Bacterial infections commonly cause bovine endometritis and infertility via innate immune pathways. However, mechanistic studies using isolated cells or chopped tissue may be compromised by the disruption of endometrial architecture and release of damage-associated molecular patterns. So, this study aimed to establish an ex vivo model of intact bovine endometrium to study innate immunity and inflammation.

To analyse the peripheral blood NK cells in women with repeated IVF failure (RIF) and a fertile control group to determine which parameters best differentiate the two populations.

The objective was to investigate placental inflammation in chromosomally normal miscarriages in vivo and in vitro.

Mucosal inflammation caused by infections of the female lower genital tract is considered to be an important cofactor for HIV transmission. We hypothesize that COX-2, a key inflammation-related enzyme, is involved in these responses and is upregulated by microbial ligands and pro-inflammatory cytokines.

Bacterial infections perturb ovarian follicle function, despite the lack of immune cells such as macrophages within healthy ovarian follicles. This study examined whether the granulosa cells that line ovarian follicles could, like macrophages, use Toll-like receptors (TLRs) to detect pathogen-associated molecular patterns (PAMPs) and initiate inflammation.

Soluble triggering receptor expressed on myeloid cells-1 (sTREM-1) is a useful biomarker of infection and inflammation.

To evaluate the effects of the anti-inflammatory and anti-angiogenic roles of LXA4 on endometriosis in mice.

Acupuncture has a positive effect on implantation obstacle, but the mechanism is still not clear, so the aim of the experiment is to explore the possible role that acupuncture plays in implantation.

This study aimed to investigate the regulation of expression, localization and physiological role of the CCL11/CCR3 axis in mouse ovary during the periovulatory period.

a2 isoform of vacuolar ATPase (Atp6v0a2) is important for maintaining the delicate immunological balance required for successful pregnancy. The objective of this investigation is to study the dynamic changes in spleen and blood that appear during spontaneous abortion in mice.

Problem  Animals deficient in Heme oxygenase-1 (HO-1, Hmox1^−/− mice) have impaired pregnancies, characterized by intrauterine fetal death. HO-1 expression has been shown to be essential for pregnancy by dictating placentation and intrauterine fetal development. Its absence leads to intrauterine fetal growth restriction and fetal loss, which is independent of the immune system. Defect in previous steps, e.g., ovulation, may, however, also count for their poor reproductive outcome.

Method of study  Here, we investigated ovulation after hormonal hyperstimulation in Hmox1 wild-type and knockout animals.

Results and Conclusions  We observed that animals lacking HO-1 produced significantly less oocytes after hormonal stimulation than wild type animals and this was mirrored by the number of corpora lutea in the ovary. Furthermore, ovulated oocytes from Hmox1^−/− animals were poorly fertilized compared with those from wild-type animals. In conclusion, we demonstrate here that HO-1 plays a pivotal role in the process of oocyte ovulation as well as fertilization, bringing to light a new and unsuspected role for HO-1.

Problem  The preventative value of progesterone in preterm labor has been recently recognized, especially when it is administered via vaginal suppository. This study was undertaken to evaluate the effect of progesterone on interleukin-6 (IL-6) production in human uterine cervical fibroblasts (UCFs) treated with lipopolysaccharides (LPS).

Method of study  Human uterine cervical tissue was obtained at term, prior to the onset of labor, during the scheduled cesarean section or cesarean hysterectomy. Primary UCF cultures were established and confirmed by immunohistochemistry. IL-6 mRNA and protein expressions were examined by reverse-transcription polymerase chain reaction and enzyme-linked immunosorbent assay, respectively.

Results  Lipopolysaccharides stimulation induced a clear time- and dose-dependent increase in IL-6 mRNA and protein levels in UCFs (P < 0.05). Progesterone treatment significantly attenuated LPS-induced increases in IL-6 mRNA and protein expressions in UCFs (P < 0.05). Estrogen exposure had no effect on LPS-induced IL-6 up-regulation and did not modulate the effects of progesterone.

Conclusion  Our preliminary results suggest that vaginal progesterone might prevent spontaneous preterm labor through a mechanism involving anti-inflammatory effects on UCFs, particularly suppression of IL-6 production.

Problem  Short-chain fatty acids (SCFAs), produced at relatively high levels by anaerobic bacteria in bacterial vaginosis (BV), are believed to be anti-inflammatory. BV, a common alteration in the genital microbiota associated with increased susceptibility to HIV infection, is characterized by increased levels of both pro-inflammatory cytokines and SCFAs. We investigated how SCFAs alone or together with Toll-like receptor (TLR) ligands affected pro-inflammatory cytokine secretion.

Method of study  Cytokines were measured by ELISA. Flow was used for phenotyping and reactive oxygen species (ROS) measurement.

Results  Short-chain fatty acids, at 20 mm, induced interleukin (IL)-8, IL-6, and IL-1β release, while lower levels (0.02–2 mm) did not induce cytokine secretion. Levels >20 mm were toxic to cells. Interestingly, lower levels of SCFAs significantly enhanced TLR2 ligand- and TLR7 ligand-induced production of IL-8 and TNFα in a time- and dose-dependent manner, but had little effect on lipopolysaccharide-induced cytokine release. SCFAs mediated their effects on pro-inflammatory cytokine production at least in part by inducing the generation of ROS.

Conclusion  Our data suggest that SCFAs, especially when combined with specific TLR ligands, contribute to a pro-inflammatory milieu in the lower genital tract and help further our understanding of how BV affects susceptibility to microbial infections.

Problem  The soluble endoglin (sEng) is an antiangiogenic protein that may inhibit TGF-β1 signaling and endothelial nitric oxide synthase activation in endothelial cells. The levels of sEng increased in sera obtained from preeclampsia. The factors that increase the sEng in preeclampsia have not been known well. To investigate the factors that may increase sEng in preeclampsia, we examined the effect of preeclampsia sera on the production of sEng from human choriocarcinoma (JEG-3) cells.

Methods  Serum samples were taken from women with normal pregnancy and from those with preeclampsia. JEG-3 cells were cultured with serum for 24 hrs, and the sEng levels in supernatants and expression of sEng and Hemo oxygenase-1 (HO-1) mRNA in cells were measured.

Results  The addition of preeclampsia sera into JEG-3 cells led to increased release of sEng and expression of Eng mRNA. Preeclampsia sera inhibited the expression of HO-1 mRNA in JEG-3 cells.

Conclusion  The results suggest that preeclampsia sera may increase the protein production of sEng and mRNA expression of Eng from JEG-3 cells like trophoblast without hypoxia and that in addition to hypoxia, preeclampsia sera may play a role of high level of serum sEng in preeclampsia patients. Decreased HO-1 activity may relate to increased sEng release.

Problem  This study investigates dose-dependent effects of melatonin on ovarian graft.

Method of Study  Vitrified-thawed whole ovaries of newborn mice were grafted into ovariectomized mature ones. Melatonin (20, 50, 100, and 200 mg/kg/day) was administrated to separate groups of host mice for 32 days. IgM and IgG antibodies, Th1 and Th2 cytokines, and melatonin in recipient’s blood were measured. Subsequent survival of the grafted ovaries was scored. An assessment of follicular morphology was performed using TUNEL assay and hematoxylin-eosin staining.

Results  The administration of melatonin did not disturb the circadian rhythm of melatonin concentration. The ovarian graft lifespan was prolonged at 200 mg/kg/day melatonin (P < 0.001). However, in doses of higher than 20 mg/kg/day melatonin, the proportion of healthy follicles and ovary size decreased. Th1 cytokines levels were reduced dose dependently. However, the effect of melatonin on Th2 cytokines was not pronounced. IgM and IgG2a decreased in recipients receiving 200 mg/kg/day melatonin in comparison with non-treated group (P < 0.001), while this variables were significantly increased at the dose of 50 mg/kg/day (P < 0.001).

Conclusion  Melatonin at 200 mg/kg/day has an immunosuppresent effect and produce prolongation of graft survival. However, the associated reduction in healthy follicles suggests that melatonin in doses of higher than 20 mg/kg/day has no preventative ischemic action.

Problem  This study investigates whether affectivity differs between mothers delivering preterm and term and whether maternal and umbilical cord serum cytokines differ between these groups. Further, whether there are associations between mothers’ emotions and maternal and cord cytokines at preterm and term birth.

Method of study  Twenty-seven mothers delivering preterm and 37 mothers delivering at term reported positive/negative affect and previous depressive symptoms during pregnancy. Blood samples from mothers in labor and cord samples (23 preterm and 33 term) were analyzed for cytokines.

Results  Maternal IL-8 was lower at preterm delivery compared with term. In the preterm group only, associations were found between negative emotions and maternal IL-6, IL-8 and cord IL-6, IL-8, IL-10, IL-13, and IL-18.

Conclusion  The findings indicate associations in preterm delivery between negative emotions and both maternal and neonate immune activity. Future studies should investigate whether such associations are part of the etiology of preterm delivery.

Problem  Up to 20% of pregnancies end in the first trimester by spontaneous abortion, but a significant number remains unexplained. The aim of this study is to investigate the role of variants within the gene for apolipoprotein E (APOE) in the genetic determination of spontaneous abortions.

Method of study  We collected DNA from 410 tissue samples of spontaneous abortions, and APOE was genotyped by PCR–RFLP method. The frequencies were compared with a population sample of adults (N = 2606) and with a positive control (1060 women with at least two children).

Results  The frequencies of the APOE genotypes in abortions (APOE2E2 + E3E2 = 0.132; APOE3E3 = 0.661; APOE3E4 + E4E4 = 0.195; APOE4E2 = 0.012) did not significantly differ (P = 0.604) from the frequencies in analyzed adult population study (APOE2E2 + E3E2 = 0.132; APOE3E3 = 0.686; APOE3E4 + E4E4 = 0.169; APOE4E2 = 0.014) or from the positive control (APOE2E2 + E3E2 = 0.133; APOE3E3 = 0.691; APOE3E4 + E4E4 =0.166; APOE4E2 = 0.010; P = 0.592).

Conclusion  Our study suggests that APOE may not be associated with spontaneous abortions in Caucasians.

Problem  CD300a is an immunomodulatory molecule of the immunoglobulin receptor superfamily expressed in the leukocytes of myeloid and lymphoid lineages. However, its biological function on CD8+ T lymphocytes remains largely unknown. This study was conducted to assess the biological significance of CD300a expression in T lymphocytes and to determine whether its expression in peripheral T lymphocytes changes in pregnant women presenting with antifetal rejection.

Methods of Study  Microarray analysis was performed using total RNA isolated from peripheral CD300a+ and CD300a− T lymphocytes. Flow cytometric analysis of the peripheral blood samples of pregnant women and pathologic examination of the placentas were conducted.

Results  A large number of genes (N = 1245) were differentially expressed between CD300a− and CD300a+ subsets of CD8+ T lymphocytes, which included CCR7, CD244, CX3CR1, GLNY, GZMB, GZMK, IL15, ITGB1, KLRG1, PRF1, and SLAMF7. Gene ontology analysis of differentially expressed genes demonstrated enrichment of biological processes such as immune response, cell death, and signal transduction. CD300a expression in CD8+ T lymphocytes was coupled to a more cytotoxic molecular signature. Of note, the proportion of CD300a+CD8+ T lymphocytes increased in pregnant women with chronic chorioamnionitis (antifetal rejection of the chorioamniotic membranes; P < 0.05).

Conclusion  The findings of this study strongly suggest an increase in systemic T-lymphocyte-mediated cytotoxicity in pregnant women with chronic chorioamnionitis as a manifestation of maternal antifetal rejection.

Problem  The extent of the vaginal immune response is not fully determined. The purpose of this study was to evaluate the vaginal immune cells from women with vulvovaginitis (VV).

Method of Study  A total of 142 volunteers diagnosed with bacterial vaginosis (BV), vulvovaginal candidiasis (VC), and BV associated with VC or normal microflora were sampled to evaluate the immune cells by flow cytometry. The immune cells were obtained by vaginal lavage and labeled with fluorochrome-conjugated monoclonal antibodies to identify neutrophil granulocytes, macrophages, CD4⁺ and CD8⁺ T lymphocytes, B lymphocytes, and NK lymphocytes.

Results  Neutrophil granulocytes were present in 84.6% of samples among the leukocyte populations. Considering samples in which neutrophils were present, the mean percentage of neutrophil granulocytes was significantly higher in women with VC than BV and normal microflora and was significantly lower in women with BV than normal microflora. Macrophages and lymphocytes were present in a lower percentage of samples. The mean percentage of CD4⁺ T lymphocytes in vaginal lavages was significantly higher in VC and BV compared with women with normal microflora.

Conclusions  Neutrophils were the predominant leukocytes and were associated with VC and inversely with BV. CD4⁺ T lymphocytes were associated with both VC and BV.

Problem  Preeclampsia is a pregnancy-specific disorder that may result from an adverse maternal response to circulating placenta-derived factors, causing a systemic inflammation including endothelial activation. Plasma from preeclamptic patients was shown to induce endothelial activation in the presence of monocytes. We investigated whether microparticles (MP) are the plasma factors causing this activation of endothelial cells.

Method of study  Monocultures and co-cultures of monocytes and endothelial cells were incubated with plasma, MP-poor plasma or isolated MP from non-pregnant and pregnant women and preeclamptic patients (each n = 8). ICAM-1 expression was analyzed with flow cytometry.

Results  The expression of ICAM-1 was significantly increased in monocytes and endothelial cells in co-cultures after the addition of isolated MP from preeclamptic patients (P = 0.017) and to a lesser extent in pregnant women (P = 0.012) compared to non-pregnant controls.

Conclusions  Microparticles from preeclamptic patients activate endothelial cells in the presence of monocytes. Whether all MP have the same effect on monocytes and endothelial cells or only a specific subgroup is the focus of future research.

Problem  Either vascular endothelial growth factor C (VEGFC) or CYR61 plays an important role in placental development and may be involved in pre-eclampsia. Decidual natural killer (dNK) cells are the main source of VEGFC in the maternal–fetal interface. However, it is unclear about CYR61 on the regulation of VEGFC secretion in dNK cells.

Method of study  Decidual natural killer cells were isolated from decidual tissues of first trimester of pregnancy with anti-human CD56–conjugated microbeads. Integrin αvβ3 was detected using immunofluorescent staining. dNK cells were cultured in the presence of CYR61, anti-human αvβ3 integrin antibody (LM609), PI3K inhibitor (LY294002), or MEK inhibitor (U0126). VEGFC mRNA and protein were evaluated by real-time PCR and ELISA, respectively.

Results  Exogenous CYR61 induced the expression of VEGFC in dNK cells in both mRNA and protein levels. Integrin αvβ3 was strongly expressed on dNK cell surface. Anti-αvβ3 integrin antibody inhibited the effect of CYR61 on VEGFC expression. LY294002, but not U0126, significantly reduced this promotion effect of CYR61 on dNK cells.

Conclusions  The upregulation of VEGFC secretion mainly depends on CYR61 binding with integrin αvβ3 on the surface of dNK cells. PI3K/AKT, rather than the ERK/MAPK signal, is involved in the regulation.

Problem  In cattle, retained placenta (RP) is suggested to arise from failure of immune-mediated rejection of the fetal membranes by the maternal immune system and is associated with major histocompatibility (MHC) class I compatibility between calf and dam.

Method of study  To study the association between RP and different MHC class I compatibilities between calf–dam–granddam combinations, massively parallel pyrosequencing was used to determine the MHC class I haplotypes of cows with and without RP.

Results  Two-way calf to dam MHC class I compatibility gave a high risk for RP. There was a tendency for a higher risk for RP with calf to dam MHC class I compatibility.

Conclusions  We concluded that in two-way compatible pregnancies, the maternal immune system fails to reject the fetal membranes, and the fetal immune system does not mount an immune response against maternal MHC class I antigens that could influence the immune-mediated rejection of the fetal membranes by the maternal immune system. The lack of immune-mediated rejection of the fetal membranes by the maternal immune system increases the risk of occurrence of RP.

Problem  Women treated with allogeneic immunization using paternal lymphocytes often request laboratory molecular tests using cell-free fetal DNA in maternal plasma. There is concern whether the treatment can interfere with its results. This study evaluated the applicability of fetal sex determination using fragments of sex-determining region Y (SRY) in the plasma of women submitted to paternal lymphocyte immunization.

Method of study  Non-pregnant women blood samples were collected at two different moments: prior to paternal lymphocyte immunization and after three doses of the immunotherapy, in a prospective study. For women who became pregnant, another sample was collect during the first trimester. Amplification of the fragment of the Y chromosome (SRY) was performed using real-time PCR.

Results  The SRY gene was not identified in any of the plasma samples of the 50 non-pregnant women submitted to paternal lymphocyte immunization at either of the two moments evaluated. For the 26 pregnant women, the results of the identification of sex -determining in maternal plasma were completely in agreement with the infant sex.

Conclusion  Paternal lymphocyte immunization does not affect the results of SRY fragment investigation in the plasma of women submitted to paternal lymphocyte immunization therapy.

Problem  One of the major challenges faced by the clinicians in preterm premature rupture of the membranes (PPROM) is to correctly identify when a significant chorioamnionitis is evolving and decide timely delivery of the fetus. Measuring interleukin-6 levels in maternal serum can be useful for the identification of asymptomatic intrauterine infections in subjects with PPROM.

Method of study  A total of 75 pregnant women, of which 45 pregnant women presenting with PPROM between 24 and 34 weeks gestation and 30 healthy pregnant women without PPROM, were included in the study. Serum IL-6 levels were determined by solid-phase sandwich enzyme-linked immunosorbent assay (Diaclone Research, Besancon, France).

Results  The mean serum IL-6 value at admission in the control group was 2.48 ± 2.7 pg/mL and in the study group was 11.86 ± 14.5 pg/mL (P = 0.001). Mean serum IL-6 concentrations at admission in subjects without histological chorioamnionitis were 3.98 ± 3.9 pg/mL and in those who had histological chorioamnionitis were 20.09 ± 16.8 pg/ml (P < 0.001).

Conclusion  Maternal serum IL-6 levels were significantly elevated in subjects with PPROM with infectious morbidity as compared to those without infectious morbidity in the present study. There was a significant rise in maternal serum IL-6 levels with increased duration of rupture of membranes and with evidence of histological chorioamnionitis and funisitis in the placenta.

Problem  Our study aimed to assess in vitro production of IL-10, IL-6, TNF-A, and adiponectin serum levels in pregnant women with and without gestational diabetes mellitus (GDM) and to investigate a possible association between GDM and IL-10−1082 A>G (rs1800896), IL-6−174 G>C (rs1800795), TNF-A−308 G>A (rs1800629), adiponectin +45 T>G (rs2241766), and adiponectin−11377 C>G (rs266729) gene polymorphisms.

Method of study  This case–control study included 79 women with GDM and 169 healthy controls (C) grouped according to pre-pregnancy BMI. IL-10, IL-6, and TNF-A culture supernatant and adiponectin serum levels were assessed by ELISA. DNA genotype was performed by PCR-RFLP.

Results  Adiponectin levels were significantly higher in C than GDM women, even within the same BMI category. Cytokines levels were similar between the groups. There were no associations between GDM and the analyzed gene polymorphisms.

Conclusions  Women with GDM have significantly lower adiponectin levels in the third trimester, regardless of BMI.

Problem We investigated the beta2-glycoprotein I and anti-annexin V antibodies as anti-phospholipid–cofactor antibodies; and factor V G1691A Leiden, prothrombin G20210A, and methylenetetrahydrofolate reductase (MTHFR) C677T mutations as hereditary thrombophilia in recurrent pregnancy losses (RPL).

Method of study Study group consisted of 84 women with recurrent pregnancy loss and control group consisted of 84 women having at least one live birth.

Results Methylenetetrahydrofolate reductase C677T homozygous mutation was detected in 28.5% of the study group and in 14.2% of the controls, and the difference was highly significant (P < 0.001). Heterozygous mutation of this gene was found in 64.3% of the study population and in 38.1% of the controls, and difference in heterozygous mutation frequency was also significant (P < 0.001). Both homozygous and heterozygous mutations of PT G20210A and factor V G1691A were not different between the groups. There was no significant difference in anti-annexin V levels and anti-beta2-gp 1 levels of the groups.

Conclusion We concluded that both homozygous and heterozygous mutations of MTHFR C677T were related with RPL in Caucasian women.

Problem  Presence of immune infiltrates in the tumor does not always correlate with an anti-tumoral immune response. We previously identified two subpopulations of epithelial ovarian cancer (EOC) cells with differential cytokine profile. We hypothesize that these two subpopulations of EOC cells may differentially regulate the immune phenotype in the tumor microenvironment and therefore affect the immune response.

Method of Study  Macrophages derived from CD14+ monocytes and naive CD4+T cells were treated with conditioned media from two subpopulations of EOC cells. Differentiation markers and phagocytic activity were measured by western blot analysis and flow cytometry. Cytokine levels were quantified using xMAP technology.

Results  Type I EOC cells are able to enhance macrophages’ capacity for tumor repair and renewal by enhancing expression of scavenger receptors and by promoting the secretion of cytokines associated with tissue repair. On the other hand, type II EOC cells are able to create a tolerant microenvironment and prevent an immune response by inducing macrophages’ to secrete IL-10 and by promoting the generation of T regs.

Conclusion  We demonstrate that each ovarian cancer cell subpopulation can induce a unique phenotype of macrophages and T cells, both associated with tumor-supportive function.