An imbalance in interleukin-17-producing T and Foxp3⁺ regulatory T cells in women with idiopathic recurrent pregnancy loss.

BACKGROUND: T cells which produce interleukin (IL)-17 are involved in chronic inflammatory processes and regulatory T (Treg) cells are possibly the most important immune regulators. We aimed to investigate peripheral blood IL-17(+) T and Foxp3(+) Treg cells in women with idiopathic recurrent pregnancy loss (RPL). METHODS: The study design is a cross-sectional evaluation of Th1, Th2, IL-17(+) T and Treg cells in women with idiopathic RPL (n = 42) and age-matched parous controls (n = 24). Flow cytometric analysis was performed to measure IL-17(+) T and Foxp3(+) Treg cells, and ratios of Th1/Th2 cells using anti-IL-17A and anti-Foxp3 antibodies, and monoclonal antibodies to tumor necrosis factor (TNF)-α, interferon (IFN)-γ and IL-10. Student's t-test and partial correlations were applied for statistical analysis. RESULTS: TNF-α-/IL-10-producing CD3(+)CD4(+) T cell ratio was higher in women with RPL than controls (P = 0.048). Levels of IL-17(+) T cells (P = 0.021) and the IL-17(+) T/CD4(+)Foxp3(+) Treg cell ratio (P = 0.001) were increased, whereas Foxp3(+) (P = 0.035), Foxp3(low) (P = 0.032) and CD4(+)Foxp3(+) T cell (P = 0.037) levels were decreased in women with RPL, compared with controls. Levels of IL-17(+) T cells were correlated with TNF-α-producing CD3(+)CD4(+) T cells (r = 0.269, P = 0.033), and with ratios of TNF-α/IL-10 (r = 0.276, P = 0.027) and IFN-γ/IL-10 (r = 0.266, P = 0.035)-producing CD3(+)CD4(+) cells. Furthermore, the ratio of IL-17(+) T cells to CD4(+)Foxp3(+) Treg cells showed a positive correlation with TNF-α-producing CD3(+)CD4(+) T cells (P = 0.047) and IFN-γ-producing CD3(+)CD4(+) T cells (P = 0.048) as well as a ratio of IFN-γ/IL-10-producing CD3(+)CD4(+) T cells (P = 0.037). CONCLUSIONS: Enhanced pro-inflammatory immune responses with suppressed immune regulation may be an important immune mechanism involved in RPL.

Reproductive outcomes of women with recurrent pregnancy losses and repeated implantation failures are significantly improved with immunomodulatory treatment.

This study investigated if immunomodulatory treatment improves the in-vitro fertilization (IVF) success rates of women with two or more recurrent pregnancy losses (RPL) and repeated implantation failures (RIF) with cellular immune abnormalities and thrombophilia. We performed a retrospective cohort study of 197 RPL patients who received immunomodulatory and anticoagulation treatment undergoing IVF cycles (fresh or frozen embryo transfer). Patients were divided into four groups; Group 1: women with RPL but without RIF, Group 2: women with RPL and RIF (≥3), Group 3: women with RPL after IVF cycles (>2) and without RIF, and Group 4: women with RPL after IVF cycles and RIF. Patients received immunomodulatory treatment with prednisone-only or prednisone and intravenous immunoglobulin G (IVIG) and anticoagulation treatment with low molecular weight heparin and low dose aspirin. IVF success rates of study groups were compared to those of the historical controls. The pregnancy rate of IVF cycles with immunomodulatory treatment was significantly increased in all patients (48.2 % vs. 33.0 %, P < 0.001), Group 1 (54.2 % vs. 30.5 %, P < 0.005) and Group 2 (33.3 % vs. 11.0 %, P < 0.005) as compared to historical controls. The live birth rates per ET cycle were significantly improved for all patients (1.8 % vs. 39.6 %, P < 0.001), and study groups compared to their historical controls (Group 1, 43.1 % vs. 0 %; Group 2, 33.3 % vs. 2.5 %; Group 3, 45.5 % vs. 2.3 %; and Group 4, 16.7 % vs. 1.2 %, P < 0.001, respectively). Immunomodulatory and anticoagulation treatment significantly improved the reproductive outcomes of IVF cycles in women with a history of RPL and/or RIF of immune etiologies.

Uterine Radial Artery Resistance Index Predicts Reproductive Outcome in Women with Recurrent Pregnancy Losses and Thrombophilia.

Uterine radial artery resistance index (URa-RI) by Doppler ultrasound may reflect the changes in the uteroplacental circulation and be associated with adverse events in early pregnancy. Recurrent pregnancy losses (RPL) are associated with thrombophilia, and anticoagulation treatment with low molecular weight heparin improves pregnancy outcome in women with RPL and thrombophilia. A retrospective cohort study was conducted in 139 pregnant women with 3 or more RPL and thrombophilia. The relationship between pregnancy outcome and dynamic changes of URa-RI was analyzed in 116 women who delivered a liveborn infant and 23 who miscarried the index pregnancy. Patients were on preconception low molecular weight heparin, low-dose aspirin (81mg per day), and prednisone treatment. URa-RI was measured during periovulation time, at the time of positive pregnancy test, and then repeated every two weeks until 32-week gestation or the time of miscarriage. The URa-RI at 8-week gestation was significantly higher in women who miscarried the index pregnancy than those who delivered alive born infant (0.51±0.08 vs. 0.42±0.03, P<0.001). Receiver operating characteristic curve analysis demonstrated that URa-RI of 8 wk gestation effectively distinguished women who miscarried from those who had a live birth with an area under the curve of 82.6% (95% CI 69.01-97.17). After adjusting for covariates including age, BMI, and number of miscarriages, multiple logistic regression models showed that each 0.1 unit increase of URa-RI of 8 wk gestation was associated with 18.70-point increase in the risk of miscarriage (OR19.70, 95%CI 4.26-91.1, P<0.001), and women with an URa-RI≥0.45 had an OR of 49.48 (95% CI 8.01-307.95; P<0.001) for miscarriage compared to those who had URa-RI<0.45. In women with RPL and inherited thrombophilia, increased URa-RI at 8-week gestation was associated with spontaneous abortion independent of other risk factors while they were on anticoagulation treatment.

Early pregnancy immune biomarkers in peripheral blood may predict preeclampsia.

We performed a prospective cohort study in 197 pregnant women. Peripheral blood was collected between 5 and 16 weeks of gestation. Intracellular cytokine analysis and immunophenotype were performed by flow-cytometry. Serum levels of cytokines and chemokines were analyzed by multiplex assay. 86 patients were eligible for the analysis and 10.5% (n=9) developed preeclampsia. Patients with preeclampsia had significantly higher percentage of CD3+CD4+TNFα+ T helper (Th) 1 cells (45.4±10.3 vs 37.1±8.5, P=0.032) and CD3+CD4+IL17+ Th 17 cells (2.4±1.3 vs 1.6±1.1, P=0.029) when compared to those of patients without preeclampsia. CD3+CD4+CD25+CD127dim/- T regulatory cells (Treg) cells (5.7±1.2% vs 7.0±1.6%, P=0.015) were significantly lower in patients with preeclampsia when compared to those without preeclampsia. Patients with preeclampsia had significantly higher TNFα/IL-10 cell ratio (43.8±10.3 vs 34.3±7.9, P=0.005) and Th17/Treg cell ratio (0.5±0.3 vs 0.2±0.2, P=0.011) when compared to those of patients without preeclampsia. IL-8 and Macrophage inflammatory protein (MIP)-1α serum levels were significantly higher in patients with preeclampsia when compared with patients without preeclampsia (Median=341.0 vs 87.6, U=152, P=0.020 and Median=35.7 vs 17.7, U=120, P=0.029 respectively). Serum MCP-1 levels were significantly lower in patients with preeclampsia when compared with patients without preeclampsia (Median=233.8 vs 390.9, U=183, P=0.021). The logistic regression predictive model combining TNFα/IL-10 ratios, IL-8 and MCP-1 serum levels had the best performance (AUC=0.886, 95%CI 0.8-0.9). We concluded that elevated Th1 and Th17 cell percentages, elevated TNFα/IL-10 and Th17/Treg cell ratios and decreased Treg cell percentages in early pregnancy are associated with preeclampsia.

Deficiency in memory B cell compartment in a patient with infertility and recurrent pregnancy losses.

Alterations in normal balance of B cell subsets have been reported in various rheumatic diseases. In this study, we report a woman with a history of recurrent pregnancy losses (RPL) and infertility who had low levels of memory B cells. A 35-year-old woman with a history of RPL and infertility was demonstrated to have increased peripheral blood CD19+ B cells with persistently low levels of memory B cell subsets. Prior to the frozen donor egg transfer cycle, prednisone and intravenous immunoglobulin G (IVIg) treatment was initiated and patient achieved dichorionic diamniotic twin pregnancies. During pregnancy, proportion (%) of switched memory B cells CD27+IgD- increased, while percent of total CD19+ B cells and CD27-IgD+ naive B cells were gradually decreased with a high dose IVIg treatment. She developed cervical incompetence at 20 weeks of gestation, received a Cesarean section at 32 weeks of gestation due to preterm labor, and delivered twin babies. B cell subset abnormalities may be associated with infertility, RPL and preterm labor, and further investigation is needed.

Status of peripheral blood natural killer cells in women with recurrent spontaneous abortions and infertility of unknown aetiology.

The aim of this study was to investigate the functional status and immunophenotypic characteristics of natural killer (NK) cells in women who suffer recurrent spontaneous abortions (RSA) or have infertility of unknown aetiology. Peripheral blood mononuclear cells (PBMC) were obtained from 40 study patients and 13 normal healthy multiparous controls. NK cells were identified using anti-CD56 and anti-CD16 monoclonal antibodies (mAb). The expression of CD69, CD25, CD122, CD30, CD154, CD128 and CD94 on NK cells was detected using specific mAb and analysed by flow cytometry. CD69 expression on NK cells after ED(27) human trophoblast cell line co-culture with PBMC was also investigated. A significant increase in CD69 expression on CD56(+) NK cells was demonstrated in women with RSA (P < 0.005) and infertility (P < 0.05) as compared with that of normal controls. Conversely, CD94 expression was significantly decreased in women with RSA (P < 0.005) and infertility (P < 0.05) in comparison with that of controls. Increased CD69 expression on NK cells was induced after 24 h co-culture with ED(27). In conclusion, peripheral blood NK cells of women with RSA and infertility of unknown aetiology have higher proportions of activated NK cells in vivo. Unbalanced CD69 and CD94 expression may explain the underlying pathology.

Increased T helper 1 cytokine responses by circulating T cells are present in women with recurrent pregnancy losses and in infertile women with multiple implantation failures after IVF.

BACKGROUND: We aimed to study T-helper 1 (Th1) and Th2 intracellular cytokine expression in peripheral blood lymphocytes of women with recurrent spontaneous abortions (RSA) or infertility with multiple implantation failures after IVF cycles. METHODS: Twenty-six women with three or more RSA and 23 with two or more IVF failures (14 with no history of spontaneous abortion (SAB) and nine with more than one SAB) comprised the two study groups. Twenty-one non-pregnant healthy multiparous women served as controls. Proportions (%) of lymphocytes containing IFN-gamma, TNF-alpha, IL-4 and IL-10 and the Th1/Th2 ratios of IFN-gamma/IL-4, IFN-gamma/IL-10, TNF-alpha/IL-4 and TNF-alpha/IL-10 in CD3+, CD3+/CD8- (T helper) and CD3+/CD8+ (T suppressor) cells were measured by 4-colour flow cytometry. RESULTS: RSA women demonstrated significantly higher Th1/Th2 ratios of IFN-gamma/IL-4 (P < 0.01), TNF-alpha/IL-4 and TNF-alpha/IL-10 (P < 0.05 each) in CD3+/CD8- T helper cells than those of controls. The proportion of TNF-alpha producing CD3+/CD8- cells (P < 0.05), and the Th1/Th2 ratios of TNF-alpha/IL-4 (P < 0.05) and TNF-alpha/IL-10 (P < 0.005) in CD3+/CD8- cells were significantly higher in women with multiple IVF failures without SAB as compared with those of controls. CONCLUSIONS: The prevalence of dominant Th1 immune responses in peripheral blood lymphocytes may reflect the systemic contribution of Th1 cytokines to RSA or multiple implantation failures in IVF cycles.