Cytokine Patterns in Maternal Serum From First Trimester to Term and Beyond.

Pregnancy implies delicate immunological balance between two individuals, with constant changes and adaptions in response to maternal capacity and fetal demands. We performed cytokine profiling of 1149 longitudinal serum samples from 707 pregnant women to map immunological changes from first trimester to term and beyond. The serum levels of 22 cytokines and C-reactive protein (CRP) followed diverse but characteristic trajectories throughout pregnancy, consistent with staged immunological adaptions. Eotaxin showed a particularly robust decrease throughout pregnancy. A strong surge in cytokine levels developed when pregnancies progressed beyond term and the increase was amplified as labor approached. Maternal obesity, smoking and pregnancies with large fetuses showed sustained increase in distinct cytokines throughout pregnancy. Multiparous women had increased cytokine levels in the first trimester compared to nulliparous women with higher cytokine levels in the third trimester. Fetal sex affected first trimester cytokine levels with increased levels in pregnancies with a female fetus. These findings unravel important immunological dynamics of pregnancy, demonstrate how both maternal and fetal factors influence maternal systemic cytokines, and serve as a comprehensive reference for cytokine profiles in normal pregnancies.

Secretory leukocyte protease inhibitor and progranulin as possible regulators of cervical remodeling in pregnancy.

Secretory leukocyte protease inhibitor (SLPI) and progranulin (PGRN) are secretory proteins with an anti-inflammatory property. Their involvement in cervical remodeling in pregnant uterus is not yet elucidated. Thus, this study aimed to explore the significance of SLPI and PGRN in the maintenance of pregnancy by investigating the factors associated with their expression levels at the cervix. Concentrations of SLPI and PGRN proteins were measured in cervical mucus samples collected from asymptomatic pregnant women at 24-26 weeks of gestation (n = 166). The concentrations of those molecules were analyzed with clinical parameters related to risk for preterm delivery (PD). In pregnant mice, we evaluated the effect of lipopolysaccharide-induced inflammation and progesterone effect modulation on cervical mRNA expression of SLPI and PGRN. The cervical PGRN level was significantly lower in women with short cervix (<35 mm) and with a history of threatened PD. In women with short cervix, cervical SLPI concentrations were positively correlated with inflammatory cytokines, interleukin-6 (R2 = 0.75) and interleukin-8 (R2 = 0.71). In pregnant mice, cervical mRNA expressions of PGRN and SLPI were increased in response to progesterone supplementation and were suppressed by a progesterone antagonist, mifepristone. Lipopolysaccharide-induced inflammation caused remarkable upregulation in cervical SLPI mRNA level but not in PGRN. Progesterone and local inflammation are the factors controlling expression levels of PGRN and SLPI at the cervix. The observed relationship of PGRN and SLPI levels in the cervical mucus with PD-related clinical parameters supports that those anti-inflammatory molecules possibly play a significant role in appropriate regulation of cervical remodeling.

Associations between maternal inflammation during pregnancy and infant birth outcomes in the Seychelles Child Development Study.

PROBLEM: Markers of maternal inflammation may determine infant birth outcomes. METHOD OF STUDY: Maternal serum samples were collected at 28 weeks gestation (n = 1418) in the Seychelles Child Development Study Nutrition Cohort 2 and analyzed for immune markers by MSD multiplex assay, including cytokines from the Th1 (IFN-γ, IL-1ß, IL-2 and TNF-α) and Th2 (IL-4, IL-5, IL-10) subsets, with IL-6, MCP-1, TARC, sFlt-1 and VEGF-D. Associations of log-transformed immune markers with birthweight, length, head circumference and gestational age were assessed by multiple linear regression models, which were adjusted for maternal age, BMI, parity, child sex, gestational age and socioeconomic status. RESULTS: Neither total Th1, Th2 nor Th1:Th2 were significantly associated with any birth outcome. However, the angiogenesis marker VEGF-D was predictive of a lower birthweight, (ß = -0.058, P = 0.017) and birth length (ß = -0.088, P = 0.001) after adjusting for covariates. Higher concentrations of CRP were predictive of a lower birthweight (ß = -0.057, P = 0.023) and IL-2 (ß = 0.073, P = 0.009) and the chemokine MCP-1 (ß = 0.067, P = 0.016) were predictive of a longer gestational age. CONCLUSIONS: In our cohort of healthy pregnant women, we found no evidence for associations between the Th1 or Th2 inflammatory markers with birth outcomes. However, VEGF-D and CRP appear to predict lower birthweight and IL-2 and MCP-1 a longer gestation. Greater understanding is required of the variation in these immune markers at different gestational stages, as well as the factors which may regulate their balance in healthy pregnancy. n = 233.

Interleukin-7, T helper 1, and regulatory T-cell activity-related cytokines are increased during the second trimester of healthy pregnancy compared to non-pregnant women.

PROBLEM: Healthy pregnancy is associated with a physiologic increase in inflammatory responses. The objective of this study was to assess changes in plasma cytokines associated with uncomplicated pregnancy. METHOD OF STUDY: To examine these changes, plasma levels of immune response mediators from healthy gravidas (N = 115, gestation weeks 23-30) were compared with those from healthy non-pregnant women (N = 42). Comparisons were performed using multiplex analysis for Th1 activity-related cytokines (IFN-γ, IL-2, sIL-2Rα, IL-12[P70], and IL-27), Th2 activity-related cytokines (IL-4, IL-5, and IL-13), other immune response mediators (GM-CSF, IL-1ß, sIL-1RI, IL-6, IL-8, IL-15, IL-17A, IL-17F, IL-21, IL-22, IL-23, TGFß1, TGFß2, TGFß3, and TNFα), regulatory T cell-related cytokines (IL-10 and sTNFRII), adipokines (adiponectin, leptin, PAI-1, and resistin), chemokines (IP-10, MCP-1, and MIP-1ß), and hematopoietic growth factor IL-7. RESULTS: Multivariate linear regression models showed increased levels of IL-7, Th1-, and Treg activity-related cytokines and decreased levels of adipokines and chemokines in healthy gravidas compared with healthy non-pregnant women. Additionally, season of the year, age, pre-pregnancy body mass index, and HLA-DR/DQ genotypes for type 1 diabetes risk showed different and sometimes reciprocal influence on cytokine levels. CONCLUSION: Our study stresses the importance of profiling immune response mediators during pregnancy to better understand the effect of healthy pregnancy on cytokine levels.

Predictive value of cervical cytokine, antimicrobial and microflora levels for pre-term birth in high-risk women.

Spontaneous preterm birth (sPTB, delivery <37 weeks gestation), accounts for approximately 10% of births worldwide; the aetiology is multifactorial with intra-amniotic infection being one contributing factor. This study aimed to determine whether asymptomatic women with a history of sPTB or cervical surgery have altered levels of inflammatory/antimicrobial mediators and/or microflora within cervical fluid at 22-24 weeks gestation. External cervical fluid was collected from women with history of previous sPTB and/or cervical surgery at 22-24 weeks gestation (n = 135). Cytokine and antimicrobial peptides were measured on a multiplex platform or by ELISA. qPCR was performed for detection of 7 potentially pathogenic bacterial species. IL-8 and IL-1ß levels were lower in women who delivered preterm compared to those who delivered at term (IL-8 P = 0.02; IL-1ß P = 0.04). There were no differences in elafin or human beta defensin-1 protein levels between the two groups. Multiple bacterial species were detected in a higher proportion of women who delivered preterm than in those who delivered at term (P = 0.005). Cervical fluid IL-8 and IL-1ß and microflora have the potential to be used as biomarkers to predict sPTB in high risk women.

Levels of Systemic Low-grade Inflammation in Pregnant Mothers and Their Offspring are Correlated.

High sensitivity C-reactive protein (hs-CRP) is a marker of systemic low-grade inflammation and associated with chronic inflammatory diseases. It is unknown whether maternal and infant hs-CRP levels are correlated and little is known about risk factors in early childhood. Hs-CRP were measured in mothers during pregnancy week 24 (N = 690), and one-week postpartum (N = 675) and in their children age 6 mo (N = 640) enrolled in the Copenhagen Prospective Studies on Asthma in Childhood2010 (COPSAC2010) cohort. The risk factor analysis included anthropometrics, environmental exposures and CRP-Genetic Risk Score (GRS). Mother's body mass index (BMI), use of antibiotics, smoking, cesarean delivery and season were associated with higher maternal hs-CRP level, whereas higher social circumstances were associated with lower hs-CRP level (p < 0.05). Child's BMI, siblings, bacterial airway colonization, current infection, CRP-genetic risk score and season were associated with higher hs-CRP at age 6 mo (all p < 0.05). Mother's hs-CRP level in pregnancy week 24 was associated with hs-CRP level in the child at 6 mo: ß-coefficient = 0.11 [95% CI: 0.01-0.20], R2 = 0.22, p = 0.03. The association was unchanged adjusted for all significant risk factors. Systemic low-grade inflammation in pregnant mothers and their offspring is correlated independently of BMI, environmental exposures and genetic risk factors.

Maternal inflammation during pregnancy and offspring psychiatric symptoms in childhood: Timing and sex matter.

OBJECTIVE: Maternal infection during pregnancy has been associated with increased risk of offspring psychopathology, including depression. As most infections do not cross the placenta, maternal immune responses to infection have been considered as potentially contributing to this relationship. This study examined whether gestational timing of maternal inflammation during pregnancy is associated with offspring internalizing and/or externalizing symptoms during childhood and, further, whether fetal sex moderated this relationship. METHOD: Participants were 737 pregnant women and their offspring who were continuously followed through late childhood. Archived first and second trimester sera were analyzed for markers of inflammation [interleukin 8 (IL-8), IL-6, IL-1 receptor antagonist (IL-1ra), and soluble tumor necrosis factor receptor-II (sTNF-RII)]. When offspring were aged 9-11, mothers completed a questionnaire assessing psychological symptoms. RESULTS: Multivariate regression analyses indicated that elevated IL-8 in the first trimester was associated with significantly higher levels of externalizing symptoms in offspring. Higher IL-1ra in the second trimester was associated with higher offspring internalizing symptoms. Further, second trimester IL-1ra was associated with increased internalizing symptoms in females only. CONCLUSION: These findings demonstrate that elevated maternal inflammation during pregnancy is associated with the emergence of separate psychological phenotypes and that timing of exposure and fetal sex matter for offspring outcomes. Given that internalizing and externalizing symptoms in childhood increase risk for a variety of mental disorders later in development, these findings potentially have major implications for early intervention and prevention work.

Human dNK cell function is differentially regulated by extrinsic cellular engagement and intrinsic activating receptors in first and second trimester pregnancy.

Decidual natural killer (dNK) cells express an array of activation receptors to regulate placental immunity and development during early pregnancy. We investigated the functional character of human dNK cells during the first and second trimester of gestation and the interaction between dNK and trophoblast cells. Although the frequency of CD56+CD16- dNK among the total CD45+ leukocytes did not change over this period, the expression of the activating receptors, NKp80 and NKG2D, was greatly upregulated. We observed a significantly higher number of extravillous trophoblast cells in proximity to the dNK cells in the first trimester in comparison with the second trimester decidua. NKG2D expression by first trimester dNK cells was decreased when co-cultured with the HTR-8 trophoblast cell line. In the second trimester, functional markers of dNK activation, i.e., angiogenic factor production (e.g., vascular endothelial growth factor, interleukin-8, interferon-gamma), remained stable despite an increase in NKp80 or NKG2D surface expression. Furthermore, the degranulation capacity of dNK cells, as assessed by CD107a, was decreased in the second trimester. We suggest that in the first trimester, trophoblast-dNK interactions generate a population of dNK cells with a suppressed activating phenotype. In the second trimester, the loss of trophoblast-dNK interactions led to the inhibition of dNK cell function, although their activating receptor expression was increased. We speculate that during pregnancy, two mechanisms operate to modulate the dNK cell activation:suppression of activating receptor levels in the first trimester by trophoblasts and disengagement of receptor-ligand coupling in the second trimester.

Comparative Analysis of Midtrimester Amniotic Fluid Cytokine Levels to Predict Spontaneous Very Pre-term Birth in Patients with Cervical Insufficiency.

PROBLEM: Few studies have investigated the roles of cytokines and chemokines in women with cervical insufficiency, and those that have done so evaluated only a limited number of cytokines in amniotic fluid. METHOD OF STUDY: A retrospective cohort study enrolled 71 patients undergoing physical examination-indicated cerclage to determine whether expanded amniotic fluid cytokine levels predict spontaneous very pre-term birth (≤32 weeks of gestation) in patients with cervical insufficiency. Analysis of multiple cytokines and chemokines was performed with the multiplex immunoassay. RESULTS: Sixty-seven amniotic fluid samples were available for analysis and assayed for 15 cytokines. Thirty-eight (56.7%) patients delivered pre-term. Of these, 26 (38.8%) were spontaneous very pre-term births. Most cytokine levels were significantly increased in the amniotic fluid from the study group when compared with those from controls. The levels of interleukin-1ß (IL-1ß), IL- 6, IL- 7, IL-15, IL-17α, tumour necrosis factor-α (TNF-α), MIP-1α, and MIP-1ß were higher in patients with a very pre-term delivery than in those with a late pre-term delivery. IL-1ß, IL-6, IL-7, IL-17α, TNF-α, and cervical dilation were independently associated with a very pre-term birth. CONCLUSION: Intra-amniotic inflammation may contribute to cervical insufficiency, and the severity of inflammation is associated with a very pre-term birth in women with cervical insufficiency.

Cervical leukocytes and spontaneous preterm birth.

The objective was to characterise cervical leukocyte populations and inflammatory mediators associated with term and recurrent spontaneous preterm birth (SPTB) in pregnant women with a history of SPTB. A prospective observational study was undertaken on 120 women with a history of SPTB. A cytobrush was used to sample cells from the cervix at 12-25 weeks' gestation. Cells were enumerated and characterised by flow cytometry. Cytokines and chemokines were also measured. Participants were then grouped according to delivery at term (>36+6 weeks), late SPTB (34-36+6 weeks) or early SPTB (<34 weeks). Differences in leukocyte sub-populations, cytokine and chemokine levels were compared with outcome. Cervical leukocytes comprised up to 60% of the host-derived cells. Most of these (90-100%) were polymorphonuclear cells (PMN). Most of the remaining cells were mucosal macrophages expressing CD68 and CD103 in addition to markers shared with blood-borne monocytes. Failure to detect cervical macrophages in at least 250,000 cervical epithelial cells was a feature of women who experienced early SPTB (6 out of 6 cases, 95% CI 61-100%) compared with 34% (30 out of 88 cases, 95% CI 25-43%, P<0.001) of women delivering after 34 weeks. CCL2 (MCP-1) was also low in SPTB before 34 weeks and levels above 75 ng/g and/or the presence of macrophages increased the specificity for birth after 34 weeks from 66% to 82% (55 out of 67 cases, 95% CI 73-91%). Absence of cervical macrophages and low CCL2 may be features of pregnancies at risk of early SPTB.

Tailored anti-TNF therapy during pregnancy in patients with IBD: maternal and fetal safety.

OBJECTIVE: Antitumour necrosis factor (TNF) during pregnancy in patients with IBD is related to high fetal anti-TNF levels. We evaluated maternal and child safety on discontinuing anti-TNF in the second trimester of pregnancy. DESIGN: Two groups of women with IBD were prospectively followed-up during pregnancy: women in sustained remission stopped anti-TNF before week 25 (stop group) and the remaining group continued anti-TNF beyond week 30 (continue group). Maternal, birth and 1-year child outcomes were compared with children of non-IBD women. RESULTS: Overall, 106 patients with 83 completed pregnancies were included. Relapse rate after week 22 did not differ between the stop (n=51) and continue (n=32) groups (5 (9.8%) versus 5 (15.6%), p=0.14). There was no difference in allergic reactions (p=1.00) or loss of response (p=1.00) postpartum between the two groups. Birth outcomes were comparable. Infants from both groups had lower birth weight (p=0.001), shorter gestational term (p=0.0001), were more often delivered via caesarean section (p=0.0001) and were less often breastfed (p=0.0001) compared with infants from non-IBD controls. Growth, infection rate, allergies, eczema and adverse reactions to vaccines were comparable across the stop and the continue groups as well as the children of anti-TNF-exposed and non-IBD women at 1 year. CONCLUSIONS: To limit anti-TNF exposure in utero, anti-TNF can be stopped safely in the second trimester in women with IBD in sustained remission. In patients not in sustained remission, anti-TNF may be continued without clear additional risks to the fetus. We observed excellent 1-year child outcomes compared with children from non-IBD controls.

Identification of a novel neutrophil population: proangiogenic granulocytes in second-trimester human decidua.

The maternal leukocytes of the first-trimester decidua play a fundamental role in implantation and early development of the fetus and placenta, yet little is known regarding the second-trimester decidual environment. Our multicolor flow cytometric analyses of human decidual leukocytes detected an elevation in tissue resident neutrophils in the second trimester. These cells in both human and murine samples were spatially restricted to decidua basalis. In comparison with peripheral blood neutrophils (PMNs), the decidual neutrophils expressed high levels of neutrophil activation markers and the angiogenesis-related proteins: vascular endothelial growth factor-A, Arginase-1, and CCL2, similarly shown in tumor-associated neutrophils. Functional in vitro assays showed that second-trimester human decidua conditioned medium stimulated transendothelial PMN invasion, upregulated VEGFA, ARG1, CCL2, and ICAM1 mRNA levels, and increased PMN-driven in vitro angiogenesis in a CXCL8-dependent manner. This study identified a novel neutrophil population with a physiological, angiogenic role in human decidua.

Role of cytokines in development of pre-eclampsia associated with periodontal disease - Cohort Study.

AIM: The present study was designed to find any association of cytokines in women with periodontal disease and development of pre-eclampsia in North Indian population. MATERIALS AND METHODS: A total of 504 consecutively registered primigravida with a single live pregnancy were recruited at 14-18 weeks of gestation from antenatal clinic of Maulana Azad Medical College & associated Lok Nayak Hospital and Maulana Azad Institute of Dental Sciences, New Delhi. One periodontist performed oral health examination of all patients at inclusion into study. Blood samples were collected to measure the level of cytokines IL-4, IL-10, TNF-α and IFN-γ. RESULTS: The profile of blood levels of cytokines from women with periodontal disease was observed. The log serum levels of TNF-α & IL-4 at 16-18 weeks of gestation were significantly higher in women with periodontal disease (4.13 ± 2.06; 0.47 ± 1.56 pg/ml respectively) than in women with healthy gums (2.16 ± 1.51; 0.02 ± 1.84 pg/ml respectively, p < 0.001). Periodontal disease is associated with log serum TNF-α levels at cut-off ≥14.43 pg/ml at sensitivity 71.2% and specificity 62% (OR = 4.04; 95%CI = 2.77-5.87). Woman with periodontal disease who later developed pre-eclampsia had lower levels of TNF-α (3.72 ± 1.33 pg/ml) than those with periodontal disease who did not develop pre-eclampsia (4.20 ± 2.15 pg/ml, p ≥ 0.05). CONCLUSION: Reduced TNF-α level secretion in the early second trimester in women with periodontal disease appears to be associated with the development of pre-eclampsia.

Influence of mid-trimester amniotic fluid on endogenous and lipopolysaccharide-mediated responses of mononuclear lymphoid cells.

PROBLEM We evaluated the influence of amniotic fluid (AF) on immune mediator production by mononuclear leukocytes. METHOD OF STUDY Thirty mid-gestation AFs were incubated with peripheral blood mononuclear cells (PBMCs) in the presence or absence of lipopolysaccharide (LPS). Supernatants were tested for interleukin (IL) - 6, 10, 12, 23, tumor necrosis factor-α (TNF-α) and monocyte chemotactic protein (MCP)-1. RESULTS Endogenous mediator production was minimal or non-detectable. AF stimulated endogenous MCP-1, IL-6 and TNF-α release. In the presence of LPS, production of MCP-1 and IL-10 by PBMCs was enhanced eight- to ninefold by AF. Release of IL-6 and IL-23 was enhanced less than twofold by the addition of AF while TNF-α production was unchanged. AF-stimulated mediator production was similar irrespective of pregnancy outcome. CONCLUSION Selective AF stimulation of LPS-mediated MCP-1 and IL-10 release may be a mechanism to promote antibody production and the influx of phagocytic cells to engulf pathogens while downregulating the production of pro-inflammatory cytokines.

[Predictive value of some parameters of immune balance in preeclampsia].

The goal of our research was to reveal correlation between pro- and anti-inflammatory cytokines in preeclamptic women. The research was conducted on pregnant women with physiologic pregnancy and with preeclampsia. Parameters of immune system - pro-inflammatory cytokines (IL-2; TNF-α) and anti-inflammatory cytokine (IL-10) were measured in venous blood by ELISA. The increase of TNF-α and decrease of IL-10 in blood of pregnant women with pre-eclampsia after 20 weeks of gestation were observed; the content of IL-2 was in norm. The highest activation of inflammatory reactions was at the 20-28 weeks of gestation. It was found that women with preeclampsia had high levels of TNF- α in their blood. The increase of TNF- α has a prognostic significance.

Reduced immunogenicity of pancreatic progenitor cells derived from first-trimester human fetal pancreas.

The relatively low immunogenic and tumorigenic nature of fetal stem cells makes them attractive candidates for transplantation. Pancreatic progenitor cells (PPCs) derived from human fetal pancreas that are amenable to growth and differentiation into transplantable insulin-producing islet-like cell clusters (ICCs) have been reported recently; however, the immunological nature of these cells has yet to be characterized. We thus investigated and compared the immunogenicity of pancreatic progenitor cells and islet-like cell clusters from first- and second-trimester human fetal pancreas. Polymerase chain reaction demonstrated that pancreatic progenitor cells and islet-like cell clusters express immune-related genes of major histocompatibility complex, MHC-I and MHC-II, complement component 3 (C3), chemokine ligand (CCL19), and tumor necrosis factor super family (TNFSF10), but no expression of the co-stimulatory genes, CD80 and CD86. Interestingly, pancreatic progenitor cells showed a differential expression of MHC-I and MHC-II with advancing gestational age with a greater expression in pancreatic progenitor cells from the second trimester. Pre-incubation of the second-trimester cells with interferon-γ (IFN-γ) increased MHC molecule expression. Functional alloreactivity of pancreatic progenitor cells was investigated via mixed lymphocyte reactions (MLRs). Relative to first-trimester pancreatic progenitor cells, second-trimester pancreatic progenitor cells induced a greater extent of proliferation of peripheral blood mononuclear cells (PBMCs) and resulted in more IFN-γ production in phytohaemagllutinin-stimulated peripheral blood mononuclear cells following co-culture. Results of the study indicated that first-trimester pancreatic progenitor cells and islet-like cell clusters have a distinctively lower immunogenicity relative to second-trimester pancreatic progenitor cells, even after a pro-inflammatory cytokine challenge.

Prevalence of Epstein-Barr virus reactivation in pregnancy.

Recent evidence suggests a link between Epstein-Barr virus (EBV) reactivation and chronic stress in nonpregnant adults, possibly due to decreased cellular immune response. Our objective was to determine the prevalence of EBV seropositivity in a diverse cohort of pregnant women and whether maternal demographic characteristics were associated with EBV reactivation. In this cross-sectional study, we evaluated midpregnancy serum specimens from 64 healthy pregnant women for presence of EBV viral capsid antigen, EBV nuclear antigen, and EBV early antigen. The subjects were reported as EBV seronegative, EBV seropositive with reactivation, and EBV seropositive without reactivation. The maternal demographics of the seropositive women with EBV reactivation were compared with their nonreactivated counterparts. Chi-square and Student T test were used for statistical analysis. In our pregnant cohort, 63 (98%) of the 64 women were EBV seropositive. Among these seropositive women, 22 (35%) women demonstrated EBV reactivation in pregnancy. EBV reactivation was not associated with maternal age, race, parity, or insurance type. In our diverse pregnant cohort, 98% of women analyzed were EBV seropositive with 35% demonstrating EBV reactivation in the pregnancy by the second trimester. The pathophysiology and clinical implications of EBV reactivation during pregnancy need further study.

Systemic reduction of functionally suppressive CD4dimCD25highFoxp3+ Tregs in human second trimester pregnancy is induced by progesterone and 17beta-estradiol.

CD4(+)CD25(high) regulatory T cells (Tregs) are implicated in the maintenance of murine pregnancy. However, reports regarding circulating Treg frequencies in human pregnancy are inconsistent, and the functionality and phenotype of these cells in pregnancy have not been clarified. The aim of this study was to determine the frequency, phenotype, and function of circulating Tregs in the second trimester of human pregnancy and the influence of progesterone and 17beta-estradiol on Treg phenotype and frequency. Based on expressions of Foxp3, CD127, and HLA-DR as determined by multicolor flow cytometry, we defined a proper CD4(dim)CD25(high) Treg population and showed, in contrast to most previous reports, that this population was reduced in second trimester of pregnancy. Unexpectedly, Foxp3 expression was decreased in the Treg, as well as in the CD4(+) population. These changes could be replicated in an in vitro system resembling the pregnancy hormonal milieu, where 17beta-estradiol, and in particular progesterone, induced, in line with the pregnancy situation, a reduction of CD4(dim)CD25(high)Foxp3(+) cells in PBMC from nonpregnant women. By coculturing FACS-sorted Tregs and autologous CD4(+)CD25(-) responder cells, we showed that Tregs from pregnant women still displayed the same suppressive capacity as nonpregnant women in terms of suppressing IL-2, TNF-alpha, and IFN-gamma secretion from responder cells while efficiently producing IL-4 and IL-10. Our findings support the view of hormones, particularly progesterone, as critical regulators of Tregs in pregnancy. Furthermore, we suggest that in the light of the results of this study, early data on circulating Treg frequencies in pregnancy need reevaluation.

Human chorionic gonadotropin attracts regulatory T cells into the fetal-maternal interface during early human pregnancy.

Regulatory T cells (Treg) expand during pregnancy and are present at the fetal-maternal interface at very early stages in pregnancy. The migration mechanisms of Treg to the pregnant uterus are still unclear. Human chorionic gonadotropin (hCG) is secreted by the blastocyst immediately after fertilization and has chemoattractant properties. Therefore, we sought to analyze whether hCG secreted by early trophoblasts attracts Treg to the uterus and hence contributes to maternal tolerance toward the fetus. Decidua and placenta tissue samples from patients having spontaneous abortions or ectopic pregnancies were employed to evaluate Treg and hCG levels. Age-matched samples from normal pregnant women served as controls. We further performed in vitro studies with primary first trimester trophoblast cells and a choriocarcinoma cell line (JEG-3) aiming to evaluate the ability of secreted hCG to attract Treg. Patients having miscarriages or ectopic pregnancy presented significantly decreased hCG mRNA and protein levels associated with decreased Foxp3, neuropilin-1, IL-10, and TGF-beta mRNA levels as compared with normal pregnant women. Using migration assays we demonstrated that Treg were attracted by hCG-producing trophoblasts or choriocarcinoma cells. Treg migration toward cells transfected with hCG expression vectors confirmed the chemoattractant ability of hCG. Our data clearly show that hCG produced by trophoblasts attracts Treg to the fetal-maternal interface. High hCG levels at very early pregnancy stages ensure Treg to migrate to the site of contact between paternal Ags and maternal immune cells and to orchestrate immune tolerance toward the fetus.