Low chorionic villous succinate accumulation associates with recurrent spontaneous abortion risk.

Dysregulated extravillous trophoblast invasion and proliferation are known to increase the risk of recurrent spontaneous abortion (RSA); however, the underlying mechanism remains unclear. Herein, in our retrospective observational case-control study we show that villous samples from RSA patients, compared to healthy controls, display reduced succinate dehydrogenase complex iron sulfur subunit (SDHB) DNA methylation, elevated SDHB expression, and reduced succinate levels, indicating that low succinate levels correlate with RSA. Moreover, we find high succinate levels in early pregnant women are correlated with successful embryo implantation. SDHB promoter methylation recruited MBD1 and excluded c-Fos, inactivating SDHB expression and causing intracellular succinate accumulation which mimicked hypoxia in extravillous trophoblasts cell lines JEG3 and HTR8 via the PHD2-VHL-HIF-1α pathway; however, low succinate levels reversed this effect and increased the risk of abortion in mouse model. This study reveals that abnormal metabolite levels inhibit extravillous trophoblast function and highlights an approach for RSA intervention.

Abnormal Expression of Indoleamine 2, 3-Dioxygenase in Human Recurrent Miscarriage.

Indoleamine 2, 3-dioxygenase (IDO), an immunosuppressive enzyme that mediates the conversion of tryptophan to kynurenine, was shown to play a key role in placental development during normal pregnancy. However, little is known about the pattern of IDO expression in the endometrium and its attendant functional significance in pregnancies complicated with recurrent miscarriage (RM). Immunohistochemical studies of IDO, Foxp3, CD56, and CD163 expression were performed in endometrial samples from women with RM and healthy fertile controls. Our study found that IDO was localized in glandular epithelial cells, surface epithelial cells, and a small number of cells within the stromal compartment (including stromal cells and leukocytes) in endometrium. Indoleamine 2, 3-dioxygenase expression in the RM group was significantly lower than control group. The Foxp3 and CD56 expression were significantly increased with the elevated IDO expression in controls but not in RM. The percentage of Foxp3 + Tregs was significantly correlated with the level of IDO expression in the control group. Comparatively, no correlation was found between the percentage of CD56 + cells, CD163 + cells, and the level of IDO expression, no matter in controls and RM patients. This study demonstrated that the downregulation of IDO expression and noncoordinated association between IDO and other endometrial immune cells were associated with RM. Our findings provide insights into the contribution of IDO in immune regulation to maintain normal pregnancy, which could be used to develop potential therapeutic methods for RM.

The association between maternal methylenetetrahydrofolate reductase C677T and A1298C polymorphism and birth defects and adverse pregnancy outcomes.

Published studies indicate the MTHFR C677T and A1298C polymorphisms are associated with abnormal homocysteine levels, which may cause various pregnancy complications and birth defects. However, the results obtained from different studies have been inconsistent. Therefore, this meta-analysis explores the association between MTHFR polymorphisms and birth defects and adverse pregnancy outcomes. The PubMed, ScienceDirect, Embase, and China Biology Medicine literature databases and ClinicalTrials were searched. Analyses of public bias, meta-regression, subgroups, and sensitivity were used to ensure the robustness of our results. MTHFR C677T was significantly associated with recurrent pregnancy loss in developing countries (odds ratio [OR], 1.34; 95% confidence interval [CI], 1.20-1.50) but not in developed countries (OR, 0.87; 95% CI, 0.68-1.11). No significant relationship was found between MTHFR A1298C and recurrent pregnancy loss (OR, 1.04; 95% CI, 0.93-1.18). MTHFR C677T and A1298C were not associated with preeclampsia (OR, 1.06; 95% CI, 0.97-1.16 and OR, 1.16; 95% CI, 0.97-1.39, respectively), and C677T was not associated with placental abruption (OR, 1.03; 95% CI, 0.87-1.21), intrauterine growth retardation (OR, 1.02; 95% CI, 0.90-1.15), or congenital heart disease (OR, 1.05; 95% CI, 0.89-1.25). MTHFR C677T, but not A1298C, was associated with neural tube defects (OR, 1.24; 95% CI, 1.08-1.42) and Down syndrome (OR, 1.65; 95% CI, 1.39-1.95). CONCLUSION: Although MTHFR C677T and A1298C are significantly associated with some types of congenital defects and adverse pregnancy outcomes, the impact of these polymorphisms is moderate.

Role of cathepsin E in decidual macrophage of patients with recurrent miscarriage.

In a previous study, we reported that the cathepsin-cystatin system caused endometrial dysfunction in early pregnancy. Here, we investigated the existence and contribution of cathepsin E in early pregnancy in patients with recurrent miscarriage (RM). The effect of cathepsin deficiency on fertility and female reproductive organs were also analyzed in CatE(-/-) mice. Human studies were conducted in a hospital setting, with informed consent. Cervical mucus was collected from RM patients in early pregnancy (4-6 gestational weeks, n = 21), and the pregnancy outcome was compared prospectively. The cathepsin E expression in decidua of RM patients (n = 49) and normal pregnant women undergoing elective surgical abortion (n = 24) was measured using SDS-PAGE, and western blot analysis. Decidual macrophages were isolated from RM patients (n = 6) and stimulated by lipopolysaccharide (LPS) and interferon gamma (IFN-γ). Results from the mouse model showed that CatE(-/-) mice were fertile, but the litter number was significantly smaller. The uterus of CatE(-/-) mice showed granulation tissue. In human samples, protease activity of cathepsin E measured with Fluorescence-Quenching Substrate (KYS-1) in cervical mucus of patients who developed miscarriage was markedly decreased compared with patients without RM. The expression of cathepsin E in decidua, semi-quantified by SDS-PAGE, western blot analysis was significantly lower in RM patients compared with patients without RM. By double staining immunofluorescence, the staining of cathepsin E was observed in CD14 or CD68 positive cells in all deciduas. Upon stimulation with LPS and IFN-γ, the expression of cathepsin E in cell lysate of decidual macrophages was markedly reduced in RM patients compared with controls. The results suggested that decreased activity of cathepsin E produced by decidual macrophages might be responsible for the induction of miscarriages in some RM patients.

Phosphodiesterase 8B gene polymorphism in women with recurrent miscarriage: a retrospective case control study.

BACKGROUND: Recurrent miscarriage affects approximately 1% of all couples. There is a known relation between hypothyroidism and recurrent miscarriage. Phosphodiesterase 8B (PDE8B) is a regulator of cyclic adenosine monophosphate (cAMP) with important influence on human thyroid metabolism. Single nucleotide polymorphism (SNP) rs 4704397 in the PDE8B gene has been shown to be associated with variations in serum Thyroid Stimulating Hormone (TSH) and thyroxine (T4) levels. The aim of this study was to investigate whether there is an association between the SNP rs 4704397 in the PDE8B gene and recurrent miscarriage. METHODS: The study was designed as a retrospective case control study. 188 cases with recurrent miscarriage were included and compared with 391 controls who had delivered at least once and with no history of miscarriage or assisted reproduction. RESULTS: No difference between cases and controls concerning age was found. Bivariate associations between homozygous A/A (OR 1.57, 95% CI 0.98-2.52) as well as G/G carriers (OR 1.52, 95% CI 1.02-2.25) of SNP rs 4704397 in PDE8B and recurrent miscarriage were verified (test for trend across all 3 genotypes, p=0.059). After adjustment for known confounders such as age, BMI and smoking the association between homozygous A/A (AOR 1.63, 95% CI 1.01-2.64, p=0.045) and G/G (AOR 1.52, 95% CI 1.02-2.27, p=0.039) carriers of SNP rs 4704397 in PDE8B and recurrent miscarriage remained. CONCLUSIONS: Our findings suggest that there is an association between homozygous A/A as well as homozygous G/G carriers of SNP rs 4704397 in PDE8B and recurrent miscarriage.

Role of cyclooxygenase-2 signaling pathway dysfunction in unexplained recurrent spontaneous abortion.

BACKGROUND: Experimental evidence indicates that cyclooxygenase-2 (COX-2) plays a critical role in blastocyst implantation; however, little is known of the role of COX-2 in unexplained recurrent spontaneous abortion (URSA). METHODS: We evaluated the expression level and potential signaling pathway of COX-2 in 30 cases of URSA who were excluded the abnormality of chromosomes, anatomy, endocrine, infectious, autoimmune diseases and in 30 normal pregnancies. RESULTS: The mRNA and the protein expression level of COX-2 in the URSA group (-0.238 +/- 0.848, 0.368 +/- 0.089, respectively) were significantly lower than that in the control group (1.943 +/- 3.845, 1.046 +/- 0.108, respectively) (both, P < 0.01). The expression of prostaglandins PGF(2a), PGD(2), PGE(2), and PGI(2), in the URSA group ((2326.0 +/- 295.6) pg/ml, (2164.0 +/- 240.5) pg/ml, (238.7 +/- 26.4) pg/ml, (2337.0 +/- 263.0) pg/ml, respectively) were significantly lower than that in the control group ((3450.0 +/- 421.7) pg/ml, (3174.0 +/- 415.6) pg/ml, (323.5 +/- 43.8) pg/ml, (3623.0 +/- 460.4) pg/ml, respectively) (P < 0.05). The mRNA expression level of PPARbeta and RXRalpha (0.859 +/- 0.653, -0.172 +/- 0.752, respectively) in URSA group was significantly lower than that in the control group (1.554 +/- 1.735, 0.777 +/- 2.482, respectively) (both P< 0.05). The mRNA and protein expression levels of vascular endothelial growth factor-A (VEGF-A) in the URSA group (2.010 +/- 1.522, 0.35 +/- 0.46) was significantly lower than that in the control group (4.569 +/- 2.430, 0.750 +/- 0.350) (both P < 0.05). CONCLUSIONS: COX-2 and the COX-2-derived PGI(2) signaling pathway possibly play an important role in successful embryo implantation, and their decreased expression may result in URSA. The decreased expression may influence the expression of VEGF-A which interferes with placental angiogenesis causing failure of embryo implantation, leading to spontaneous abortion.

Feto-maternal ACP1 activity ratio and intrauterine survival.

OBJECTIVE: Genetic differences in the activity of phosphotyrosine phosphatases between mother and embryo could result in a differential activation of signals induced by growth factors in the two sides of placenta. Previous observations suggest that this may have important effects on intrauterine development and survival. The aim of the present study is to confirm previous observations and show new data. STUDY DESIGN: We have studied 573 mother/newborn pairs, 169 wife/husband couples with repeated spontaneous abortion and 34 fertile wife/husband couples RESULTS: In mother/newborn pairs, the analysis of joint mother/infant ACP1 distribution has shown a deficit of pairs with the mother having low ACP1 S isoform concentration and the infant having high S isoform concentration, and an excess of pairs with the mother having high S isoform concentration and the infant having low S isoform concentration. In RSA couples there is an excess of couples in which the wife has low S isoform concentration and the husband has high S isoform concentration and a deficit of couples in which the wife has high S isoform concentration and the husband has low S isoform concentration. In fertile couples the pattern is reversed. CONCLUSION: The data suggest that when the mother to fetus S isoform concentration ratio is in favour of the mother, the probability of survival of the fetus is greater than in the opposite situation.

Tumor necrosis factor-alpha and oxidant status are essential participating factors in unexplained recurrent spontaneous abortions.

BACKGROUND: Many factors have been implicated in the pathogenesis of unexplained recurrent spontaneous abortion (URSA). The current study was conducted to determine the possible role of antioxidant status and tumor necrosis factor-alpha (TNF-alpha) in URSA. METHODS: Reduced glutathione (GSH), glutathione reductase (GSH-R), glutathione peroxidase (GSH-PX), catalase (CAT), superoxide dismutase (SOD), nitric oxide (NO), malondialdehyde (MDA) and TNF-alpha were assayed in women suffering unexplained first-trimester abortions. Two groups were included, the first represented by 24 women with URSA (number of abortions 3-5) and the second included 16 women with URSA (number of abortions >5). The control group included 20 women within their first trimester of pregnancy and 20 non-pregnant healthy females within their follicular phase. RESULTS: We observed that the antioxidant levels measured were significantly lower in URSA groups than in the control group (p<0.05 for each comparison). Higher TNF-alpha, MDA and NO production were detected in URSA groups compared to controls (p<0.05 for each comparison). URSA 3-5 was associated with significantly higher levels of antioxidants and lower levels of TNF-alpha compared to levels in URSA >5. CONCLUSIONS: Impaired antioxidant defense and an increase in oxidative reactive species may be responsible for recurrent abortion due to possible damage produced by their generation. In addition, the level of TNF-alpha apparently contributes to the pathogenesis of URSA.

Role of cathepsins and cystatins in patients with recurrent miscarriage.

In the implantation, trophoblasts penetrate maternal decidua by secreting proteases. It has been reported that cathepsins are highly expressed in the mouse villi, and play an important role in normal embryonal growth and decidualization. In this study, we evaluated cathepsins and their endogenous inhibitors, cystatins, in tissue and serum of patients with recurrent miscarriage. Decidua and villi were surgically collected from 22 patients and 12 healthy women. Immunohistochemistry was performed with antibodies against cathepsins, stefin A (cystatin A), stefin B (cystatin B) and cystatin C. The concentrations of cathepsins, stefins and cystatin C were measured by Enzyme-linked immunosorbent assay. In addition, we measured the serum level of cystatin C in 85 Japanese women with recurrent miscarriage. Staining of cathepsin B, D, H, L, stefin B and cystatin C was observed in the cytoplasm of epithelial cells in decidua. Stefin A was expressed on the surface of the trophoblast. The concentration of cathepsin B and H in patients' decidua was significantly higher than in control individuals. The serum level of cystatin C was significantly lower in patients than in control individuals. Our findings suggest that the regulation of the cathepsin-cystatin system may play an important role in patients with recurrent miscarriage.

Association of anticardiolipin antibody and C677T in methylenetetrahydrofolate reductase mutation in women with recurrent spontaneous abortions: a new path to thrombophilia?

CONTEXTO: O aborto espontâneo recorrente já foi associado a vários fatores etiológicos e à trombose materna. Fatores de trombofilia adquiridos e hereditários podem ser causas destes eventos. OBJETIVO: Avaliar a associação entre fatores de trombofilia e aborto espontâneo recorrente. IPO DE ESTUDO: Caso-controle. LOCAL: Centro de Atenção Integral à Saúde da Mulher, Universidade Estadual de Campinas, São Paulo, Brasil. MÉTODOS: Foram retirados 40 ml de sangue de 88 mulheres com aborto espontâneo recorrente do Ambulatório de Aborto Recorrente e 88 mulheres férteis do Ambulatório de Planejamento Familiar, pareadas por idade e raça, para pesquisar fatores de trombofilia adquiridos e hereditários. O anticorpo anticardiolipina (ACA), anticoagulante lúpico (LA) e a deficiência das proteínas C, S e antitrombina III foram pesquisados por ELISA (enzyme-linkek immunosorbent assay), dRVVT (dilute Russel Viper Venom Time, dRVVT) e métodos coagulométrico e cromogênico. O ácido desoxirribonucléico (DNA) foi amplificado pela técnica de reação em cadeia da polimerase (PCR) para o estudo das mutações fator V de Leiden, G20210A no gene da protrombina e C677T no gene da enzima metilenotetrahidrofolato redutase (MTHFR). RESULTADOS: O ACA foi encontrado em 11 mulheres com RSA e em uma mulher fértil [OR 12.4 (IC 95% 1.5 a 98.5)]. A mutação C677T heterozigota foi encontrada em 59 mulheres com RSA e em 35 mulheres férteis [OR 3.1 (IC 95% 1.7 a 5.7)]. A presença concomitante do ACA e da mutação C677T heterozigota foi encontrada em oito mulheres com aborto espontâneo recorrente e em nenhuma mulher fértil (p < 0,01). DISCUSSÃO: O significado da associação entre a mutação heterozigota C677T no gene da MTHFR e o anticorpo anticardiolipina não está claro. Pode-se supor que um fator hereditário que, isolado, não predisporia fortemente um indivíduo à trombose poderia, em associação com um fator adquirido, deflagrar o processo e intensificar a expressão da trombose. CONCLUSÕES: O ACA e a mutação C677T heterozigota no gene da MTHFR apresentaram associação estatística com RSA. A concomitância destas duas alterações é um novo achado no estudo de fatores trombogênicos em aborto espontâneo recorrente.

Adenosine deaminase activity and its isoenzyme pattern in women with recurrent spontaneous abortions.

Normal pregnancy is characterized by suppressed cell-mediated immunity. Adenosine deaminase (ADA) is a purine metabolic enzyme enriched in trophoblast cells of the placenta. It is an early marker of trophoblast cell differentiation. Also, the activation of ADA gene expression in the placenta is crucial and essential for proper fetal development. The activity of ADA shows changes in diseases characterized by the alteration of cell-mediated immunity. The purpose of this study was to assess the possible role of the alteration of cell-mediated immunity in women with recurrent spontaneous abortions (RSA) as a cause of changes in tADA activity, and also to evaluate the extent of the contribution of ADA1 and ADA2 to changes of tADA activity in serum and peripheral blood lymphocytes (PBLs). We measured in serum and in PBLs activities of tADA, ADA1 and ADA2 of 25 married women with RSA (group A) and of 28 healthy non-pregnant women (group B). According to our results in women with RSA, mean serum tADA, ADA1 and ADA2 activities were significantly higher than those of non-pregnant women (p < 0.001, p < 0.05 and p < 0.05 respectively). In women with RSA, mean PBLs tADA, ADA1 and ADA2 activities were significantly higher than those of non-pregnant women (p < 0.001, p < 0.05 and p < 0.05 respectively). The findings of this study show a marked increase of serum and PBLs ADA activities, which is derived from an increase of ADA2 and ADA1 activity in women with RSA. These changes reflect cell-mediated immunological changes.

Changes in regulation of oxidase activity of peripheral blood granulocytes in women with habitual abortions.

Generation of active oxygen forms by blood granulocytes was studied in women with a history of habitual abortions (2-3 spontaneous abortions in the first trimester, undeveloped pregnancies). The level of spontaneous luminol-dependent chemiluminescence of nonfractionated peripheral blood was increased in this patient population (study group) in comparison with women with normal reproductive function (reference group). The two groups differed by the level of activation of respiratory burst induced by opsonized zymosan and by activity of isolated granulocytes in response to chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (1-50 microM). Differences in the effects of inhibitor of tyrosine protein kinases and protein phosphatases and inhibitor of mitogen-activated proteinkinase p38 MAPK were detected. The results attest to predisposition to oxidative stress and poor cytotoxic functions of granulocytes in women with habitual abortions, which can be due to specific features of regulation of oxidase activity by tyrosine protein kinases and protein phosphatases and by p38 MAPK.

Neonatal and fetal methylenetetrahydrofolate reductase genetic polymorphisms: an examination of C677T and A1298C mutations.

Methylenetetrahydrofolate reductase (MTHFR) mutations are commonly associated with hyperhomocysteinemia, and, through their defects in homocysteine metabolism, they have been implicated as risk factors for neural tube defects and unexplained, recurrent embryo losses in early pregnancy. Folate sufficiency is thought to play an integral role in the phenotypic expression of MTHFR mutations. Samples of neonatal cord blood (n=119) and fetal tissue (n=161) were analyzed for MTHFR C677T and A1298C mutations to determine whether certain MTHFR genotype combinations were associated with decreased in utero viability. Mutation analysis revealed that all possible MTHFR genotype combinations were represented in the fetal group, demonstrating that 677T and 1298C alleles could occur in both cis and trans configurations. Combined 677CT/1298CC and 677TT/1298CC genotypes, which contain three and four mutant alleles, respectively, were not observed in the neonatal group (P=.0402). This suggests decreased viability among fetuses carrying these mutations and a possible selection disadvantage among fetuses with increased numbers of mutant MTHFR alleles. This is the first report that describes the existence of human MTHFR 677CT/1298CC and 677TT/1298CC genotypes and demonstrates their potential role in compromised fetal viability.

Phosphotyrosine-protein-phosphatases and human reproduction: an association between low molecular weight acid phosphatase (ACP1) and spontaneous abortion.

ACP1 (low molecular weight acid phosphatase) genetic polymorphism has been studied in 173 women with a history of two or more consecutive spontaneous abortions and in 1508 control subjects, including 482 normal pregnant women. The proportion of carriers of ACP1*C allele (*A/ *C, *B/*C) in women with a history of repeated spontaneous abortion is lower than in normal pregnant women and other control groups. Women with repeated spontaneous abortion show a specific decrease of ACP1 S isoform concentration as compared to normal pregnant women. The other component of ACP1 activity, the F isoform, does not show a significant difference between the two groups. The data suggest that women with ACP1 genotypes showing a high concentration of S isoform are relatively 'protected' against spontaneous abortion. Preliminary analysis of a sample of 352 normal puerperae along with their newborn babies supports this hypothesis.

[Contents of prostaglandins E2 and F2alpha and cAMP in the endometrium of women with habitual abortion during late pregnancy terms]. / Soderzhanie prostaglandinov E2 i F2 alfa i tsAMF v éndometrii zhenshchin, stradaiushchikh privychnym nevynashivaniem beremenosti pozdnikh srokov.

Measurements of prostaglandins E2 and F2 alpha in the endometrium of 35 women of a reproductive age, suffering from habitual abortions in late pregnancy terms, have shown elevation of these prostaglandin levels in the secretory phase as against the reference group and their normal values during the proliferative phase. It is possible, that disordered prostaglandin synthesis in the endometrium, manifesting in pregnancy, is one of the causes of habitual abortions. cAMP levels were somewhat decreased in the secretory phase in this patient population; this permits a hypothesis on disorders in the adenylate cyclase system.