indicating different immune functions within various parts of the oviduct.

Murine vaginal infection with the obligate intracellular bacterium Chlamydia muridarum is commonly used as a model for ascending Chlamydia infections of the human female genital tract. Gamma interferon-producing Th1 cells, in concert with other mononuclear infiltrates, primarily mediate antichlamydial immunity. However, many factors modify this response, including the bacterial load. To investigate the manner in which the inoculating dose of C. muridarum modulates a genital infection, we measured innate and adaptive cell numbers, CD4+ lymphocyte cytokine profile, chemokine expression, course of infection, and pathological sequelae in genital tracts of BALB/c mice infected with doses of C. muridarum ranging from 10(4) to 10(7) inclusion-forming units. We found that the influx of both innate and adaptive immune cells responded similarly in the lower genital tract (cervical-vaginal tissues) and upper genital tract (oviduct tissues) to increasing inoculating doses. However, cells expressing the innate markers Gr-1 and CD11c were affected to a greater degree by increasing dose than lymphocytes of the adaptive immune response (Th1, CD4+, CD8+, CD19+), resulting in a change in the balance of innate and adaptive cell numbers to favor innate cells at higher infecting doses. Surprisingly, we detected greater numbers of viable chlamydiae in the oviducts at lower inoculating doses, and the number of organisms appeared to directly correlate with hydrosalpinx formation after both primary infection and repeat infection. Taken together, these data suggest that innate immune cells contribute to control of ascending infection.

Human genital infection caused by Chlamydia trachomatis is thought to be immunologically mediated, resulting in local recruitment of lymphocyte subsets and inducing the production of cytokines. Little information is available about the role of lymphocyte recruitment and the regulation of cytokine production in the genital tract of C. trachomatis positive infertile women. We have evaluated the recruitment of lymphocyte subsets in the genital tract and production of Th1/Th2 cytokines in cervical secretions and laparoscopic specimens from the fallopian tubes of C. trachomatis positive infertile women (n = 17) and compared them with controls, viz. C. trachomatis negative infertile women (n = 20) using ELISA and flow cytometry. None of these patients were found to be infected either with Candida sps., bacterial vaginosis, Trichomonas vaginalis, Neisseria gonorrhoeae, Mycoplasma hominis or Ureaplasma urealyticum in the cervix. Flow cytometric analysis of cervical secretions in Chlamydia positive women revealed recruitment of both CD4 and CD8 lymphocytes to the genital tract was up-regulated and a variation in the production rates of different cytokines in cervical secretions and fallopian tube was observed. We found that the immune responses in cervical secretions were of Th0 type, since all the analysed cytokines, viz. IFN-gamma, TNF-alpha, IL-10 and IL-12 were up-regulated. As, both CD4 and CD8 cells contribute to the production of IFN-gamma and IL-10, these results suggest that along with CD4 cells, CD8 lymphocytes also may be important for local regulation of Th1/Th2 responses in the genital tract during C. trachomatis infection.

The role of immune disturbances on the systemic and local levels is evaluated and the immunological mechanisms of tuboperitoneal sterility as the basis for the development of the methods of immunocorrection is grounded.

Cytokines are believed to play a critical role as mediators between the oviduct and the developing embryo. A synchronous development of embryo and endometrium is essential to successful implantation. It seems to be beneficial for embryo development to rest for some time in the Fallopian tube. Expression of cytokines in the human Fallopian tube and the effect of mifepristone were investigated. Fourteen women with regular menstrual cycles and proven fertility, admitted to the hospital for tubal ligation, were randomly allocated to control or treatment groups. Mifepristone 200 mg was given on day LH+2. Surgery was performed on day LH+3 to LH+5. Biopsies were obtained from the ampullar and isthmic regions of the tubes. Expression of interleukin 8 (IL-8), tumour necrosis factor alpha (TNFalpha), transforming growth factor beta (TGFbeta) and leukaemia inhibitory factor (LIF) was analysed using immunohistochemistry. All cytokines except IL-8 showed the same staining intensity both in the ampullar and isthmic region, while IL-8 was more pronounced in the ampullar region in both epithelial and stromal cells. Exposure to mifepristone made the spatial difference in IL-8 disappear and increased the expression of TNFalpha in the epithelium of the isthmus, but had no effect on the expression of TGFbeta1 or LIF. Changes in cytokine expression in the Fallopian tube are likely to influence embryo development, which could contribute to the contraceptive effect of mifepristone.

The luminal epithelium of the porcine oviduct is composed of ciliated cells and secretory cells, but it is assumed for several species that under the control of steroid hormones secretory cells are able to be transformed into ciliated cells. In order to better understand such physiological changes during the different stages of the oestrous cycle, we evaluated epithelial cell proliferation together with oestrogen receptor alpha (ERalpha) expression of porcine ampullary oviducts. To identify the immunophenotype of proliferating cells, double immunohistochemistry was performed using anti-chromogranin A antibody (anti-CgA) as the second primary antibody. Anti-CgA, recently shown to be an immunocytochemical marker of ciliated cells of the cow, also labelled specifically the luminal surface of ciliated cells of the pig. Double labelling of sections with the monoclonal antibody MIB-1 against the proliferation-associated nuclear epitope Ki-67 and anti-CgA clearly demonstrates that MIB-1 was selectively localised in the nuclei of secretory cells. Proliferative activity was not observed in CgA-positive ciliated cells in all examined stages of the oestrous cycle. The percentage of Ki-67-positive epithelial cells was higher at pro-oestrus, compared with the other stages of the oestrous cycle. Furthermore, ERalpha immunoreactivity was exclusively detected in the nuclei of the epithelial cells, which were negative for CgA. We conclude, therefore, that oestrogen may induce the initial proliferation of secretory cells and promote the differentiation into ciliated cells., (Copyright 2004 Springer-Verlag)

The roles of Toll-like receptor (TLR) 2 and TLR4 in the host inflammatory response to infection caused by Chlamydia trachomatis have not been elucidated. We examined production of TNF-alpha and IL-6 in wild-type TLR2 knockout (KO), and TLR4 KO murine peritoneal macrophages infected with the mouse pneumonitis strain of C. trachomatis. Furthermore, we compared the outcomes of genital tract infection in control, TLR2 KO, and TLR4 KO mice. Macrophages lacking TLR2 produced significantly less TNF-alpha and IL6 in response to active infection. In contrast, macrophages from TLR4 KO mice consistently produced higher TNF-alpha and IL-6 responses than those from normal mice on in vitro infection. Infected TLR2-deficient fibroblasts had less mRNA for IL-1, IL-6, and macrophage-inflammatory protein-2, but TLR4-deficient cells had increased mRNA levels for these cytokines compared with controls, suggesting that ligation of TLR4 by whole chlamydiae may down-modulate signaling by other TLRs. In TLR2 KO mice, although the course of genital tract infection was not different from that of controls, significantly lower levels of TNF-alpha and macrophage-inflammatory protein-2 were detected in genital tract secretions during the first week of infection, and there was a significant reduction in oviduct and mesosalpinx pathology at late time points. TLR4 KO mice responded to in vivo infection similarly to wild-type controls and developed similar pathology. TLR2 is an important mediator in the innate immune response to C. trachomatis infection and appears to play a role in both early production of inflammatory mediators and development of chronic inflammatory pathology.

Infection of the Fallopian tubes (FT) by Neisseria gonorrhoeae can lead to acute salpingitis, an inflammatory condition, which is a major cause of infertility. Challenge of explants of human FT with gonococci induced mRNA expression and protein secretion for the proinflammatory cytokines interleukin (IL)-1alpha, IL-1beta, and tumor necrosis factor alpha (TNF-alpha) but not for granulocyte-macrophage colony-stimulating factor. In contrast, FT expression of IL-6 and of the cytokine receptors IL-6R, TNF receptor I (TNF-RI), and TNF-RII was constitutive and was not increased by gonococcal challenge. These studies suggest that several proinflammatory cytokines are likely to contribute to the cell and tissue damage observed in gonococcal salpingitis.

Unlabelled: The aim of the study was to determine wether Gram-negative bacterial cell membrane lipopolysaccharides (endotoxine) can change the IL-6 and TNF-alpha cytokine concentration synthetized by fallopian tube endothelial cells. For the study 5 normal fallopian tubes from females at their reproductive age who underwent total hysterectomy due to uterine myoma were used. The fallopian tubes specimens (endothelial tissue) 2 mm2 fixed in 0.5 ml DMEM/HAM F-12 (GibcoBRL) solution with 15% FCS, Gentamycin, Fungizone, ITS (GibcoBRL) were incubated at 37 degrees C with 5% CO2. The explants were stimulated by Escherichia coli lipopolysaccharide (LPS) at ascending concentrations 1 ng/mL, 10 ng/mL and 100 ng LPS/mL incubation media. Tissue specimen incubated in a media without LPS were used for control test. IL-6 activity in the supernatants were determined by Van Sinc method, TNF-alpha activity were determined against WEHI-164.13. cells according to Espevik and Nisser-Mayer. The presence of TNF-alpha and IL-6 were confirmed in all the supernatants of the incubated fallopian tubes explants. The LPS stimulation caused a concentration increase in both cytokines. The maximum cytokine concentration level was observed in the incubation stimulated at 1 and 10 ng LPS/mL incubation media. The use of the highest LPS concentration retarded the cytokine production., Conclusion: The TNF-alpha and IL-6 cytokines dose-dependent production is caused by the fallopian tubes LPS stimulation.

The development of a protective vaccine against the sexually transmitted disease caused by Chlamydia trachomatis may prevent complications associated with insidious infection. Vaccination via the vaginal route may not be practical, and other routes should be investigated. To this end, the adhesion molecules induced on the fallopian tube endothelium during infection with C. trachomatis were characterized. Adhesion molecules were identified in fallopian tube biopsy specimens cultured with 5 x 10(6) infection-forming units of C. trachomatis serovar E. Frozen sections were prepared from these tissues and were stained by immunohistochemical techniques. Infection with live, but not UV-inactivated, C. trachomatis induced a significant increase in levels of vascular cell adhesion molecule-1 and the mucosal addressin cell adhesion molecule-1 but not of other adhesion molecules. Therefore, infection with C. trachomatis induces adhesion molecules that are associated with other mucosal tissues and inflammatory sites, which suggests that mucosal routes of immunization may be effective.

Objective: To assess the expression of integrin alpha v and beta 3 in the serosal tissue of intraperitoneal organs and adhesions in persons with and without adhesions., Design: Prospective study., Setting: Academic research centers., Patient(s): Fifty-seven patients undergoing abdominal or pelvic surgery., Main Outcome Measure(s): Integrin alpha v and beta 3 messenger RNA (mRNA) expression was evaluated by quantitative reverse transcription polymerase chain reaction., Result(s): The serosal tissue of the parietal peritoneum, uterus, fallopian tubes, ovary, and the large and small bowel, as well as peritoneal adhesions, skin, fascia, subcutaneous tissue, and omentum, expresses integrin alpha v and beta 3 mRNA. The level of alpha v and beta 3 mRNA expression varied among these tissues; expression of the former substance was highest in uterine serosa and lowest in the skin and small bowel, and expression of the latter substance was highest in the fallopian tubes and skin and lowest in the uterine serosa. Parietal peritoneum and adhesions express equal levels of integrin alpha v; however, integrin beta 3 expression was >100-fold lower in adhesions than in peritoneum. The level of integrin beta 3 expression in omentum, small and large bowels, and subcutaneous tissue was 100-fold to 10,000-fold lower than in other tissues., Conclusion(s): Serosal tissue of peritoneal organs and adhesions express variable levels of integrin alpha v and beta 3 mRNA. On the basis of such variation and the knowledge that tissue injury alters local integrin expression, integrins may play a key role in adhesion development, particularly in tissue with higher integrin expression.

Embryo-maternal dialogue starts shortly after fertilization and is exerted through both local and systemic signaling. We have discovered specific embryo derived pre-implantation factors (PIF), novel peptides that are secreted already at the two cell stage and which modulate cellular immunity. In the fallopian tube the embryo, a partial allograft, is tolerated by the mother. Embryo derived peripheral signaling (PIF) is also detected prior to implantation in maternal sera. This signal may also help prime the endometrium to facilitate implantation. Upon implantation, embryo-endometrial communication becomes direct and highly amplified. When the immune privilege appears to be secured, embryogenesis proper initiates. This requires proliferation/differentiation to be tightly controlled. Knowledge of proliferation promoters is ample while the factors involved in its control remain less understood. We have identified a class of novel proteins/peptides, developmental proteins (DPs), that are present in the embryo before a mature immune system has developed. Their role is to create a balance between pro and antiproliferative forces, to promote normal proliferation while controlling abnormal cell proliferation (i.e. due to carcinogens, toxins, viruses, and ionic radiation). DPs, may also redirect growth towards functionality through differentiation. DPs appear to act through a specific receptor negating growth factors action through promotion of tumor suppressors and inhibition of tumor promoters at 2 minutes, blocking DNA synthesis at ~24 hours, and promoting apoptosis at ~48 hours. When an embryo becomes incompatible with life, DPs may lead to growth arrest, PIF-like compounds decline, the immune system to be restored and the pregnancy is rejected. Final identification and use of PIF and DPs is likely to help both managing early pregnancy disorders and aid in treatment of proliferative disorders due to cancer and viral infection.

The nonclassical HLA-G class I gene is expressed by extravillous cytotrophoblast that invades decidua in uterine pregnancy, suggesting that it may contribute to the immunological mechanisms that protect the fetus against maternal alloimmune response and/or pathogen infections. We first addressed the question of whether HLA-G expression was dependent on maternal tissue environment by comparing uterine and ectopic tubal pregnancies. Using HLA-G-specific mAb on placental cryosections, we found by immunohistochemistry that all subtypes of extravillous cytotrophoblast similarly expressed HLA-G in pregnant tubes, demonstrating that its expression was independent of the site of implantation. We next compared by immunohistochemistry the phenotype of maternal leukocytes recruited in both pregnant tissues. In contrast to decidua, pregnant tubes were characterized firstly, by the lack of natural killer (NK) cells and of cells expressing CD94 receptor specific for HLA-E, secondly, by a prominent increase of CD8+ T cells, dendritic cells, and macrophages, the latter co-expressing the LIR1/ILT2 killer immunoglobulin-like receptor (KIR), and finally, by the presence of cells expressing LIR2/ILT4 KIR or BY55 NK receptors, known to bind to HLA-G. Such cell types may favor a unique innate defense in pregnant tubes. These observations also suggest that trophoblast HLA-G expression does not influence the recruitment of particular maternal leukocytes in pregnant tissues.

Host defense mechanisms against vaginal Candida albicans infections are poorly understood. Despite the protective role of T helper (Th)1-type cell-mediated immunity (CMI) against mucosal C. albicans infections, studies using an estrogen-dependent murine model of vaginal candidiasis have shown a lack of effect of systemic Th1-type CMI against a vaginal C. albicans infection, and a lack of changes in local T cells during infection. In the present study, the local Thl- (interleukin [IL]-2, interferon [IFN]-gamma and IL-12) and Th2- (IL-4, IL-10 and transforming growth factor [TGF]-beta1) type cytokines were evaluated in vaginal tissue during an experimental C. albicans infection. Results showed constitutive expression of TGF-beta1 in vaginal tissue of naive mice that was two-fold higher than the levels of the other cytokines examined. These high levels of TGF-beta1 were further increased as a result of pseudoestrus and/or infection, and were corroborated at the messenger RNA level. Furthermore, the levels of TGF-beta in naive or infected mice were significantly higher in the vagina compared to other areas of the genital tract. Finally, TGF-beta1 predominated as well in the draining, but not non-draining, lymph nodes during infection. These results suggest that TGF-beta1, a potent immunoregulatory cytokine, may be important in the lack of demonstrable CMI at the vaginal mucosa against C. albicans.

The molecular backgrounds of the anti-phospholipid syndrome and immunisation against the Rhesus proteolipid antigens are still largely unknown. In the present study, expression of (1) CD1, a major histocompatibility complex class l-like lipid antigen presenting molecule, (2) IL-10, a cytokine promoting induction of clonal anergy, and (3) CD80 and CD86, two T-cell costimulators preventing induction of clonal anergy when present, was investigated in frozen sections of cervix, corpus and the fallopian tube (FT) of 25-day-old BALB/c mice injected with FSH, progesterone or medium and of pregnant mice from each trimester (days 7, 14 and 19). CD1 was expressed by all endometrial epithelial cells. Enhanced immunostaining of the endometrial epithelial cells was observed after FSH treatment, and cervix and FT expressed generally more than the corpus of the uterus. After treatment with medroxy progesterone acetate (MPA), expression of CD1 by the endometrial epithelia was weak. During pregnancy CD1 was absent from the endometrium adjacent to the foetus, but was unaltered in the cervix and FT. IL-10 was expressed by the endometrial glands and also by the endometrial surface epithelium. MPA treatment increased the intensity of the IL-10 immunofluorescence. There were also chains of positive cells between the muscle bundles within the pregnant myometrium. CD80 and CD86 were usually absent from the female reproductive tract, but were occasionally found in the cervix during pregnancy. The present study demonstrates definite differences in the expression of both CD1 and IL-10 between the FSH and MPA treated mice, suggesting differences during the oestrous cycle. As IL-10 is expressed more in the secretory phase, it is probably involved in making the endometrium more acceptable for implantation by inducing clonal anergy. This is supported by the absence of CD80 and CD86. These results also suggest that abnormal expression of CD1d during pregnancy may predispose the mother for immunisation against lipid antigens such as membrane phospholipids and Rhesus-antigens.

The epithelium of the uterine tube consists of ciliated cells and secretory cells. Basal cells are a third cell type observed in tubal epithelium and they are located principally in the basal part of the epithelium. The objectives of this study were to characterize these basal cells in normal and superovulated heifers and to determine whether they participate in the replacement of the ciliated and secretory cell populations. All heifers received cloprostenol (PG) to induce oestrus (day 0). Superovulated heifers received 24 mg pFSH at doses of 4.5, 3.5, 2.5 and 1.5 mg given twice daily. Control and superovulated heifers were slaughtered on days 1, 3, 5 and 7 of the oestrous cycle. Another group of normal cycling heifers was slaughtered on days 2-3 and 11-13 of the oestrous cycle and used for immunocytochemistry. Samples from ampulla, pre-isthmus and isthmus of the uterine tube were collected and processed for light and transmission electron microscopy. Quantitative examination by light microscopy showed that there was a significant difference in the number of basal cells between the regions of the heifers' uterine tube. On the basis of ultrastructure two populations of basal cells were observed. One (type I) had a nucleus with much condensed heterochromatin and very sparse cytoplasmic organelles. The second cell (type II) had a nucleus with heterochromatin typically clumped around the nuclear envelope. Its cytoplasm contained many organelles including a number of lysosomes. The ultrastructural features of these cells were similar in all regions and at all days of the oestrous cycle examined. Immunocytochemistry revealed that type I basal cells were lymphocytes and type II basal cells were macrophages., (Copyright 2000 S. Karger AG, Basel)

A primate model of chlamydial pelvic inflammatory disease was used to characterize serum antibody responses to the 60 kDa chlamydial heat shock protein (CHSP60). Forty monkeys were infected in the fallopian tubes with Chlamydia trachomatis and then were treated. Twenty-three (58%) monkeys developed antibodies against CHSP60, of whom 6 (15%) had CHSP60 responses that persisted throughout the study and 17 (42.5%) had a transient response. A persistent CHSP60 antibody response was correlated with being culture- or ligase chain reaction-positive in the fallopian tubes (P=.004), but not in the cervix pretreatment, and with being tubal-positive posttreatment (P=. 02). Compared with tubal-negative monkeys, tubal-positive monkeys had more intense CHSP60 responses (P=.006) that lasted longer (P=. 002). Among CHSP60 responders, an OD>0.5 was correlated with more severe salpingeal pathology before treatment (P=.04). CHSP60 antibody response may be useful as a marker of persistent chlamydial infection in the fallopian tubes.

Fertile female Wistar rats were immunised against rat and mouse seminal vesicle secretion (SVS) to test the production of allo-antibodies and the effect of the antibodies elicited on fertility. Twenty-six per cent of the rat and mouse SVS-immunised females were infertile after the treatment. The sera were titrated by ELISA and used in Western blots to detect the proteins recognised. Although neither the antibody titres nor the proteins recognised by the sera showed a close relation with the degree of fertility, in all females the highest antibody titre in the fluids from the genital tract was found in the oviductal fluid and during the night of oestrus. This fact suggested that the site of action of the antibody could be the oviduct. Similar results were obtained using mouse SVS as immunogen--a fact that can be related to the antigenic similarity between the SVS of the two species. The antibodies react with the spermatozoa but not with eggs or embryos. Analyses performed on embryos collected from sterile females showed that there was a delay in fertilisation and normal embryogenesis. Our results suggest that SVS proteins are antigenic and that these antigens are bound to the spermatozoa and could take part in early pre-fertilisation events such as capacitation or sperm transport.

The role of the human fallopian tube in reproduction is more than a passive conduit. This organ is involved in oocyte pick-up, fertilization, and embryo transport. Metabolic substrates, cytokines, and immunoglobulins have been identified within the tubal lumen. Complex and coordinated endocrine secretions, neuromuscular activity, and cilial action are required for successful tubal function.

Endometriosis is characterised by the presence in abnormal situation of a tissue resembling the endometrium with glands and stroma. Several assumptions try to explain the development of this tissue. The oldest, the theory of the metaplasia, suggests that under various influences the coelomic tissues would be transformed into endometrium. Most often agreed, the theory known as the implantation theory, explains why the endometrial cells and fragment desquamated during menstrual period transported through the fallopian tubes into the peritoneal cavity (physiological phenomenon) could under certain conditions implant, proliferate and develop overcoming the local mechanisms of defence. The peritoneal cavity of the normal women have the capacity to prevent the evolution towards endometriosis. The reasons of an evolution towards the endometriosis and its symptoms (pain, sterility, adherences are probably numerous implying the immune system, the endometrium, the macrophages, the cells natural killer, the peritoneum the fallopian tubes. The failure to removes the peritoneal cavity of the fragments would induce an inflammatory local state with hyperactivation of the macrophages which secrete many molecules, of which some could lead the metaplasia of the peritoneum or the development of müllerian residues.

Unlabelled: The aim of this study was to investigate the presence of a local immunological defense mechanism of the secretory IgA class in the female genital tract., Material and Methods: We studied by a streptavidin-biotin method the secretory component (SC) and IgA distribution in paraffin-embedded sections of 90 formalin-fixed specimens. We studied 10 normal and 5 neoplastic cervical specimens, 20 normal, 10 hyperplastic endometrial specimens and 10 endometrial adenocarcinomas, 5 normal ovarian tubes and 30 ovarian epithelial neoplasms, serous and mucinous. A polyclonal SC and (Dako) and a mab IgA (Dako) was used and the reaction was scored from 1-3., Results: Normal cervical mucosa and atrophic endometria were negative, while the basal portion of the endometrium, focally the proliferative glands, most of the secretory glands and most of the hyperplastic glands were positive for SC. IgA showed a similar distribution and a perivascular stromal reaction. Adenocarcinomas were positive for SC, but the intensity of the reaction was dependent on the differentiation of the tumors. Mucous and most serous neoplasms were negative for SC. IgA showed a similar reaction., Conclusion: There is evidence that the female genital tract has a local defensive immune system that may be hormone dependent. SC is a valuable marker of glandular differentiation.

The co-expression of alpha1beta1, alpha4beta1 and alphavbeta3 integrins in the human endometrium coincides with the implantation window. The alphavbeta3 integrin is expressed in the apical surface of the luminal epithelium and may serve to anchor trophoblast cells in the adhesion phase of implantation. Using immunohistochemistry, we compared the expression of alphav, alpha1, alpha4 and beta3 integrin subunits in samples of normal human Fallopian tube and endometrium obtained from five women in the non-receptive period (luteal phase days 2-4) and from another five women in the receptive period (luteal phase days 6-8). The staining was quantified visually on a scale of 0 to ++, according to the intensity and density of stained cells. The alphav subunit is expressed in the Fallopian tube epithelium during both periods in a pericellular distribution. The beta3 subunit is also expressed in the same location, but it is up-regulated during the period of endometrial receptivity. The other subunits are expressed in localizations which are not relevant to trophoblast adhesion and exhibit little or no difference in the level of expression between the non-receptive and receptive periods. Based on these results we postulate that the expression of the beta3 subunit in the human tubal epithelium is under the same systemic controlling signals as in the endometrium and that the normal tubal epithelium may have an implantation window, at about the same time as the endometrium, that affords the opportunity for trophoblast attachment should a 5-7 day embryo be unduly retained in the tube.

The epithelium is the first barrier against pathogens invading the lumen of the human oviduct. Its expression of class II major histocompatibility complex (MHC class II) proteins suggests that it might play a role in antigen presentation during the local immune response. To study the role of the oviductal epithelium in antigen processing, its endocytic properties and MHC class II expression were examined. For assay of endocytosis, fluorescein isothiocyanate-labelled bovine serum albumin (BSA-FITC) or Escherichia coli (E. coli-FITC) was infused into the lumen. One-centimetre pieces of oviduct were incubated for 2 h and processed for fluorescence and confocal microscopy, and transmission electron microscopy. Incorporation into secretory and ciliated epithelial cells was observed, which was unrelated to the phase of the menstrual cycle. Small pieces of the organs were frozen and processed for immunohistochemistry. Most oviducts expressed MHC class II (HLA.DR) in the epithelium and in some cases this was coincident with endocytosis, but there was no statistically significant association between this expression and either endocytotic activity or the phase of the menstrual cycle. Results demonstrate that the epithelium of the human oviduct exhibits endocytic properties towards luminal soluble and particle antigens, which is not related either to MHC class II expression or to the phase of the menstrual cycle.

Objective: To compare and quantify leukocyte populations at the tubal implantation site, in the tubal mucosa away from the implantation site and in the intrauterine decidua in women who have had an ectopic tubal pregnancy with leukocyte populations in the decidualized endometrium in women who have had a normal intrauterine pregnancy., Design: Retrospective immunohistochemical study., Setting: Department of Pathology, University of Newcastle, Royal Victoria Infirmary, Newcastle upon Tyne, Tyne and Wear, United Kingdom., Patient(s): Paraffin-embedded tissue blocks from 30 women who had had an ectopic pregnancy (EP) and control tissue blocks from 19 women who had undergone elective first trimester pregnancy termination., Intervention(s): None., Main Outcome Measure(s): Quantitative analysis of defined leukocyte subpopulations in the fallopian tube (at the implantation site and in the tubal mucosa away from the implantation site) and intrauterine decidua., Result(s): Endometrial granulated lymphocytes were absent from both the tubal mucosa away from the implantation site and the tubal implantation site. T lymphocytes and macrophages formed the predominant leukocyte subpopulations in both tubal areas. In the intrauterine decidua from women who had had an EP, endometrial granulated lymphocytes were the most abundant leukocyte population and macrophages and T cells formed the second and third major leukocyte subpopulations, respectively. No differences were detected in the number or proportion of these decidual leukocytes in women who had had a normal pregnancy vs. in those who had had an EP., Conclusion(s): The absence of endometrial granulated lymphocytes from the tubal implantation site suggests that the local presence of the cells is not essential for implantation and early placental development events.

The oviduct of most mammalian species is the site where spermatozoa first encounter the oocyte and the process of fertilization is initiated. It should therefore be considered an important focus of study for the development of an effective gamete immunocontraceptive with the goal of immunologically interfering with spermatozoon function. The study reported here used the cynomolgus macaque as a nonhuman primate model in which to analyze the serum and oviductal fluid immune response to immunization with the human sperm protein Sp17 and synthetic peptides derived from Sp17. Human and macaque Sp17 were shown to share a very high degree of identity (96.7%), confirming this species as a suitable model in which to study the antibody response and recognition of spermatozoa. The oviductal fluid antibody titer varied with respect to serum titer both over time for individual monkeys and between different monkeys. In all cases, however, the antibody response was restricted solely to the immunoglobulin G class. Finally, both serum and oviductal fluid antibodies were directed against identical Sp17 B-cell epitopes and both recognized native macaque Sp17 in spermatozoa.

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.

The oviduct is a key component of the reproductive system where essential states such as spermatozoa capacitation, fertilization and early embryo development take place. Recently, an additional role for the entire female reproductive tract has emerged with important implications for our understanding and management of reproductive health, namely, its role in mounting local immune responses against microbial pathogens. Most of the evidence about mucosal immune responses in the female reproductive tract is related to the vagina, with less information available for the uterus. The less known segment in this regard is the oviduct, which prompted us to review and summarize the current state of knowledge on the immune system at the level of the mammalian oviduct. A comprehensive search was conducted in Medline and--for the last two years--also in Current Contents. Result demonstrate that the mammalian oviduct is endowed with many of the elements that characterize a mucosal immune system. However, there are gaps in our knowledge that, in addition to important interspecies differences, make it essential to explore further some fundamental questions regarding this system.

Assessment of immune responses in the oviduct is of importance in understanding reproductive tract responses to infections, vaccination against reproductive tract pathogens, or contraceptive immunogens. This review discusses a technique that permits repeated sampling of oviductal fluid from the same monkey at intervals spanning up to several years, and the analysis of antigen-specific immunoglobulins in the fluid. This technique is important to immunocontraceptive development because previous studies in primates have lacked information on oviductal immune responses and contraceptive efficacy may not correlate well with serum antibody titers. Thus, a reliable method of sampling oviductal fluid before and after immunization with a defined antigen is required to determine the quantity and type of local immune responses necessary to achieve contraceptive effects. Implantation of access ports proved useful for repeatedly aspirating oviductal fluid in vivo from cynomolgus monkeys that was free from artifactual contaminants and with no observable changes in the behavior or health of the animals. Subsequent assays of relative and absolute concentrations of antibodies in oviductal fluid and serum demonstrated the presence of IgA and IgG specific for the recombinant sperm immunogen SP-10 in fluid collected from the periovulatory oviduct of primates after intramuscular inoculations. The antibodies evoked by the recombinant sperm vaccinogen recognized the endogenous antigen target on both human and macaque sperm, lending support for the possibility of developing a contraceptive immunogen that prevents fertilization.

Macaque oviductal fluids were assayed for specific antibodies to the intra-acrosomal sperm protein SP-10 after immunizations with recombinant macaque SP-10 (re-mqSP-10), a candidate contraceptive vaccinogen. Access ports, consisting of a subcutaneous collecting reservoir and a catheter to cannulate the oviduct, were implanted into monkeys for repeated aspiration of oviductal fluid. Monkeys were inoculated i.m. once a month with an emulsion consisting of 2 mg re-mqSP-10 in a vehicle of squalene and mannin monooleate. Oviductal fluids and serum were collected during the periovulatory period for six menstrual cycles, and IgG and IgA antigen-specific antibodies in preimmune and immune fluids were compared by ELISA. Both relative and absolute concentrations of SP-10-specific immunoglobulins (Ig) were determined. Oviductal fluids from immunized animals showed significant increases in anti-SP-10 IgG at cycle 2 and at all subsequent intervals. Anti-SP-10 IgA significantly increased in oviductal fluid at cycles 4, 5, and 6. Serum anti-SP-10 IgG increased at cycle 2 and remained significantly elevated through cycle 6, while serum anti-SP-10 IgA was higher than in preimmune samples at cycle 4. Serum antibodies generated to the recombinant SP-10 recognized SP-10 extracted from macaque sperm on Western blots. Immunocytochemical staining of macaque and human sperm showed acrosomal immunofluorescence with both immune oviductal fluids and serum using both anti-IgG and anti-IgA secondary antibodies. This study demonstrates for the first time 1) IgG and IgA antibodies to a defined recombinant sperm-specific antigen in primate oviductal fluids after systemic immunization and 2) the recognition by primate oviductal fluid IgG and IgA of the endogenous contraceptive target on both human and macaque sperm.

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.

The objective of this study was to detect and characterize a secreted oviduct-specific glycoprotein (OGP) in the rhesus macaque (Macaca mulatta) and to compare the characteristics of this OGP to those previously characterized in baboons and women. Oviducts were obtained from untreated ovariectomized rhesus and from ovariectomized rhesus either treated with estradiol (E2) for 14 days or treated sequentially with E2 for 14 days and then with E2 plus progesterone (P4) for an additional 14 days. Segments of oviducts were either fixed for morphological analysis, cultured for OGP synthesis and release, or frozen for RNA analysis. The proteins present in the culture media were separated by one-dimensional SDS-PAGE, and OGP was detected on Western blots using polyclonal antibodies generated against the reduced form of baboon OGP or a 17-amino acid segment of the baboon core protein. Cross-reacting antigens were present in the 120-kDa region, identical to what was observed for baboon and human OGP. Indirect immunogold localization of OGP on thin sections demonstrated specific clustering of gold particles over the apical secretory granules of the secretory cells of the oviductal epithelium. A cDNA was generated using RT-PCR and 5' and 3' rapid amplification of cDNA ends (RACE), and sequenced. The total transcript was 2237 nucleotides in length plus a poly(A) tail. The largest open reading frame was 624 amino acids, which would produce a protein of 69.3 kDa. The nucleotide sequence was more than 95% identical to the nucleotide sequences of baboon and human OGP. Northern blots revealed a single message at 2.4 kilobases (kb) in oviduct samples obtained from E2-treated rhesus. This message was absent in oviducts obtained from untreated ovariectomized and from sequential E2 plus P4-treated rhesus macaques. In summary, the rhesus oviduct synthesizes and secretes an OGP in the presence of E2 that is immunologically and structurally similar to the baboon and human OGP. The presence of a highly homologous glycoprotein in several primates suggests a similar function for OGP in the reproductive process.

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.

Vitamin D3, via its active metabolite 1,25-dihydroxyvitamin D3, plays a critical part in male and female reproduction in the rat. 1,25-Dihydroxyvitamin D3 activity is mediated by an intracellular receptor (VDR). VDR distribution in reproductive tissue has not been studied using antibodies against the receptor. We developed a polyclonal antibody against the VDR and used it to examine VDR distribution in male and female rat reproductive tissues. In rat testes, VDR epitopes were observed in seminiferous tubules, specifically in spermatogonia, Sertoli cells and spermatocytes. Spermatozoa stained faintly. Epithelial cells of the epididymis, seminal vesicles and prostate also expressed VDR epitopes. In the female rat reproductive tract, immunostaining for VDR was seen in ovarian follicles, specifically in granulosa cells. Weaker VDR immunostaining was observed in follicular thecal cells and in the ovarian stroma and germinal epithelium. Corpus luteal cells stained intensely for VDR. Epithelium of fallopian tubes and the uterus also contained VDR epitopes. Both nuclear and cytoplasmic VDR immunostaining was observed in male and female rat reproductive tissues. We conclude that the VDR is widely distributed in male and female reproductive tissues and that it is likely to mediate actions of 1,25-dihydroxyvitamin D3 in the tissues.

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.