Diminished miR-374c-5p negatively regulates IL (interleukin)-6 in unexplained recurrent spontaneous abortion.

Unexplained recurrent spontaneous abortion (URSA) is commonly observed, and seriously affects women's reproductive health. Excessive interleukin-6 (IL-6) production has been shown to frequently occur and relate to URSA pathogenesis. In this study, the miRNA expression profile in peripheral blood mononuclear cells (PBMCs) from URSA patients and normal pregnant (NP) women was assessed by miRNA microarray and real-time quantitative reverse-transcription polymerase chain reaction (qPCR). MiRNA target prediction tools and luciferase reporter assay were used to detect direct binding between miRNAs and IL6. Functional study of administering anti-IL-6 neutralizing antibody and miR-374c-5p mimics to an URSA animal model was performed to evaluate embryo resorption rates. In the results, compared with NP women, the expression of IL-6 increased markedly in PBMCs and decidual tissues at both mRNA and protein levels, while miR-374c-5p expression decreased significantly. Prediction software and luciferase reporter assay showed that miR-374c-5p binds with IL6 3'UTR via the complementary bases. Transfection of miR-374c-5p mimics into an in vitro HeLa cell line significantly downregulated the expression of IL-6, while transfection of the miR-374c-5p inhibitor induced an opposite result. In the URSA mouse model, miR-374c-5p overexpression reduced the embryo resorption rate significantly, accompanied with decreased expression of IL-6 in the decidua. To sum up, downregulated miR-374c-5p was involved in the pathogenesis of URSA by enhancing IL-6 expression. Modulation of miR-374c-5p expression may be used to regulate IL-6 production for the treatment of URSA.

Vitamin D insufficiency as a risk factor for reproductive losses in miscarriage.

OBJECTIVE: To study the relationship between vitamin D deficiency, VDR gene polymorphism rs10735810 (A > G), and a missed abortion in the first trimester of gestation; to determine the predictors of its risk. RESEARCH METHODS: 178 women aged between 18 and 41 were surveyed. The main group consisted of patients with miscarriage (n = 101), verified at the hospital stage (O02.0; O02.1), which were stratified by I group (n = 58, patients with the first miscarriage) and II groups (n = 43, patients with repeated miscarriage). The control group (n = 77) consisted of women with a successful pregnancy (Z34.0), which subsequently ended in delivery at term with a live fetus. Patients were surveyed and data was extracted from primary medical records. The level of 25(OH)D in the blood serum was investigated by mass spectrometry (n = 99). Genotyping for the vitamin D receptor gene polymorphism rs10735810 (VDR A > G) was performed for 177 patients. Statistical data analysis was performed via Statistica 10 and SAS JMP 11 application packages, using single-factor prediction for quantitative and binary factors, ROC analysis, and CHAID decision tree construction. RESULTS OF THE STUDY: WE found that patients with miscarriage in the first trimester of gestation (n = 60) more frequently than those in the control group (n = 39) had vitamin D insufficiency (93.3% versus 76.9%, p = .0183) including its deficiency, occurring at 25(OH)D of blood <20 ng/ml (71.7% versus 51.3%, p = .0392). This pattern was found in patients with the first miscarriage, where significant differences in the frequency of vitamin D deficiency were also detected in comparison with the control group (80.0% versus 51.3%, p = .0026). No direct correlation was found between the frequency of miscarriages in the first trimester and the variant of the polymorphism of the vitamin D receptor gene (VDR A > G [rs10735810]); the GG genotype in patients with repeated miscarriages was even less frequent compared to the control group (14.0% versus 23.7%, p = .3344). However, the decision tree has identified four risk classes and has determined that the highest risk of missed abortion in the cohort studied is formed by three predicates: smoking, serum level 25(OH)D < 6.5 ng/ml and VDR AA and GG genotypes. CONCLUSION: The data obtained show that vitamin D insufficiency plays a pathogenetically significant role in early reproductive losses associated with miscarriages, both first and recurrent.

Association of reduced maternal sHLA-G5 isoform levels and elevated TNF-α/IL-4 cytokine ratio with Recurrent Pregnancy Loss: A study on South Indian women.

Inflammation is of critical importance in successful implantation during pregnancy. However, the establishment of maternal immune tolerance towards semi-allograft foetus is more exigent and is achieved predominantly by human leukocyte antigen-G (HLA-G) isoforms with a special emphasis on soluble HLA-G5 (sHLA-G5). Constant inflammation and lack of resolution by anti-inflammatory milieu, due to aberrant expression of critical immunoregulatory molecules such as sHLA-G5 and dysfunctional T helper cells 1 and 2 (Th1-Th2) cytokine shift, can lead to adverse pregnancy outcomes including recurrent pregnancy loss (RPL). Serum samples of 270 pregnant women (135 healthy parous and 135 with a history of RPL) were evaluated for the concentrations of sHLA-G5, interleukin-4 (IL-4) and tumour necrosis factor-alpha (TNF-α) using sandwich enzyme-linked immunosorbent assay (ELISA) and found elevated levels of sHLA-G5 and IL-4 in controls and higher TNF-α levels and TNF-α:IL-4 ratio in patients (P < .05). Stratified data analysis based on the time of sample collection, that is the first and second trimesters exhibited higher sHLA-G5 and IL-4 in both first and second trimesters in controls than patients, while they displayed lower levels concerning TNF-α and TNF-α:IL-4 ratio (P < .05). However, within patients and controls in the first or second trimesters, there was a significant variation concerning sHLA-G5 alone. Further, the outcome of pregnancies studied in the present investigation revealed a significant elevation in sHLA-G5 levels among women with successful pregnancies compared with women who experienced pregnancy loss, therefore, concluding the potential application of sHLA-G5 isoform as a marker in assisting improved pregnancy outcomes.

Decidual CXCR4+ CD56bright NK cells as a novel NK subset in maternal-foetal immune tolerance to alleviate early pregnancy failure.

Natural killer (NK) cells preferentially accumulate at maternal-foetal interface and are believed to play vital immune-modulatory roles during early pregnancy and related immunological dysfunction may result in pregnant failure such as recurrent miscarriage (RM). However, the mechanisms underlying the establishment of maternal-foetal immunotolerance are complex but clarifying the roles of decidual NK (dNK) cells offers the potential to design immunotherapeutic strategies to assist RM patients. In this report, we analysed RNA sequencing on peripheral NK (pNK) and decidual NK cells during early pregnancy; we identified an immunomodulatory dNK subset CXCR4+ CD56bright dNK and investigated its origin and phenotypic and functional characteristics. CXCR4+ CD56bright dNK displayed a less activated and cytotoxic phenotype but an enhanced immunomodulatory potential relative to the CXCR4 negative subset. CXCR4+ CD56bright dNK promote Th2 shift in an IL-4-dependent manner and can be recruited from peripheral blood and reprogramed by trophoblasts, as an active participant in the establishment of immune-tolerance during early pregnancy. Diminished CXCR4+ dNK cells and their impaired ability to induce Th2 differentiation were found in RM patients and mouse models of spontaneous abortion. Moreover, adoptive transfer of CXCR4+ dNK cells to NK-deficient (Nfil3-/-) mice showed great therapeutic potential of CXCR4+ dNK via recovering the Th2/Th1 bias and reducing embryo resorption rates. The identification of this new dNK cell subset may lay the foundation for understanding NK cell mechanisms in early pregnancy and provide potential prognostic factors for the diagnosis and therapy of RM.

Extensive serum biomarker analysis in the prethrombotic state of recurrent spontaneous abortion.

The prethrombotic state (PTS) is a possible cause of recurrent spontaneous abortion (RSA). The aim of this study was to identify serum biomarkers for the detection of RSA with PTS (PSRSA). A Quantibody array 440 was used to screen novel serum-based biomarkers for PSRSA/NRSA (RSA without PTS). Proteins differentially expressed in PSRSA were analysed using bioinformatics methods and subjected to a customized array and enzyme-linked immunosorbent assay (ELISA) validation. We used receiver operating characteristic to calculate diagnostic accuracy, and machine learning methods to establish a biomarker model for evaluation of the identified targets. 20 targets were selected for validation using a customized array, and seven targets via ELISA. The decision tree model showed that IL-24 was the first node and eotaxin-3 was the second node distinguishing the PSRSA and NRSA groups (an accuracy rate of 100% and an AUC of 1). Epidermal growth factor (EGF) as the node distinguished the PSRSA and NC groups (an accuracy rate of 100% and an AUC of 1). EGF as the node distinguished the NRSA and NC groups (an accuracy rate of 96.5% and an AUC of 0.998). Serum DNAM-1, BAFF, CNTF, LAG-3, IL-24, Eotaxin-3 and EGF represent a panel of promising diagnostic biomarkers to detect the PSRSA.

Differences in the Expression of KIR, ILT Inhibitory Receptors, and VEGF Production in the Induced Decidual NK Cell Cultures of Fertile and RPL Women.

RESULTS: KIR2DL1 and ILT-2 expression on idNK cells was higher in healthy women than in RPL patients. Sildenafil enhanced NKG2A expression in RPL patients. VEGF concentration was higher in fertile woman idNK cell cultures. idNK cells were more sensitive for necrosis in RPL than in fertile women. SC did not influence VEGF production or idNK cell apoptosis. CONCLUSIONS: A combination of hypoxia, IL-15, and AZA promotes the conversion of pbNK into idNK cells CD56+CD16--expressing KIR receptors and produces VEGF. Alterations in KIR2DL1 and ILT-2 expression as well as impaired VEGF production were associated with RPL. SC affects NKG2A expression on RPL idNK cells. SC had no effect on VEGF release or idNK cell apoptosis.

Functional NK surrogate biomarkers for inflammatory recurrent pregnancy loss and recurrent implantation failure.

PROBLEM: Expansion of circulating NK cells has been related to pregnancy complications. This study aims at investigating several surface NK cell markers to identify a baseline inflammatory profile in women with recurrent pregnancy loss (iRPL) and recurrent implantation failure (iRIF). METHOD OF STUDY: Expression of NKp30, TIGIT, NKp46, and DNAM-1 on total peripheral blood NK subsets, regulatory (CD56bright CD16neg ), and cytotoxic (CD56dim CD16pos/neg ) NK cells was measured. RESULTS: Eighty-three women were recruited and classified into two groups, 58 women with RPL and 25 with RIF. A control group of 31 fertile women was included. Expression of NKp30 on cytNK was significantly higher in RPL (p = .019) and RIF (p < .001) than HC. TIGIT on cytNK cells was also higher in both RPL (p < .001) and RIF (p < .01). An optimal cutoff of 70% for NKp30+ cytNK disclosed a sensitivity of 82%, a specificity of 55%, and 83% PPV for RPL diagnosis. A cutoff level of 83% for TIGIT+ cytNK was chosen to discriminate between healthy controls and RPL women, with PPV of 84%. CONCLUSION: Our preliminary data on this RPL and RIF cohorts suggest a simple diagnostic tool by combining NKp30 and TIGIT on cytNK cells to better identify a subgroup of RPL and RIF patients with a baseline inflammatory profile. A more rigorous selection of these patients through phenotyping peripheral cytNK cells may better define patients that could benefit from an immunomodulatory treatment to prevent further pregnancy losses. The performance of these biomarkers requires further investigation and validation in independent cohorts.

Evaluation of paternal lymphocyte immunotherapy and potential biomarker mixed lymphocyte reaction-blocking factor in an Argentinian cohort of women with unexplained recurrent spontaneous abortion and unexplained infertility.

PROBLEM: Analyze the effect of paternal immunotherapy treatment (PIT) in primary and secondary unexplained recurrent spontaneous abortion (URSA) and unexplained infertility (UI). METHODS OF STUDY: A retrospective study analyzed a two-year follow-up between the generation of MLR-Bfs after PIT treatment (or controls first consultation) and a live birth. Recruited patients included primary URSA with two or more miscarriages at <12 weeks gestation, secondary URSA with previous live birth before two or more miscarriages, and UI with inability to conceive after 2 years of regular unprotected intercourse or in vitro fertilizations (IVF). PIT treated were compared with untreated controls. RESULTS: Primary URSA: live birth was 241/416 (58%) versus 64/282 (23%) controls (p < .0001). Up to age 35, success was 158/217 (73%) and 37/144 (26%) controls (p < .0001). With 3 or more previous URSA, success was 90/135 (67%) versus 17/79 (22%) controls (p < .0001). Between ages 36 and 40, success was 69/147(47%) versus 22/98 (22%) controls (p < .0003), with 3 or more previous URSA live birth was 45/95 (47%) versus 6/46 (13%) controls (p < .0001). In UI, live birth was 99/298 (33%) versus 54/263 (21%) in controls (p < .0009) that increased under age 35 to 53/116 (46%) in treated versus 26/101 (26%) controls (p < .0056). In PIT treated, IVF success required a median of 1 (1.37 ± 0.67) versus a median of 3 IVF procedures (2.75 ± 0.84) in controls. CONCLUSION: PIT is a successful treatment for primary and secondary URSA, and UI. PIT reduced the number of IVF required for achieving pregnancy.

Foxp3 TSDR Hypermethylation Is Correlated with Decreased Tregs in Patients with Unexplained Recurrent Spontaneous Abortion.

A decline of T regulatory cell (Treg) number and function is associated with unexplained recurrent spontaneous abortion (URSA). However, the mechanism of downregulation of Tregs in URSA patients is still unknown. This study aimed to investigate the changes of Tregs in URSA patients and the epigenetic regulation for these changes. Venous blood samples were collected from 20 patients with URSA and 20 healthy control subjects. Treg number and inhibitory capacity, and Foxp3 mRNA expression and Foxp3 TSDR methylation were compared between the 2 groups. Correlations between Treg frequency and inhibitory function and TSDR methylation status were examined by Spearman's correlation. The proportion of Tregs within the population of CD4+ T cells and the expression of Foxp3 mRNA was significantly lower in URSA patients than in healthy control subjects. Tregs from URSA patients and healthy controls both significantly inhibited the cytotoxic activity of natural killer (NK) cells toward K562 targets; however, the inhibitory ability of Tregs from URSA patients was significantly lower than that from healthy controls. The methylation level of the Treg-specific demethylated region (TSDR) in the Foxp3 gene was significantly greater in URSA patients than in the controls, and the level of methylation was inversely correlated with the proportion of Tregs and Foxp3 mRNA expression in the peripheral blood. However, the methylation level was not correlated with the inhibitory function of Tregs. A decrease of Treg number and function may be related to the pathogenesis of URSA, and Foxp3 hypermethylation may be associated with the decreased Treg number.

Two of a kind? Immunological and clinical risk factors differ between recurrent implantation failure and recurrent miscarriage.

Recurrent miscarriage (RM) and recurrent implantation failure (RIF) are unsolved challenges in reproductive medicine. Whether RIF patients share the same risk factors as RM patients is a matter of debate. Besides clinical factors, immune alterations are discussed in both conditions. The scope of this study was to compare the prevalence of clinical and immunological risk factors in a large cohort of RM and RIF patients. Between 11/2011 and 02/2019, 613 RM and 185 RIF patients were included. A screening for anatomical malformations, endocrine, autoimmune, prothrombotic and parental chromosomal disorders was performed. The immune status was assessed using flow cytometry analysis of peripheral lymphocyte subpopulations and uterine natural killer cells (uNK cells) using immunohistochemistry. RM patients showed a higher rate of intrauterine adhesions and elevated antinuclear antibodies ≥ 1:160 (p < 0.05). A higher prevalence of submucous fibroids and increased factor VIII levels were observed in RIF patients (p < 0.05). The prevalence of an antiphospholipid syndrome (APLS) was low and did not differ between the two groups. RIF patients had higher numbers of peripheral regulatory T-cells (p < 0.05). Significant more RIF patients were diagnosed with elevated uNK cells (p < 0.05). Differences in clinical and immunological risk factors of RM and RIF patients reflect different entities. Lower Tregs in RM and higher uNK cells in RIF patients might be related to the previous exposure of the immune system to fetal cells. The low prevalence of an APLS indicates a potential overestimation of this factor in the pathophysiology of RM and RIF.

Predicting first-trimester outcome of embryos with cardiac activity in women with recurrent spontaneous abortion.

OBJECTIVE: This study was performed to evaluate the capability of routine clinical indicators to predict the early outcome of embryos with cardiac activity in women with recurrent spontaneous abortion (RSA). METHODS: A retrospective cohort study of pregnant women with a history of RSA in a Chinese tertiary hospital was performed using unadjusted and multivariable logistic regression. RESULTS: Of 789 pregnant women with RSA, 625 (79.21%) had ongoing pregnancy, whereas 164 (20.79%) developed abortion before 20 full weeks of gestational age even after embryonic heart motion was detected. The final model had an area under the curve of 0.81 (95% confidence interval, 0.78-0.84) with a sensitivity of 74.39%, a specificity of 76.00%, and a false-positive rate of 52.32% at a fixed detection rate of 90%. CONCLUSIONS: The combination of multiple routine clinical indicators was valuable in predicting the early outcome of embryos with cardiac activity in viable pregnancies with RSA. However, this model might result in a high false-positive rate with a fixed detection rate of 90%; other markers must be investigated to identify first-trimester RSA once positive embryonic heart motion is established.

Weighted gene correlation network analysis reveals novel regulatory modules associated with recurrent early pregnancy loss.

At present, the etiology and pathogenesis of recurrent early pregnancy loss (REPL) are not completely clear. Therefore, identifying the underlying diagnostic and prognostic biomarkers of REPL can provide new ideas for the diagnosis and treatment of REPL. The chip data of REPL (GSE63901) were downloaded from the NCBI Gene Expression Omnibus (GEO) database. Weighted Gene Co-Expression Network Analysis (WGCNA) was used to construct a co-expression module for studying the relationship between gene modules and clinical features. In addition, functional analysis of hub genes in modules of interest was performed. A total of 23 co-expression modules were identified, two of which were most significantly associated with three clinical features. The MEbrown module was positively correlated with cyclin E level and the out-of-phase trait while the MEred module was positively correlated with the effect of progesterone. We identified 17 hub genes in the MEred module. The functional enrichment analysis indicated that such hub genes were mainly involved in pathways related to cellular defense response and natural killer (NK) cell-mediated cytotoxicity. In the MEbrown module, we identified 19 hub genes, which were mainly enriched in cell adhesion molecule production, regulation of cellular response to growth factor stimulus, epithelial cell proliferation, and transforming growth factor-ß (TGF-ß) signaling pathway. In addition, the hub genes were validated by using other datasets and three true hub genes were finally obtained, namely DOCK2 for the MEred module, and TRMT44 and ERVMER34-1 for the MEbrown module. In conclusion, our results screened potential biomarkers that might contribute to the diagnosis and treatment of REPL.

Association of IL-1ß, IL-6, TNF-α, and TGFß1 Gene Polymorphisms with Recurrent Spontaneous Abortion in Polycystic Ovary Syndrome.

Recurrent spontaneous abortion (RSA) is a common pregnancy-associated complication of polycystic ovary syndrome (PCOS) which is an endocrine malfunction disease. Patients with PCOS may have several underlying contributing and interrelated factors, which have been reported in women with RSA. The incidence rate between PCOS and RSA remains uncertain. The aim of this study is to determine the possible association of IL-1ß-511C/T, IL-6-174G/C, TNF-α-1031T/C, and TGFß1-509T/C with RSA patients with or without PCOS. A total of 140 RSA patients, 70 of which were PCOS patients, and 140 healthy females with no history of RSA or PCOS were included in this study. PCR amplification, genotyping, and sequence analysis were employed to investigate the presence of the polymorphisms. The genotypic and allelic frequencies were calculated separately for each subject. Out of the four studied polymorphisms, the IL-1ß-511C/T genotype in RSA without PCOS patients (12.7%) was significantly different compared with that in control subjects (p = 0.047). For IL-6-174C/G, there was a tendency towards more CC carriers among RSA with PCOS patients (10%) than in controls (3%). The GG genotype in RSA women with PCOS (60%) was significantly different compared with that in control subjects (p = 0.033), and the GC genotype in RSA with PCOS patients (30%) showed a marginal significant difference compared with that in control subjects (p = 0.050). Significant difference was identified in the allelic frequencies in RSA patients with PCOS compared to controls (p = 0.025). IL-6-174G/C and TNF-α-1031T/C polymorphisms are significantly associated with RSA patients in Saudi patients with PCOS, while the IL-1ß-511C/T polymorphism is significantly associated with RSA patients without PCOS.

Evaluation of 1,25(OH)2D3 Effects on FOXP3, ROR-γt, GITR, and CTLA-4 Gene Expression in the PBMCs of Vitamin D-Deficient Women with Unexplained Recurrent Pregnancy Loss (URPL).

Background: Vitamin D insufficiency and deficiency can be associated with adverse effects on fetus and pregnancy outcomes. This study aimed at evaluating the effect of 1,25VitD3 on specific transcription factor and markers of Tregs and T helper 17 (Th17) cells in peripheral blood mononuclear cells (PBMCs) of women with unexplained recurrent pregnancy loss (URPL) as a case group and PBMCs of healthy women as a control group. Methods: Samples from 20 non-pregnant patients with a history of URPL were compared to 20 normal non-pregnant women. PBMCs were divided into three wells for each subject in the presence of 1,25VitD3 (50 nM, for 16 hours), phytohemagglutinin (10 µM; positive control), and without any treatment (negative control). By Real-time PCR (Taqman assay), specific transcription factors of Tregs and Th17 cells, forkhead box P3 (FOXP3), retinoic acid-related orphan receptor γt (ROR-γt), glucocorticoid-induced tumor necrosis factor receptor-related (GITR), and CTLA-4 mRNA expressions in two groups were measured. Results: FOXP3/ROR-γt mRNA expression in PBMCs decreased significantly in women experiencing URPL compared to the control group (p = 0.0001). Although 1,25VitD3 (50 nM) increased FOXP3 gene expression (p = 0.0001), it did not significantly affect ROR-γt gene expression. Besides, 1,25VitD3 treatment significantly increased FOXP3/ROR-γt mRNA expression from baseline in PBMCs of the fetal loss group compared to that of the control group (p = 0.01). The 1,25VitD3 also increased GITR gene expression (p = 0.017) in PBMCs of URPL women compared to the controls. Conclusion: Vitamin D deficiency may be a contributor to recurrent pregnancy loss and suggests that the supplementation of women with Vitamin D pre-pregnancy may be protective against URPL via affecting Tregs signature genes, FOXP3 and GITR.

Diminished ovarian reserve in recurrent pregnancy loss: a systematic review and meta-analysis.

OBJECTIVE: To evaluate the association between diminished ovarian reserve (DOR) in women at risk of recurrent pregnancy loss (RPL) using ovarian reserve tests. DESIGN: Systematic review and meta-analysis. SETTING: University medical schools. PATIENT(S): Women with a history of RPL. INTERVENTION(S): Systematic reviews of major electronic databases (MEDLINE, EMBASE, Web of Science, and Scopus) for studies that evaluated the incidence of DOR in women with RPL. MAIN OUTCOME MEASURE(S): Association between RPL and DOR. RESULT(S): In studies up to May 2019 we assessed quality using the Newcastle-Ottawa Scale and meta-analyzed data using a random-effect model. We included 15 studies (n = 3,082 women) reporting on six ovarian reserve tests: antimüllerian hormone [AMH], antral follicle count, follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol, and FSH:LH ratio. More women with RPL seemed to have DOR compared with women who did not have RPL as measured by low AMH levels (odds ratio [OR] 2.77; 95% confidence interval [CI], 1.41-5.46) and AFC (OR 2.45; 95% CI, 1.16-5.19). Women with unexplained RPL also seemed to have a higher association with DOR compared with women whose RPL had a known etiology, as measured by low AMH levels (OR 3.23; 95% CI, 1.81-5.76). No statistically significant differences were found in the levels of any of the remaining ovarian reserve tests between those groups of women. CONCLUSION(S): There is an apparent association between DOR and RPL. Low AMH and AFC levels could predict higher odds for pregnancy loss, but more studies are needed to evaluate their prognostic value in the management of women with RPL. SYSTEMATIC REVIEW REGISTRATION NUMBER: Prospero CRD42018114673.

NK cell subsets in idiopathic recurrent miscarriage and renal transplant patients.

The present review article compares NK cell subsets and cytokine patterns determined in the peripheral blood as well as results of functional in-vitro assays using peripheral NK cells of idiopathic recurrent miscarriage (iRM) patients with corresponding results obtained in female healthy controls and female renal transplant recipients with good long-term graft function. Immune mechanisms, inducing transplant rejection in long-term transplant recipients might also be able to induce rejection of semi-allogeneic fetal cells in patients with iRM. Consequently, the immune status of transplant recipients with good stable long-term graft function should be different from the immune status of iRM patients. iRM patients show a strong persistent cytotoxic NK cell response in the periphery. Simultaneously, immunostimulatory Th1 as well as immunosuppressive Th2 type lymphocytes in the blood are strongly activated but plasma levels of immunosuppressive Th2 type cytokines are abnormally low. In-vitro, unstimulated NK cell cultures of iRM patients show a strong spontaneous TGF-ß1 release in the supernatant but lower TGF-ß1 levels after stimulation with tumor cell line K562, suggesting strong consumption of TGF-ß1 by pre-activated NK cells of iRM patients that might contribute to the low systemic Th2 type plasma levels. iRM patients do not show a systemic switch to a Th2 type cytokine pattern and one might hypothesize that low TGF-ß plasma levels indicate low TGF-ß levels in the micromilieu immediately before fetal rejection. Persistent TGF-ß deficiency implies a persistent unfavorable micromilieu for pregnancy resulting in failing tolerance induction due to lack of TGF-ß, a condition that might contribute to iRM.

Maternal history of recurrent pregnancy loss and long-term risk of thromboembolic events.

BACKGROUND: Recurrent pregnancy loss is a multifactorial disorder with potential underlying maternal hypercoagulability. OBJECTIVE: We aimed to investigate whether a history of recurrent pregnancy loss poses an independent risk for future maternal long-term thromboembolic morbidity. STUDY DESIGN: A population-based study compared the incidence of long-term thromboembolic morbidity in a cohort of women with and without a history of recurrent pregnancy loss. Data were collected from two databases that were cross-linked and merged: a computerized hospitalization database and a computerized obstetrics and gynecology perinatal database. Deliveries occurred between the years 1991-2017. The risk for long-term thromboembolic morbidity was based on the hospital's database and a pre-defined set of ICD codes of thromboembolic related hospitalizations. A Kaplan-Meier survival curve was constructed to compare the cumulative incidence of significant thromboembolic morbidity in the exposed and unexposed groups, and a Cox proportional hazards model, to control for confounders. RESULTS: During the study period, 123,791 parturients met the inclusion criteria; 6.7 % n = 8247 of which experienced previous recurrent pregnancy loss-the exposed group. The rate of thromboembolic related hospitalizations was almost double in exposed women as compared to non-exposed 1.1 % vs. 0.6 %, OR = 1.8; 95 % 1.4-2.2; p < 0.001). Cumulative thromboembolic event incidence, as depicted in the survival curves, was also significantly higher among patients with recurrent pregnancy loss history (Log rank p < 0.001). The Cox model confirmed recurrent pregnancy loss history to be an independent risk factor for later maternal thromboembolic morbidity, while controlling for maternal age, known thrombophilia, ethnicity, smoking, hypertension, diabetes, and fertility treatment (aHR 1.27, 95 %CI 1.02-1.59, p=0.034). CONCLUSION: A history of recurrent pregnancy loss is an independent risk factor for long-term thromboembolic morbidity, even in the absence of known maternal thrombophilia.

Placenta specific protein-1 in recurrent pregnancy loss and in In Vitro Fertilisation failure: a prospective observational case-control study.

Observations from studies have provided evidence that Placenta-specific protein1 (PLAC1) is important for the establishment and maintenance of pregnancy and suggest it as a potential biomarker for gestational pathologies. The aim of this study is to investigate whether maternal serum PLAC1 levels have any impact on etiopathogenesis of recurrent pregnancy loss (RPL) and repeated implantation failure after In Vitro Fertilisation (RIF). We conducted a prospective observational case-control study in a Research Hospital. Twenty-eight patients with RPL (group 1), 30 patients with unexplained infertility and RIF (group 2), 29 fertile patients (group 3) were included. The demographic features and serum PLAC1 levels were compared. There was a significant difference in PLAC1 levels between the groups (group 1 = 19.71 + 16.55 ng/ml; group 2 = 4.82 + 1.44 ng/ml; group 3 = 0.89 + 0.62 ng/ml, respectively) (p=.001). Positive correlation was found between serum PLAC1 levels and abortion rates (r = 0.64; p=.001), a negative correlation was found between serum PLAC1 levels and live birth rates (r = -0.69; p=.001). PLAC1 might have a negative effect on implantation in RPL and RIF. There may be a subgroup of PLAC with different bioactivity. There are no relevant studies conducted among these populations, further large-scale studies are needed to assess the molecular role of PLAC1 on implantation.IMPACT STATEMENTWhat is already known about this subject? PLAC1 (placenta-specific protein-1) gene is located on the X chromosome which encodes for a protein that is thought to be important for placental development although its role has not been clearly defined. Studies in the literature have provided evidence that PLAC1 has an important role in the establishment and maintenance of pregnancy and suggest it as a potential biomarker for gestational pathologies. Several reports over the past few years have demonstrated PLAC1 expression in a variety of human tumours including lung cancers, breast cancer, hepatocellular and colorectal cancers, gastric cancers and uterine cancers.What do the results of this study add? There have been no previous studies conducted among patients with recurrent pregnancy loss (RPL) or repeated implantation failure after In Vitro Fertilisation (RIF) that have searched for any association between PLAC1 levels and implantation failure. This study has demonstrated higher PLAC1 levels in infertile women with RIF and RPL for the first time; suggesting that it could have a negative effect on implantation in these populations. PLAC1 could be detected in the serum as a biomarker that is associated with RIF and RPL. What are the implications of these findings for clinical practice and/or further research? Defining the precise role of PLAC1 during implantation will provide new insight into understanding of poor reproductive outcomes such as RIF and RPL and help in developing treatment strategies. Further large-scale studies with more patients are needed to uncover the clinical value of PLAC1 as a biomarker to predict repeated implantation failure and RPL.

Evaluation of peripheral and uterine immune status of chronic endometritis in patients with recurrent reproductive failure.

OBJECTIVE: To investigate whether chronic endometritis (CE) affects the immune status of peripheral blood and endometrium in patients with recurrent reproductive failure (RRF). DESIGN: Retrospective study. SETTING: Private fertility center. PATIENTS(S): A total of 524 RRF patients, including 324 women with recurrent miscarriage (RM) and 200 women with recurrent implantation failure (RIF). INTERVENTION(S): Peripheral blood and endometrium samples were collected in the midluteal phase before in vitro fertilization treatment or pregnancy. The number of peripheral T, natural killer (NK), and B cells, as well as cytotoxicity of NK cells and expression of TH1 cytokines were analyzed with the use of flow cytometry, and uterine immune cells were subjected to immunohistochemistry. MAIN OUTCOME MEASURE(S): Peripheral immune cells, cytokines, NK cytotoxicity, and endometrial immune cells were compared in RRF patients with versus without CE. RESULT(S): The proportion and function of the analyzed immune cell subsets in peripheral blood as well as the percentages of CD56+ NK cells, CD163+ M2 macrophages, and CD1a+ immature dendritic cells in the endometrium were not significantly altered between non-CE and CE patients, whereas the proportions of uterine CD68+ macrophages, CD83+ mature dendritic cells, CD8+ T cells, and Foxp3+ regulatory T cells were significantly elevated in CE patients. After antibiotic treatment, the percentage of CD68+ macrophages, CD83+ mature dendritic cells, CD8+ T cells, and Foxp3+ regulatory T cells in endometrium were significantly reduced in patients with cured CE. CONCLUSION(S): CE contributes to elevated endometrial infiltration levels of immune cells. The excessive presence of endometrial immune cells in CE patients may be involved in reduced endometrial receptivity and recurrent pregnancy failures.

CORRELATION BETWEEN LEVELS OF HOMOCYSTEINE, ANTI-MÜLLERIAN HORMONE AND INSULIN RESISTANCE IN PCOS PATIENTS WITH RECURRENT MISCARRIAGE.

There are controversial opinions regarding the impact of hyperhomocysteinemia (HHcy) and insulin resistance in PCOS patients who experience Recurrent Pregnancy Loss (RPL). Nowadays the correlation between levels of anti-müllerian hormone (AMH), homocysteine and insulin resistance (IR) have become the main subject of interest in PCOS patients for predicting RPL. Objective - investigate the relationship between level of homocysteine, anti-müllerian hormone and insulin resistance in PCOS patients with Recurrent Pregnancy Loss. 80 Georgian young women (<30 years) with PCOS were involved in the prospective study. The diagnosis of PCOS was based on the criteria of Rotterdam Consensus 2003. Patients were divided into two groups: group I-50 patients, who experienced RPL, and group II-30 patients with live births in anamnesis (control group). Patients with RPL were divided into two subgroups: subgroup A-with insulin resistance (n=28), and subgroup B-without insulin resistance (n=22). All patients underwent hormonal investigation from day 2 to 3 of menstrual cycle. Plasma levels of anti-müllerian hormone, homocysteine, follicle-stimulating hormone, luteinizing hormone (LH), total testosterone (T), free testosterone (FT), sex hormone binding globulin (SHBG) were determined. Between 2-3 days of the menstrual cycle all participants underwent an ultrasound examination using Voluson E10. This was done to determine ovarian volume and antral follicle count. Average homocysteine (Hcy) level in PCOS patients with RPL (11.5±2.24µmol/l) was significantly higher than in controls (7.55±2.45 µmol/l, p<0.001). Incidence of (HHcy) and IR in patients with RPL was 70% and 56% respectively, which was significantly higher than in controls (HHcy-54.3%; IR- 9.4%; p<0.0001). HOMA-IR in patients with RPL was significantly higher compared with controls (p<0.001). Average AMH level in patients with RPL and live births did not differ significantly. In the group of PCOS with RPL significant positive correlation between Hcy and HOMA-IR, BMI, AMH and FT levels was found (p<0.001). Serum homocysteine level is elevated in PCOS patients with RPL. This elevation is correlated with the degree of obesity, BMI, Insulin Resistance status, AMH and androgen levels. The treatment of hyperhomocysteinemia and insulin resistance in women with PCOS might become the bases for prevention of pregnancy losses and improving reproductive outcomes.