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Introduction: Vitamin D catabolizing enzymes, along with vitamin D receptor (VDR) and vitamin D binding protein (DBP) are expressed in the decidua and placenta during pregnancy and capable of synthesizing active vitamin D. Vitamin D plays roles in immunoregulation and trophoblast invasion, key features of a successful pregnancy. Epidemiological data suggests that vitamin D deficiency is associated with both spontaneous and recurrent miscarriage but few studies have investigated the expression of the key vitamin D catabolizing enzymes in miscarriage., Methods: Placenta and decidua were collected after termination of apparently normal pregnancies (controls, n = 22) or spontaneous miscarriage (n = 20). Immunohistochemical staining, Western Blot and qRT-PCR were performed for CYP27B1, CYP24A1, CYP2R1, VDR and DBP (not qRT-PCR). HTR-8/SVneo cells were cultured in CoCL 2 (hypoxic mimetic) or LPS (bacterial infection mimetic) for 24 h, RNA extracted and qRT-PCR performed for CYP27B1, CYP24A1, CYP2R1 and VDR., Results: In spontaneous miscarriage, placental and decidual expression of CYP27B1 was reduced, while expression of CYP24A1, VDR and DBP was increased. When a trophoblast cell line was treated with CoCL 2 expression of CYP27B1 was increased and CYP24A1 was reduced, while LPS induced expression of VDR., Discussion: This is the first report of altered utero-placental vitamin D catabolism in spontaneous miscarriage. It is becoming accepted that women who are undergoing assisted reproductive technologies should ensure they have sufficient vitamin D levels prior to pregnancy, these data support that all women should ensure they are vitamin D replete before planning to get pregnant., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Prostaglandins (PGs) have critical signaling functions in a variety of processes including the establishment and maintenance of pregnancy, and the initiation of labor. Most PGs are non-enzymatically degraded, however, the two PGs most prominently implicated in the termination of pregnancy, including the initiation of labor, prostaglandin E2 (PGE 2 ) and prostaglandin F2α (PGF 2α ), are enzymatically degraded by 15-hydroxyprostaglandin dehydrogenase (15-HPGD). The role of PG metabolism by 15-HPGD in the maintenance of pregnancy remains largely unknown, as direct functional studies are lacking. To test the hypothesis that 15-PGDH-mediated PG metabolism is essential for pregnancy maintenance and normal labor timing, we generated and analyzed pregnancy in 15-HPGD knockout mice (Hpgd -/- ). We report here that pregnancies resulting from matings between 15-HPGD KO mice (Hpgd -/- X Hpgd -/- KO mating) are terminated at mid gestation due to a requirement for embryo derived 15-HPGD. Aside from altered implantation site spacing, pregnancies from KO matings look grossly and histologically normal at days post coitum (dpc) 6.5 and 7.5 of pregnancy. However, virtually all of these pregnancies are resorbed by dpc 8.5. This resorption is preceded by elevation of PGF 2∝ but is not preceded by a decrease in circulating progesterone, suggesting that pregnancy loss is a local inflammatory phenomenon rather than a centrally mediated phenomena. This pregnancy loss can be temporarily deferred by indomethacin treatment, but treated pregnancies are not maintained to term and indomethacin treatment increases maternal mortality. We conclude that PG metabolism to inactive products by embryo derived 15-HPGD is essential for pregnancy maintenance in mice, and may serve a similar function during human pregnancy.

Uterine angiogenesis and vascular remodeling play critical roles in determing the normal menstrual cycle and successful pregnancy. Poor uterine angiogenesis usually results in pregnancy failure. Protein O-fucosyltransferase 1 (poFUT1) is the key enzyme responsible for O-fucosylated glycan biosynthesis on glycoproteins. However, the dynamic expression and regulation of poFUT1 on the uterine angiogenesis and vascular remodeling remain unknown. Here, we showed that the enlargement of the vascular lumen in the secretory phase was greater than that in the proliferative phase of the uterine endometrium during menstrual cycle; whereas there was a narrower vessel lumen and fewer blood vessels in the decidua from miscarriage patients than in that from healthy pregnancy women. Additionally, the expression of poFUT1 was increased in the uterine endometrium during the secretory phase compared with that in the proliferation phase, and its expression was decreased in the uterus of miscarriage patients compared with that of the healthy pregnancy women. Using hESCs and a mouse model, we demonstrated that poFUT1 increased the O-fucosylation on uPA, and activated of the RhoA signaling pathway, thus facilitating uterine angiogenesis and vascular remodeling. We also provide evidence that poFUT1 promotes hESCs angiogenesis by the decreased stemness of hESCs. These findings reveal a new insight into the uterine angiogenesis and vascular remodeling. The study suggests that poFUT1 could be seen as a novel potential diagnostic and therapeutic target for miscarriage.

Here we examine recent evidence suggesting that many drugs and diet supplements (DS), experimental AMP-activated protein kinase (AMPK) agonists as well as energy-depleting stress, lead to decreases in anabolism, growth or proliferation, and potency of cultured oocytes, embryos, and stem cells in an AMPK-dependent manner. Surprising data for DS and drugs that have some activity as AMPK agonists in in vitro experiments show possible toxicity. This needs to be balanced against a preponderance of evidence in vivo that these drugs and DS are beneficial for reproduction. We here discuss and analyze data that leads to two possible conclusions: First, although DS and drugs that have some of their therapeutic mechanisms mediated by AMPK activity associated with low ATP levels, some of the associated health problems in vivo and in vitro fertilization/assisted reproductive technologies (IVF/ART) may be better-treated by increasing ATP production using CoQ10 (Ben-Meir et al., Aging Cell 14:887-895, 2015). This enables high developmental trajectories simultaneous with solving stress by energy-requiring responses. In IVF/ART, it is ultimately best to maintain handling and culture of gametes and embryos in the quietest state with low metabolic activity (Leese et al., Mol Hum Reprod 14:667-672, 2008; Leese, Bioessays 24 (9):845-849, 2002) using back-to-nature or simplex algorithms to identify optima (Biggers, Reprod Biomed Online 4 Suppl 1:30-38, 2002). Stress markers, such as checkpoint proteins like TRP53 (aka p53) (Ganeshan et al., Exp Cell Res 358:227-233, 2017); Ganeshan et al., Biol Reprod 83:958-964, 2010) and a small set of kinases from the protein kinome that mediate enzymatic stress responses, can also be used to define optima. But, some gametes or embryos may have been stressed in vivo prior to IVF/ART or IVF/ART optimized for one outcome may be suboptimal for another. Increasing nutrition or adding CoQ10 to increase ATP production (Yang et al., Stem Cell Rev 13:454-464, 2017), managing stress enzyme levels with inhibitors (Xie et al., Mol Hum Reprod 12:217-224, 2006), or adding growth factors such as GM-CSF (Robertson et al., J Reprod Immunol 125:80-88, 2018); Chin et al., Hum Reprod 24:2997-3009, 2009) may increase survival and health of cultured embryos during different stress exposure contexts (Puscheck et al., Adv Exp Med Biol 843:77-128, 2015). We define "stress" as negative stimuli which decrease normal magnitude and speed of development, and these can be stress hormones, reactive oxygen species, inflammatory cytokines, or physical stimuli such as hypoxia. AMPK is normally activated by high AMP, commensurate with low ATP, but it was recently shown that if glucose is present inside the cell, AMPK activation by low ATP/high AMP is suppressed (Zhang et al., Nature 548:112-116, 2017). As we discuss in more detail below, this may also lead to greater AMPK agonist toxicity observed in two-cell embryos that do not import glucose. Stress in embryos and stem cells increases AMPK in large stimulation indexes but also direness indexes; the fastest AMPK activation occurs when stem cells are shifted from optimal oxygen to lower or high levels (Yang et al., J Reprod Dev 63:87-94, 2017). CoQ10 use may be better than risking AMPK-dependent metabolic and developmental toxicity when ATP is depleted and AMPK activated. Second, the use of AMPK agonists, DS, and drugs may best be rationalized when insulin resistance or obesity leads to aberrant hyperglycemia and hypertriglyceridemia, and obesity that negatively affect fertility. Under these conditions, beneficial effects of AMPK on increasing triglyceride and fatty acid and glucose uptake are important, as long as AMPK agonist exposures are not too high or do not occur during developmental windows of sensitivity. During these windows of sensitivity suppression of anabolism, proliferation, and stemness/potency due to AMPK activity, or overexposure may stunt or kill embryos or cause deleterious epigenetic changes.

During embryo implantation, crosstalk between the endometrial epithelium and the blastocyst, especially the trophoblasts, is a prerequisite for successful implantation. During this crosstalk, various molecular and functional changes occur to promote synchrony between the embryo and the endometrium as well as the uterine cavity microenvironment. In the past few years, growing evidence has shown that endometrium-derived exosomes play pivotal roles in the embryonic-maternal crosstalk during implantation, although the exact mechanism of this crosstalk has yet to be determined. The presence of metalloproteinases has been reported in endometrium-derived exosomes, implying the importance of these enzymes in exosome-based crosstalk. Thus, in this review, we describe the potential roles of the metalloproteinases of endometrium-derived exosomes in promoting embryo attachment and implantation. This study could provide a better understanding of the potential roles of exosomal metalloproteinases in embryo implantation and pave the way for developing novel exosome-based regulatory agents to support early pregnancy., (© 2017 Wiley Periodicals, Inc.)

Aim: The objective of this study was to investigate a possible correlation between the plasminogen activator inhibitor-1 (PAI-1) and methylene tetrahydrofolate reductase (MTHFR) polymorphisms and unexplained spontaneous miscarriages (SM)., Materials and Methods: PAI-1 polymorphisms were evaluated in 150 women with pregnancy in their history. One hundred women with a history of SM formed the study group and 50 women with normal pregnancies served as the control group. Also, the combination of PAI-1 and MTHFR polymorphisms were evaluated in 138 women out of a total of 150, which included 92 women with SM in their history compared to 46 women in the control group. For statistical analysis, χ2 test, Phi, and Cramer V tests were used; p < 0.05 was taken as a statistically significant result., Results: Our findings show: (a) the correlation between SM and PAI-1 mutations reaches statistical significance (p = 0.026); (b) there was a statistically significant difference between heterozygous PAI-1 in women with only 1 SM compared to the control group (p = 0.047); (c) the comparison of combinations of both mutations, PAI-1 and MTHFR, with the control group demonstrates statistical significance in favor of women with SM and both mutations (p = 0.022)., Conclusion: PAI-1 and MTHFR polymorphisms may play an important role in pregnancy complications because heterozygous PAI-1 mutations and a combination of both PAI-1 and MTHFR mutations might contribute to SM., (© 2017 S. Karger AG, Basel.)

In the present study, the frequencies of genotypes and alleles of candidate genes with respect to polymorphisms associated with increased pregnancy loss in the first trimester of pregnancy, including MMP1–1607insG, MMP9 A–8202G, and TIMP1 С536T, were reported. The frequency of homozygotes for allele MMP9 A–8202 was increased by a factor of two among women with miscarriage in the first trimester compared to the control. Significant models of interaction of genes MMPs and TIMP1 were revealed. The genotypes of genes MMP1 (rs1799750), MMP9 (rs11697325), and TIMP1 (rs11551797) increasing the risk of pregnancy loss in the first trimester were determined.

Oxidative stress is associated with some forms of both male and female infertility. However, there is insufficient knowledge of the influence of oxidative stress on the maintenance of a viable pregnancy, including pregnancy complications and fetal development. There are a number of animal models for understanding age-dependent decrease of reproductive ability and diabetic embryopathy, especially abnormal spermatogenesis, oogenesis and embryogenesis with mitochondrial dysfunctions. Several important processes occur in mitochondria, including ATP synthesis, calcium ion storage, induction of apoptosis and production of reactive oxygen species (ROS). These events have different effects on the several aspects of reproductive function. Tet-mev-1 conditional transgenic mice, developed after studies with the mev-1 mutant of the nematode C. elegans, offer the ability to carefully regulate expression of doxycycline-induced mutated SDHC(V69E) levels and hence modulate endogenous oxidative stress. The mev-1 models have served to illuminate the effects of complex II deficiency-dependent mitochondrial ROS production, although interestingly they maintain normal mitochondrial and intracellular ATP levels. In this review, the reproductive dysfunctions are presented focusing on fertility potentials in each gamete, early embryogenesis, maternal conditions with placental function and neonatal development., (Copyright © 2016. Published by Elsevier Ireland Ltd.)

Triclosan (TCS), an antibacterial agent, is identified in serum and urine of humans. Here, we show that the level of urinary TCS in 28.3% patients who had spontaneous abortion in mid-gestation were increased by 11.3-fold (high-TCS) compared with normal pregnancies. Oral administration of TCS (10 mg/kg/day) in mice (TCS mice) caused an equivalent urinary TCS level as those in the high-TCS abortion patients. The TCS-exposure from gestation day (GD) 5.5 caused dose-dependently fetal death during GD12.5-16.5 with decline of live fetal weight. GD15.5 TCS mice appeared placental thrombus and tissue necrosis with enhancement of platelet aggregation. The levels of placenta and plasma estrogen sulfotransferase (EST) mRNA and protein in TCS mice or high-TCS abortion patients were not altered, but their EST activities were significantly reduced compared to controls. Although the levels of serum estrogen (E2) in TCS mice and high-TCS abortion patients had no difference from controls, their ratio of sulfo-conjugated E2 and unconjugated E2 was reduced. The estrogen receptor antagonist ICI-182,780 prevented the enhanced platelet aggregation and placental thrombosis and attenuated the fetal death in TCS mice. The findings indicate that TCS-exposure might cause spontaneous abortion probably through inhibition of EST activity to produce placental thrombosis.

Study Question: Do N-acetylglucosaminyltransferase (GnT-V) and N-acetylglucosaminyltransferase III (GnT-III) play an important role in early spontaneous miscarriage (ESM) in humans., Summary Answer: The dynamic balance between GnT-V and GnT-III expression in chorionic villi differed between early normal pregnancy and ESM and was associated with altered β1,6-N-acetylglucosamine (β1,6-GlcNAc) and bisecting N-acetylglucosamine (bis-GlcNAc) branched N-glycans on integrin β1., What Is Known Already: GnT-V contributes to metastasis, while GnT-III is recognized as a metastasis suppressor. It has been reported that GnT-V contributes to placentation in the early phase of pregnancy, possibly regulating trophoblast invasion. However, the expressions of GnT-V and GnT-III in ESM have not been reported., Study Design, Size, Duration: Villous samples from 6 to 9 weeks of gestation were collected in the First Affiliated Hospital of Chongqing Medical University from May 2013 to September 2014 from 60 normal pregnant women undergoing elective termination of pregnancy and from 40 patients with a clinical diagnosis of ESM., Participants, Materials, Setting, Methods: Quantitative PCR and western blots were used to examine the GnT-V and GnT-III mRNA (Mgat5 and Mgat3) and protein expression, respectively, of chorionic villi in both the ESM group and the normal group from week 6 to week 9. We used immunofluorescence and immunohistochemistry to detect the location of GnT-V and GnT-III. Lectin fluorescence and histochemistry were used to test the location of β1,6-GlcNAc and bis-GlcNAc branching in the normal and ESM groups. To assess the functional capacity of GnT-V and GnT-III in the chorionic villi between the two groups, we used an enzyme-linked immunosorbent assay kit to measure the activity of these enzymes. Using co-precipitated integrin α5β1 followed by phytohaemagglutinin (PHA)-L and PHA-E blotting, we investigated whether GnT-V and GnT-III could modify the N-glycosylation profile in terms of the β1,6-GlcNAc and bis-GlcNAc structures in integrin α5β1 during the first trimester in both groups., Main Results and the Role of Chance: In the normal group expression and activity of GnT-V and the concentration of its product, β1,6-GlcNAc were higher at week 9 than at weeks 6, 7 and 8 (P < 0.05). In contrast, the expression and activity of GnT-III and the concentration of its product, bis-GlcNAc were higher at week 6 than at weeks 7, 8 and 9 (P < 0.05). Compared with the normal group, the ESM group exhibited a lower expression of GnT-V and β1,6-GlcNAc (P < 0.05) and a higher expression of GnT-III and bis-GlcNAc (P < 0.05) with consistent changes in enzymatic activity. Immunofluorescence showed that GnT-V was located mainly in the cytoplasm of syncytiotrophoblasts (STBs) and chorionic villous cytotrophoblasts (CTBs), in both the ESM group and the normal group. β1,6-GlcNAc N-glycan was mainly located outside of the STB and CTB layer in normal villi and was expressed only rarely in the ESM villi. GnT-III was expressed primarily in the cytoplasm of STBs and expressed only very weakly in the CTBs of normal villi, whereas it was highly expressed in both the STBs and CTBs in the ESM group. bis-GlcNAc was primarily located outside of the STBs in the normal villi, whereas it was expressed much more abundantly outside of both the STBs and CTBs in the ESM group at each week of gestation. Moreover, decreased β1,6-GlcNAc-branched N-glycans and increased bis-GlcNAc-branched N-glycans on integrin β1 (P < 0.05) were observed in the ESM group., Wider Implications of the Findings: Our findings provide a new insight for studying the mechanism of clinical ESM in humans and it might be valuable for the clinical diagnosis and treatment of ESM., Limitations, Reasons for Caution: The study lacks experiments in vitro to disclose the precise mechanism by which GnT-V and GnT-III regulate ESM. In some cases, degradation of the tissues after the miscarriage event cannot be ruled out., Study Funding/competing Interests: This study was supported by grants from the National Natural Science Foundation of China (31271546). The authors have no competing interests., (© The Author 2015. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

We aimed to explore the correlation between unexplained recurrent spontaneous abortion and polymorphisms in the methylene tetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) genes. A case control study was conducted in 118 patients with unexplained recurrent spontaneous abortion (abortion group) and 174 healthy women (control group). The genetic material was extracted from the oral mucosal epithelial cells obtained from all subjects. The samples were subjected to fluorescence quantitative PCR to detect the single nucleotide polymorphisms (SNPs) in the MTHFR (C677T and A1298C) and MTRR (A66G) gene loci. The distribution frequency (18/118, 15.3%) of the MTHFR 677TT genotype was significantly higher in the abortion group (χ2 = 11.006, P = 0.004) than in the control group (2/174, 1.1%); on the other hand, the distribution frequency of the MTHFR A1298C genotype did not significantly differ between the abortion and control groups (χ(2) = 0.441, P = 0.507). The distribution frequency of the MTRR A66G genotype was also significantly higher in the abortion group (14/118, 11.9%; χ(2) = 10.503, P = 0.005) than in the control group (8/174, 4.6%). The MTHFR C677T and MTRR A66G polymorphisms are significantly correlated with the occurrence of spontaneous abortion.

Fertility is a quantitative, complex character governed by a considerable number of genes. Despite clinical and scientific advances, several cases of human infertility remain unexplained. In the present study, using a positional cloning approach in a mouse model of interspecific recombinant lines, a candidate gene, ALPP, encoding the placental alkaline phosphatase, was identified as being potentially involved in recurrent spontaneous abortion. We then analyzed patients for detecting putative associations between ALPP polymorphisms, in vitro fertilization failures, and miscarriages. ALPP was sequenced in 100 controls and 100 patients affected by recurrent spontaneous abortion, from the same ethnic background. The frequency of several alleles and allelic combinations were different between recurrent spontaneous abortion and control women. One polymorphism induced a coding substitution (Ile89Leu) that was associated with a decreased risk of abortion and in vitro fertilization failure. Thereafter, the population was increased by the analysis of 92 additional controls and 612 additional patients for the coding polymorphism Ile89Leu. We finally show, by functional analysis, that the 89Leu placental alkaline phosphatase has an enhanced alkaline phosphatase activity. This study suggests that ALPP genotyping could be a strong predictor of implantation success., (Copyright © 2014 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Matrix metalloproteinases, which remodel the extracellular matrix, are involved in all physiological and pathophysiological processes. In particular, they contribute to the success of a pregnancy: from embryo implantation in the endometrium to uterine cervical ripening and uterine involution. A misregulation of their expression and/or of their activity is observed in two major diseases in pregnancy such as spontaneous abortion and preeclampsia.

Introduction: As one of the key molecules in the extracellular matrix in human conceptus, hyaluronan (HA) has been receiving particular attention. Here, we have investigated the expression and regulation of different molecular weight HA on the biological behaviors of primary human trophoblasts during the first trimester of pregnancy., Methods: The expression of HA and HA synthetase (HAS) by human first trimester trophoblasts was analyzed in placentae from normal pregnancy or miscarriage by immunochemistry and real-time RT-PCR, respectively. ELISA was used to measure the secretion of HA by primary trophoblasts. The effects of HA on the proliferation, apoptosis and invasiveness of trophoblasts were examined. We also investigated the signaling pathways involved in HA activation in human trophoblasts., Results: The higher HAS2 expression and HA secretion were observed in normal villi than that of miscarriage, and the primary trophoblasts secreted HA continuously. High molecular weight HA (HMW-HA) and medium molecular weight HA (MMW-HA) promoted proliferation and invasiveness while inhibited apoptosis of trophoblasts. However, low molecular weight HA (LMW-HA) had no obvious effect on the growth or invasiveness of human trophoblasts. In addition, HMW-HA showed more efficiently than MMW-HA on the growth while MMW-HA displayed a more obvious effect on the invasiveness of trophoblasts than HMW-HA. HMW-HA activated PI3K/AKT and MAPK/ERK1/2 signaling pathways in trophoblasts. Blocking PI3K/AKT or MAPK/ERK1/2 signaling inhibited the HA-upregulated growth and invasiveness of human trophoblasts., Conclusion: Our results suggest that higher level and greater molecular mass of HA can promote trophoblast growth and invasion in an autocrine manner, which was beneficial to placentation and maintenance of human early pregnancy., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Selenium is an essential nutrient for human health, and maternal selenium concentration has been reported to be associated with pregnancy outcome. To further investigate the possible role of selenium (Se) in miscarriage, we conducted a case-control study to evaluate the correlations among selenium status, glutathione peroxidase activity, and spontaneous abortion. A total of 46 subjects with normal pregnancies and 25 subjects with spontaneous abortion were recruited, and their serum selenium concentrations and serum glutathione peroxidase activities were analyzed. The total serum selenium concentrations in subjects with normal pregnancies were significantly higher than those of subjects with spontaneous abortion; however, the glutathione peroxidase activities were similar in both groups. We further separated the subjects into smoking and nonsmoking groups, and the logistic regression analysis suggested that total serum selenium concentration, but not serum glutathione peroxidase activity or smoking, was significantly correlated with the incidence of miscarriage. The present study thus reaffirms that low serum selenium levels are associated with miscarriage and that selenium plays an important role in pregnancy maintenance.

Adenosine deaminase (ADA) is an enzyme of purine salvage pathway and has two important isoenzymes ADA1 and ADA2. The activity of ADA has been changed in diseases characterized by altered cell-mediated immunity. It was observed that total serum ADA activity was decreased during normal pregnancy compared with non-pregnant women. However, total serum ADA activity and serum ADA2 activity was increased in hyperemesis gravidarum and pre-eclampsia in pregnant women. Less information is available regarding role of ADA in abortions (recurrent and missed) and anembryonic pregnancies. Here, we review the activity of ADA and its isoenzymes. Despite these findings, it will be interesting to know whether activity of ADA will be same if ADA is estimated throughout the pregnancy and in pregnancy related complications from early first trimester to third trimester, as all studies until now were carried out at a particular stage of pregnancy.

Background: Trophoblast cell (CTB) invasion into the maternal endometrium plays a crucial role during human embryo implantation and placentation. This invasion is facilitated by the activity of matrix metalloproteinases, which are regulated by tissue inhibitors of MMPs (TIMPs)., Methods: This study compares the serum levels of MMP-9, MMP-2/TIMP-2 complex, TIMP-1 and TIMP-2 in 129 patients with ongoing pregnancy (n = 40) or spontaneous early pregnancy failure (n = 89)., Results: MMP-9 was markedly (p < 0.0001) elevated in missed abortions, as was MMP-2/TIMP-2 complex (p < 0.0005). However, the serum levels of TIMP-1 and TIMP-2 were markedly elevated (p < 0.0001) in ongoing pregnancies., Conclusions: Human placentation is mediated by fetal trophoblastic cells that invade the maternal uterine endometrium. Trophoblast invasion requires a precisely regulated secretion of specific proteolytic enzymes able to degrade the endometrial basement membrane and extracellular matrix. The elevated levels of MMP-9 and MMP-2/TIMP-2 complex may play a role in spontaneous termination of pregnancy.

Glutamate carboxypeptidase II (GCPII) is predominantly expressed in brain, intestinal mucosa and prostate cancer in the form of three splice variants i.e. N-acetylated-α-linked acidic dipeptidase (NAALADase), folyl poly-γ-glutamate carboxypeptidase (FGCP) and prostate specific membrane antigen (PSMA) respectively. Its inhibition was found to confer protection against certain neurological disorders and cancer. Despite the pivotal role of this enzyme, the most common polymorphism i.e. H475Y has not been explored comprehensively in all its splice variants. In this study, we have determined the role of this variant in different disease conditions such as breast and prostate cancers, autism, coronary artery disease (CAD) and miscarriages (N=1561). Genotyping was done by PCR-RFLP and dideoxy sequencing. Plasma folate levels were estimated by Axysm folate kit. GCPII expression was studied by semi-quantitative RT-PCR. In silico model was developed using PYMOL. We observed the protective role of H475Y variant in cancers [breast cancer; OR (95% CI): 0.81 (0.55-1.19), prostate cancer: OR (95% CI): 0.00 (0.00-0.66)], and in autism (OR (95% CI): 0.47 (0.21-1.03), whereas inflated risk was observed in CAD (OR (95% CI): 1.69 (1.20-2.37) and miscarriages [Maternal OR (95% CI): 3.26 (2.11-5.04); Paternal OR(95% CI): 1.99 (1.23-3.21)]. Further, this variant was found to impair the intestinal folate absorption in subjects with dietary folate intake in the lowest tertile (CC vs. CT in lowest tertile; 7.56±0.85ng/ml vs. 2.73±045ng/ml, p=0.005). In silico model of GCPII showed steric hindrance with H475Y resulting in stereochemical alteration of catalytic site, thus interfering with ligand binding. Statistically significant association was not observed between dietary folate levels and GCPII expression. However, a positive correlation was seen between plasma folate levels and GCPII expression (r=0.70, p<0.05). To conclude, our data suggests that GCPII H475Y variant shows inverse association with autism and cancer while showing positive association with CAD and miscarriages., (Copyright © 2012 Elsevier B.V. All rights reserved.)

We investigated the expression of methyl transferase G9a and methylated histone H3-K9 in fresh human decidual/endometrial tissue of 12 normal early pregnancies and 15 unexplained recurrent spontaneous abortions (URSA). The samples were obtained through dilatation and curettage and collected as per strict inclusion-exclusion criteria. The tissue was subjected to immunohistochemical analysis (IHC), western blotting (WB) and RT-PCR analysis. The results demonstrated methyl transferase G9a to have a lower expression in abortions when compared with that in normal pregnancy (P < 0.05). The sensitivity of RT-PCR, IHC and WB were respectively 66.67, 75 and 71.43%, while specificity of the same were 66.67, 60 and 78.92%, respectively. Methylated histone H3-K9 was significantly lower (P < 0.0001) in URSA tissues than in controls. This study suggests that methylation may cause URSA and indicates the need for further work to explore the role of methylation in URSA and its possible prevention through locally acting methylating/demethylating agents.

The a2 isoform of vacuolar ATPase (ATP6V0A2 referred to as a2V) plays a pivotal role in successful pregnancy and provides a microenvironment to maintain the delicate immunological balance at the feto-maternal interaction. We studied the expression of a2V mRNA in embryos and placenta of abortion-prone (female CBA × male DBA) murine matings or LPS (lipopolysaccharide)-treated mice. The expression of a2V was significantly higher in the placentas of nonabortion-prone (female BALB/c × male BALB/c and female CBA × male BALB/c) matings compared with the abortion-prone (female CBA × male DBA) mating. The expression of a2V was significantly decreased in the placentas treated with LPS in both female CBA × male DBA and female BALB/c × male BALB/c mating combinations with increased Lif, Il1b, and Tnf expression in the placenta. Decreased expression of a2V in the placenta is directly correlated with high percentages of pregnancy loss in abortion-prone mating (female CBA × male DBA) as well as in LPS-treated animals. The normal expression of placental a2V on Day 16 in the nonabortion-prone matings correlated with higher Mcp1 (monocyte chemotactic protein 1) gene expression, markedly higher infiltration of M1 and M2 macrophages, and no significant polarization patterns (M1/M2 = 1.2-1.6). However, in the abortion-prone mating, decreased placental a2V expression correlated with significantly lower Mcp1 gene expression with less infiltration of M1 and M2 macrophages and with polarization patterns skewed to M1 phenotypes (M1/M2 = 3.9-4.2). These data indicate that the higher expression of placental a2V is associated with dynamic infiltration of M1 and M2 macrophages through the induction of Mcp1 expression. This strengthens our hypothesis that a2V regulates the delicate cytokine and chemokine networks that coordinate the recruitment of macrophages for successful placental development and growth at the feto-maternal interface.

It is well-known fact that various pathogens, including bacteria, virus, and protozoa, induce abortion in humans and animals. However the mechanisms of infectious abortion are little known. In this study, we demonstrated that Listeria monocytogenes infection in trophoblast giant cells decreased heme oxygenase (HO)-1 and B-cell lymphoma-extra large (Bcl-XL) expression, and that their overexpression inhibited cell death induced by the infection. Furthermore, HO-1 and Bcl-XL expression levels were also decreased by L. monocytogenes in pregnant mice. Treatment with cobalt protoporphyrin, which is known to induce HO-1, inhibited infectious abortion. Taken together, our study indicates that L. monocytogenes infection decreases HO-1 and Bcl-XL expression and induces cell death in placenta, leading to infectious abortion.

Background: Heparanase cloned from and abundant in the placenta is implicated in cell invasion, tumor angiogenesis and metastasis. Recently, we demonstrated that heparanase is involved in the regulation of the hemostatic system. Heparanase was found to up-regulate tissue factor (TF) expression (Nadir et al., JTH, 2006) and interact with tissue factor pathway inhibitor (TFPI) on the cell surface, leading to dissociation of TFPI from the cell membrane resulting in increased cell surface coagulation activity (Nadir et al., TH, 2008). Herein, we investigated the role of heparanase in the placenta, focusing on its effect on TF, TFPI, TFPI-2, and vascular endothelial growth factor (VEGF)-A., Methods: Twenty formalin embedded placenta samples of abortions (weeks 6-10) were studied applying real time RT-PCR and immunostaining. Ten cases were miscarriages of women with thrombophilia and recurrent fetal losses, and ten control cases were pregnancy terminations. JAR (human choriocarcinoma trophoblasts) cells were transfected with full-length heparanase cDNA or incubated with active (50+8 kDa) recombinant heparanase and the effects on TF, TFPI, TFPI-2 and VEGF-A were examined using real time RT-PCR and immunoblotting., Results: Sections obtained from miscarriages revealed increased (2-3-folds) levels of heparanase, VEGF-A and TFPI-2 compared to placentas from controls in maternal as well as in fetal placenta elements. JAR cells overexpressing heparanase or incubated with exogenous recombinant heparanase exhibited a 2-3-fold increase in TFPI and TFPI-2 in cell lysates both at the protein and mRNA levels, with no detectable effect on VEGF-A and TF levels. Accumulation of TFPI and TFPI-2 in the cell culture medium was increased 4-6-folds, exceeding the observed induction of TFPI and TFPI-2 gene transcription., Conclusions: These results indicate a regulatory effect of heparanase on TFPI and TFPI-2 in trophoblasts, suggesting a potential involvement of heparanase in early miscarriages., (Copyright (c) 2009 Elsevier Ltd. All rights reserved.)

Aims: To compare the levels of serum prolidase activity and oxidative stress markers including total oxidative status (TOS), total antioxidant capacity (TAC), lipid hydroperoxide (LOOH) and total free sulfhydryl (-SH) in healthy pregnant women without early pregnancy loss (EPL) and women with EPL., Methods: Serum samples were obtained from 45 healthy first-trimester pregnancies and 45 pregnancies complicated with EPL. We have measured serum prolidase activity, TAC, TOS, -SH and LOOH levels spectrophotometrically., Results: Serum prolidase, TAC and -SH levels were significantly lower in the women with EPL than in the women without EPL (p <0.001, p = 0.001, p < 0.001, respectively), whereas TOS and LOOH levels were significantly higher (p < 0.001, p = 0.004, respectively). Prolidase activity was negatively correlated with TOS and LOOH levels (r = -0.299, p = 0.004; r = -0.323, p = 0.002, respectively), while positively correlated with TAC and -SH levels (r = 0.232, p = 0.028, and r = 0.418, p <0.001, respectively), Conclusion: Findings of this study have shown that serum prolidase activity and oxidative stress are significantly associated with the presence of EPL, and the correlation between serum prolidase activity and the markers of oxidative stress was reflected in increased serum TOS and LOOH levels and decreased serum TAC and -SH levels., (Copyright (c) 2009 S. Karger AG, Basel.)

Objective: To assess prevalent polymorphisms of 5,10-methylenetetrahydrofolate reductase (MTHFR) and thymidylate synthase enhancer region (TSER) and methylation patterns of p16(INK4A) and hMLH1 genes in spontaneously aborted embryos (SAEs) with normal chromosomal integrity., Design: Retrospectively analyzed, prospectively obtained database., Setting: Bundang CHA General Hospital in South Korea., Patient(s): Fifty-nine SAEs (<20 wk of gestational age) with normal chromosomal integrity., Intervention(s): None., Main Outcome Measure(s): Genotype frequency of MTHFR, TSER polymorphisms, and methylation status of p16(INK4A) and hMLH1 genes in SAEs with normal chromosomal integrity., Result(s): The distribution of the MTHFR 677C>T polymorphism differed significantly between SAEs with normal chromosomal integrity and the two control groups. Also, the frequency of combined MTHFR 677 and TSER genotypes was significantly different between the aborted embryos and the adult control group. However, the MTHFR 677C>T and 1298A>C and TSER polymorphisms were not associated with the methylation status of p16(INK4A) and hMLH1 genes in SAEs with normal chromosomal integrity., Conclusion(s): Association between the MTHFR 677C>T polymorphism and the risk of SAEs with normal chromosomal integrity in the Korean population.

This study has evaluated the hypothesis that activity of the detoxifying enzyme butyrylcholinesterase (BuChE) correlates with levels of serum anti-cardiolipin antibodies (ACA) and T lymphocytes in peripheral blood of women experiencing recurrent spontaneous abortion (RSA). Peripheral venous blood from 16 non-pregnant, RSA-afflicted women and 8 healthy non-pregnant women was analyzed for frequency of T lymphocyte subpopulations by two-color flow cytometry and for serum BuChE using butyrylthiocholine iodide/spectrophotometry. RSA-afflicted women with high serum ACA, but not those with normal ACA levels, exhibited significantly increased percentages of CD4+CD25+ cells (p<0.01) and CD4+HLA-DR+ cells (p<0.05) relative to healthy women. CD4+CD25+(high) cells were significantly lower (p<0.05), while CD4+CD25+(low) cells were significantly higher (p<0.01), in women with elevated ACA compared to healthy women and to RSA women with normal ACA. Relative to healthy, non-pregnant subjects, serum BuChE activity in RSA patients was elevated, both for those with normal ACA (p<0.001) and elevated ACA levels (p<0.01). Among healthy controls, a significant positive correlation was observed between frequency of CD3+NK cells and BuChE activity (p<0.01), but not for RSA-afflicted subjects. A positive correlation between BuChE activity and frequency of CD4+CD25+ cells, as well as CD4+CD25+(high) cells, was observed in the RSA-afflicted subject group with elevated ACA (p<0.05), which may be related to induction of BuChE by toxic metabolites resulting from pathogenic T cell activity. It is concluded that, among RSA patients, high serum ACA correlates with elevated levels of activated T cells and reduced CD4+CD25+(high)/CD4+CD25+(low) cells in comparison to healthy women or those afflicted with RSA but with normal ACA. BuChE activity is observed to be elevated in RSA patients irrespective of serum ACA status.

Objective: In pregestational diabetes, the placenta at term of gestation is characterized by various structural and functional changes. Whether similar alterations occur in the first trimester has remained elusive. Placental development requires proper trophoblast invasion and tissue remodeling, processes involving matrix metalloproteinases (MMPs) of which the membrane-anchored members (MT-MMPs) such as MT1-MMPs are key players. Here, we hypothesize a dysregulation of placental MT1-MMP in the first trimester of type 1 diabetic pregnancies induced by the diabetic environment., Research Design and Methods: MT1-MMP protein was measured in first-trimester placentas of healthy (n = 13) and type 1 diabetic (n = 13) women. To identify potential regulators, first-trimester trophoblasts were cultured under hyperglycemia and various insulin, IGF-I, IGF-II, and tumor necrosis factor-alpha (TNF-alpha) concentrations in presence or absence of signaling pathway inhibitors., Results: MT1-MMP was strongly expressed in first-trimester trophoblasts. In type 1 diabetes, placental pro-MT1-MMP was upregulated, whereas active MT1-MMP expression was only increased in late first trimester. In isolated primary trophoblasts, insulin, IGF-I, IGF-II, and TNF-alpha upregulated MT1-MMP expression, whereas glucose had no effect. The insulin effect was dependent on phosphatidylinositol 3-kinase, the IGF-I effect on mitogen-activated protein kinase, and the IGF-II effect on both., Conclusions: This is the first study reporting alterations in the first-trimester placenta in type 1 diabetes. The upregulated MT1-MMP expression in type 1 diabetes may be the result of higher maternal insulin and TNF-alpha levels. We speculate that the elevated MT1-MMP will affect placental development and may thus contribute to long-term structural alterations in the placenta in pregestational diabetes.

BACKGROUND Polymorphisms C677T and A1298C of the MTHFR gene have been implicated in fetal viability. In this study, we determined the allele and genotype frequencies of these polymorphisms in different populations, including spontaneous abortion (SA) fetal tissues, with the objective of evaluating their impact on fetal viability. METHODS 342 samples of fetal tissues, selected from SA occurring during the 1980s, 230 samples from subjects born in the 1980s and a third set of samples from 204 subjects born in the 1950s, were genotyped by using TaqMan probes. RESULTS The wild CC genotype of the C677T polymorphism showed a strong protective effect against abortion (0.03 in SA versus 0.47 in 1950s and 0.43 in 1980s) (P < 0.0001). Genotypes of three mutations in the combinations of polymorphisms for C677T and A1298C showed a very low frequency in the living population; however, the three mutations genotypes were over expressed in the SA group (0.02 in 1950s; 0.03 in 1980s and 0.17 in SA) (P < 0.0001). Samples with four mutations (n = 2) were found only in the SA group. CONCLUSIONS There is no linkage disequilibrium between C667T and A1298C polymorphisms. Fetal viability is directly related to the CC genotype as a protector while the three and four mutation MTHFR genotypes appear to be a determinant on fetal non-viability and SA.

Problem: In consideration of the effect of adenosine deaminase (ADA) and ACP1 (a low-molecular-weight protein tyrosine phosphatase) on T-cell receptor activity, we have analysed the joint distribution of these polymorphisms in a sample of women with primary repeated spontaneous abortion (RSA) to search for possible interactive effects on susceptibility to RSA., Method of Study: ACP1 and ADA phenotypes were determined in 170 women with primary RSA in 79 healthy consecutive puerperae and in 160 female newborns from the Caucasian population of Rome and in 357 healthy consecutive puerperae from the Caucasian population of Penne. Chi-square test of independence and three way contingency table analysis by a log-linear model were performed., Results: Women with low-ADA activity and high-ACP1 activity show the lowest susceptibility to RSA. Women with high-ADA activity and low-ACP1 activity, on the contrary, show the highest susceptibility to RSA and also the highest incidence of auto antibodies and of A blood group incompatibility., Conclusion: The data are in agreement with those expected on the basis of the effects of ACP1 and ADA genetic variability on T-cell receptor activity and suggest a cooperative effect of the two polymorphic systems in the susceptibility/resistance to repeated spontaneous abortion.

The semi-allogeneic fetus has to be tolerated by the maternal immune system. In mice, it has been shown that inhibiting indoleamine-dioxygenase (IDO) leads to fetal rejection, suggesting a central significance for IDO in establishing maternal tolerance. Consequently, we have analyzed IDO expression in human endometrium and decidua to determine whether it may be of significance in human reproduction. Endometrial (n=60) and decidual (n=68; first and second trimester) tissue samples and isolated cells were analyzed for IDO mRNA and protein expression by real-time PCR, Western blot and immunohistochemistry. IDO expression in the decidua of proven fertile women (n=34) was compared to women presenting with their first pregnancy (n=22) and women with a history of miscarriages (n=12). Expression of IDO was localized in glandular epithelial cells and scattered stromal leukocytes. Expression started at the mid-luteal phase in the menstrual cycle and was high until the second trimester of pregnancy. However, glandular expression of IDO decreased during the second trimester, whereas expression in villous trophoblast started at this time. There were no significant differences in decidual IDO expression between proven fertile women and women presenting with their first pregnancy or women with a history of miscarriages. From the expression pattern we conclude that IDO may play a central role in human pregnancies for the establishment of maternal tolerance of fetal antigens. Thereby, IDO expression may be needed in each pregnancy independently from prior pregnancies, and a history of miscarriage may not reflect a general deficiency in IDO expression.

Objective: To assess prevalent 5,10-methylenetetrahydrofolate reductase (MTHFR) polymorphisms in spontaneously aborted embryos., Design: A retrospectively analyzed, prospectively obtained database., Setting: Bundang CHA General Hospital and Kangnam CHA Hospital., Patient(s): Ninety-four spontaneously aborted embryos at <20 weeks of gestational age., Intervention(s): None., Main Outcome Measure: Genotype frequency of MTHFR C677T and A1298C polymorphisms in spontaneously aborted embryos., Result(s): The aborted embryos exhibited both a significantly high frequency of the MTHFR 677CC genotype and a low frequency of the MTHFR 677CT genotype, compared to both the child and the adult control groups, respectively, whereas no significant change in the fetal MTHFR A1298C genotype frequency was observed. The combinative genotype of MTHFR 677CC/1298AC from the abortus had a high prevalence compared to the child controls. Further, the chromosomal integrity of the abortus did not affect the frequency of the MTHFR 677CC and 1298AC genotypes., Conclusion: Spontaneously aborted embryos have a unique distribution of MTHFR C677T and A1289C polymorphisms, regardless of their chromosomal integrity.

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.

Unlabelled: The incidence of spontaneous abortions in women with type 1 diabetes mellitus varies between 10-30%. The etiology of this is still unclear despite numerous experimental studies. Pregnancy is a condition of increased oxidative stress due to impaired balance between pro- and antioxidants. Glutathion and related enzymes perform the best antioxidant protection. Some authors point to a possible correlation between spontaneous abortions and low plasma Se levels as well as low intracellular activity of glutathion peroxidase enzyme. Others report that Hb A1-c, values over 1SD above normal increase the risk of spontaneous abortions with 3% and Hb A1-C values between 10-12% are critically high for the occurrence of spontaneous abortions. The purpose of the study was to evaluate the levels of Se and glutathion peroxidase enzyme (Gl-Px) in pregnant women with type 1 diabetes mellitus in the first trimester of pregnancy and to find out is there a correlation between glycemic control of diabetes and the incidence of spontaneous abortions., Description of Project: 75 pregnant women enrolled in an- 1 year prospective study divided in 3 groups according to pregnancy outcome: gr. 1 - n = 30 with type diabetes mellitus, no abortions, gr.2 - n = 16 with type diabetes mellitus with first trimester spontaneous abortion and gr. 3 - n = 29 healthy pregnant women. Women with type 1 diabetes mellitus were divided into three subgr. according to glycemic control - subgr. 1 - n = 12 (Hb A1-c < 7%), subgr.2 - n = 18 (Hb A1-c > 7< 8%), subgr.3 - n = 16 (Hb A1-c > 8%). Gl-Px activity was determined in Er hemolisate with test reagents of Randox Ransel, with ref.values 27.5 - 73 U/g Hb. Selen concentration was determined in whole blood sample by atomic absorption spectrophotometry with ref. values 0.12-1.1 micromol/l. HbA 1-C was measured by affinity chromatography with ref. values 4.5-6.3%. Statistical methods used were: dispersion, correlation analysis - SPSS package version 11.01.01., Results: Basic Se levels were low in all pregnant women in early pregnancy. The metabolic control level did not influence the levels of Se in pregnant women with diabetes mellitus type1. Gl -Px activity was within the normal limits in all women. There was no correlation between Se levels and Gl -Px activity in pregnant diabetics with and without abortions. There was a correlation between Se levels and Gl -Px activity only in healthy pregnant women. Pregnant women with poor glycemic control had higher incidence of spontaneous abortions., Conclusions: We could not support the hypothesis of reduced antioxidant protection (low Se and Gl-Px levels) as a causative factor in the pathogenesis of spontaneous abortions in diabetic patients. Our study results showed that poor metabolic control of diabetes (high Hb A1-c) in the first trimester of pregnancy had a primary role in the occurrence of early abortions. We could speculate that the early hyperglycemic maternal-fetal environment most probably plays a role of an additional stress to the developing embryo.

Recent data demonstrated that CD4+CD25+ regulatory T (Treg) cells and an enzyme called indoleamine 2,3-dioxygenase (IDO) mediate maternal tolerance to the fetus. Interestingly, Treg cells express the CTLA-4 molecule on their surface, and B7 (CD80/86) ligation by CTLA-4 enhanced IDO activity of dendritic cells (DCs) and monocytes by the induction of interferon gamma (IFN-gamma) production. In this study, we studied the IDO expression on peripheral blood monocytes and decidual monocytes or DCs after treatment with CTLA-4/Fc fusion protein or IFN-gamma using flow cytometry. IDO expressions on both peripheral blood DC and decidual DC and monocytes were up-regulated during normal pregnancy. On the other hand, both IDO expression on DC and monocytes after IFN-gamma treatment or CTLA-4 treatment were decreased in spontaneous abortion cases. The expression of CD86 on peripheral blood and decidual monocytes and DC in spontaneous abortion cases was lower compared with those in normal pregnancy subjects. Also, IFN-gamma production by decidual and peripheral blood mononuclear cells after CTLA-4/Fc treatment in spontaneous abortion cases was significantly lower than those in normal pregnancy subjects. These data suggest that CTLA-4 on Treg cells up-regulates IDO expression on decidual and peripheral blood DC and monocytes by the induction of IFN-gamma production.

Problem: Indoleamine 2,3-dioxygenase (IDO) expression in fetal trophoblast and decidual antigen-presenting cells has been proposed to inactivate maternal T cells and thereby prevent rejection of the "fetal allograft" in early pregnancy. Psychic stress has been proposed to cause miscarriages as well as infertility, at the same time in pregnancy when blockade of IDO causes loss, but the suggested mechanism of stress-triggered loss has been an increased ratio of pro-rejection Th1-type cytokines to anti-rejection Th2/3 cytokines. Could stress act by reducing IDO expression?, Methods: Using DBA/2-mated A/J mice where stress causes early pregnancy failure, we examined the role of stress in reducing IDO versus increasing Th1/Th2 ratio in deciduas. IDO loss was also examined in human decidua associated with pregnancy failure., Results: A post-implantation sonic stress increased the pregnancy failure rate, increased the Th1/Th2 ratio, but did not reduce IDO. IDO was reduced, and Th1/Th2 ratios increased in A/J mice pre-immunized against paternal DBA/2 antigens, and concomitant stress increased these effects. The rate of pregnancy failure was not further increased consistent with recent discoveries of factors that limit the impact of Th1 cytokines at the feto-maternal interface. In deciduas from spontaneous miscarriage patients, IDO(+) cell frequencies were low in only 30% of patients. CD3(+) T-cell numbers and percentage terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-digoxigenin nick end-labelling (TUNEL)(+) apoptotic T cells were increased, but the level of IDO did not correlate with likelihood of apoptosis., Conclusions: Loss of an allogeneic embryo in early pregnancy is more likely to be due to a high Th1/Th2 ratio than loss of putative protection by IDO.

The allogeneic fetus has been considered to be an allograft and the tolerance mechanisms involved in pregnancy maintenance resemble those leading to graft acceptance. Up-regulation of Heme Oxygenase-1 (HO-1) promotes graft acceptance. Additionally, HO-1 has been proposed to have tissue-protective properties. Previous studies reported the presence of HO-1 and HO-2 in mammalian placenta and postulated a protective role for HO during pregnancy. Here, we analyze HO-1 and HO-2 expression at the feto-maternal interface from normal pregnant and abortion mice and correlate these findings with inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) expression as well as with Th1/Th2 cytokine production by immune cells. DBA/2-mated CBA/J females undergoing abortion and BALB/c-mated CBA/J females having normal pregnancies were included in our study. The mice received no treatment. On day 14 of pregnancy, the mice were sacrificed, the abortion rate was calculated and the ex vivo Th1/Th2 production by decidual immune cells was analyzed by flow cytometry. The expression of HO-1 and HO-2, iNOS and eNOS was analyzed by immunohistochemistry (IHC) and Western blot in placenta samples. The Th1/Th2 cytokines ratio was augmented in decidua from abortion mice. We further observed a significant down-regulation of HO-1, HO-2, iNOS and eNOS molecules in placental tissue from mice undergoing abortion when compared to normal pregnant mice. Since we found diminished HOs and nitric oxide synthase (NOS) levels at the feto-maternal interface from abortion mice when compared to normal pregnant mice, which were further associated with increased Th1/Th2 cytokine production, we propose HO as a putative therapeutic target in immunological abortions. Up-regulation of HO-1 or HO-2 would favour the Th2-cytokine production, which could avoid abortion onset.

Background: SCO2 is a cytochrome c oxidase (COX) assembly gene that encodes a mitochondrial inner membrane protein that probably functions as a copper transporter. Mutations in SCO2 have been associated with severe COX deficiency and early-onset fatal infantile hypertrophic cardiomyopathy, encephalopathy, and neurogenic muscle atrophy. Fetal wastage has not been described in association with mutations of SCO2., Objective: To investigate a case of early spontaneous abortion in a family carrying mutations in SCO2., Design: Case report. Patients Spontaneous abortion in the first trimester occurred in a woman whose first pregnancy had also resulted in a miscarriage in the first trimester and whose only child had died at 53 days of life from cardioencephalomyopathy. This child was a compound heterozygote for mutations in SCO2, and her parents were heterozygous for each mutation., Main Outcome Measures: Mutations in the abortus by sequencing the SCO2 gene and confirmation of the point mutations as determined by restriction fragment length polymorphism analysis., Results: As in the previous affected child, we found a missense mutation (E140K) and a nonsense mutation (Q53X) in the abortus., Conclusions: The typical clinical presentation of SCO2 mutations is severe, rapidly progressive hypertrophic cardiomyopathy that presents in the neonatal period and is often associated with respiratory difficulties, metabolic acidosis, and hypotonia. The experience in this family suggests that mutations in SCO2 may also be associated with early spontaneous abortions and fetal wastage.

Problem: We previously reported a diminished expression of the heme-degrading enzymes heme oxygenases (HO)-1 and HO-2 in decidua and placenta from mice undergoing Th1-mediated abortion, strongly indicating the protective effect of HO in murine pregnancy maintenance. Here we investigated whether the expression of HO-1 and HO-2 is also reduced at the feto-maternal interface of pathologic human pregnancies., Method of Study: Immunohistochemistry was used to detect HOs expression in placental and decidual first-trimester tissue from patients with: spontaneous abortion (n = 14), choriocarcinoma (n = 14), hydatidiform mole (H-mole) (n = 12), compared with normally progressing pregnancies (n = 15). Further, we investigated early third-trimester decidual and placental tissue from patients with pre-eclampsia (n = 13) compared with fetal growth retardation (n = 14) as age-matched controls., Results: In first trimester tissue, we observed a significant reduction of HO-2 expression in invasive trophoblast cells, endothelial cells, and syncytiotrophoblasts in samples from patients with spontaneous abortion compared with normal pregnancy. H-mole samples showed a diminished expression of HO-2 in invasive trophoblast cells and endothelial cells in comparison with NP, whereas choriocarcinoma samples showed no significant differences compared with the control. In third trimester tissue, HO-2 was also reduced in syncytiotrophoblasts and invasive trophoblast cells from pre-eclampsia compared with samples from fetal growth retardation. HO-1 expression was diminished in all pathologies investigated; however, the differences did not reach levels of significance., Conclusions: Our data indicate that HOs play a crucial role in pregnancy and low expression of HO-2, as observed in pathologic pregnancies, may lead to enhanced levels of free heme at the feto-maternal interface, with subsequent upregulation of adhesion molecules, allowing enhanced inflammatory cells migration to the feto-maternal interface.

Problem: While activated/phagocytosing phagocytes infiltrating to the chorioamnion are considered to be one of the causal agents of preterm labor onset, whether placental villous macrophages (Hofbauer cells) are activated/phagocytosing in this condition is not known., Method of Study: We concomitantly localized two important phagocytosis-related enzymes, acid phosphatase (ACP) and glucose-6-phosphate dehydrogenase (G6PD), in Hofbauer cells in second trimester placental villi, and compared them with those from infection-related second trimester-spontaneous abortion (miscarriage) placentas., Results: There were two types of Hofbauer cells. The first cells exhibited ACP stainings confined to the lysosomes, suggesting that they are dormant/non-activated cells. Approximately two-thirds of these cells showed weak G6PD labeling on the cytosolic side of endoplasmic reticula, and G6PD labeling was hardly recognizable in the remaining one-third. The second cells, possessing large phagosomes, showed marked ACP labeling in the phagosomes, suggesting that they are activated/phagocytosing cells. All these cells exhibited G6PD labeling, and in 'bursting cells' (possibly hyperactivated cells) G6PD deposits were marked. The percentage of activated cells in miscarriage placentas was significantly higher (44.8 +/- 6.0%) than that in gestational age-matched controls (17.4 +/- 5.3%)., Conclusions: These observations indicated that (1) G6PD activity increased in activated/phagocytosing Hofbauer cells, and (2) the percentage of phagocytosing cells increased in infection-related miscarriage placentas. Hofbauer activation and G6PD may play an role in the pathogenesis/pathophysiology of preterm labor onset.

Inhibition of DPP IV has been shown to abrogate the stress-related increase in murine abortions and a concomitant increase in gamma-interferon. The aim of the present study was to investigate a potential impact of the DPP IV inhibitor Isoleucine Cyanopyrrolidide on the expression of surface antigens involved in T-cell responses. DBA/2-mated CBA mice were stressed on day 5.5 of pregnancy and received injections of a DPP IV inhibitor. On day 13 of gestation, the animals were sacrificed and the percentage of abortions was determined. As shown before, stress failed to boost the abortion rate in mice receiving the DPP IV inhibitor. In stressed animals, a lower surface density of CTLA-4 on decidual CD26-positive lymphocytes was observed than in non-stressed animals. Inhibition of DPP IV restored CTLA-4 surface density to normal and decreased surface expression of CD26 and CD28 on decidual lymphocytes irrespective of stress exposure. These observations suggest that a modulation of T-cell surface antigens expression due to inhibition of DPP IV activity may contribute to the potent anti-abortogenic effect observed here.

Background: The aim of this study was to evaluate telomerase activity in tissue from cases of gestational trophoblastic disease (GTD) and in placental tissue from early and late human pregnancies., Methods: We used a telomeric repeat amplification protocol assay to measure telomerase activity in 132 tissue samples from normal early pregnancies, spontaneous abortions, normal late pregnancies, cases of late-pregnancy intrauterine fetal death, and GTD., Results: Telomerase activity was detected more often in normal early pregnancies and cases of GTD than in spontaneous abortions and normal late pregnancies (P < 0.001). During early gestation, no significant difference in detection rates was found between normal pregnancies and complete hydatidiform mole. As gestational age increased, detection rates for normal pregnancies decreased significantly (P = 0.0001), while for complete hydatidiform mole no significant changes occurred., Conclusions: Our findings indicate that placental tissue from normal early pregnancies and neoplastic tissue from GTD possess similar levels of telomerase activity. Decreasing regulation of telomerase activity is present in normal pregnancies but not in complete hydatidiform mole. The fact that telomerase activity decreases in cases of fetal demise, and as pregnancy progresses, also suggests that placental senescence may play a role in the development and ageing of the placenta.

We report a Turkish family with parental consanguinity and at risk for sialidosis type II, an inherited autosomal recessive disorder caused by lysosomal alpha-N-acetyl-neuraminidase (sialidase, NEU1) deficiency. The proband was a premature male infant that presented with hydrops, hepatomegaly, respiratory distress syndrome, and anemia and that died of respiratory insufficiency 2 months after birth despite intensive care. An abnormally increased [14C]methylamine incorporation and an isolated deficiency of lysosomal alpha-N-acetyl-neuraminidase were found in cultured skin fibroblasts. A previous pregnancy of the mother terminated in a spontaneous abortion in the 13th week of gestation. A successive pregnancy showed hydrops fetalis, and an enzymatic assay of cultured amniotic fluid cells indicated a deficiency of alpha-N-acetyl-neuraminidase. Following pregnancy termination at 20 weeks gestation, light microscopy of fetal tissues revealed classic vacuolation not only in liver, bone marrow, brain, and kidney, but also in endocrine organs such as the thyroid gland, adrenal gland, hypophysis, and testes, and in the thymus. DNA analysis of the family showed that both the proband and the third sibling had a novel homozygous nonsense point mutation at nucleotide 87 in exon 1 of the alpha-N-acetyl-neuraminidase (neu1) gene causing a substitution of tryptophan at codon 29 by a termination codon (W29X). DNA sequencing of polymerase chain reaction products identified the parents as heterozygous carriers. To detect neu1 mRNA expression, a real-time reverse transcription/polymerase chain reaction was performed, and similar rates of neu1 mRNA expression were found in the fibroblasts of the fetus, the 2nd sibling, and in controls. The very early termination codon with complete loss of neuraminidase activity is probably the molecular basis of the unusually severe vacuolation pattern in this form of congenital sialidosis.

Problem: Impaired trophoblast invasion during the first trimester of pregnancy is linked to spontaneous abortion, and defective invasion in the second trimester to hypertension + proteinuria (pre-eclampsia). Hypertension developing during the third trimester of human pregnancy represents, in part, a corrective response in the mother to provide adequate placental perfusion for fetal growth when trophoblast has not to invaded and converted the myometrial porprtion of maternal spiral arteries into to low resistance-high capacitance conduits. Deportation of vesicles from hypoxemic trophoblast is thought to cause hypertension plus proteinuria, vascular damage and a systemic coagulopathy. Trophoblast invasion may be inhibited by local cytokines, such as TGF-betas but Thl-type cytokines associated with pre-eclapmsia and spontaneous abortions (e.g., IL-1, TNF-alpha, IFN-gamma) are not known to inhibit migration at in situ concentrations. Trophoblast invasion is also inhibited by the binding of surface integrins to fibronectin and fibrin, and fibrin production is stimulated by these Th1 cytokines via up-regulation of prothrombinases(s) such as fg12 which directly and via TNF-alpha-facilitated inflamation compromise trophoblast cell integrity. We, therefore, asked if fg12 expression and TNF-alpha are increased in first trimester human miscarriage and in third trimester pre-eclampsia., Methods: fg12 mRNA was detected using in situ hybridization and fg12 protein by immunohistochemistry. TNF-alpha mRNA and protein were similarly tested. The techniques were validated using uterine sections from day 8.5 of CBA x DBA/2 pregnancies, and then were applied to sections of placentae from normal and pre-eclamptic pregnancies with and without intrauterine fetal growth restriction (IUGR). Fibrin was detectectd by immunohistochemistry., Results: Expression of fg12 protein correlated with fg12 mRNA expression in mouse uteri and in placentae from normal human pregnancies. Increased expression of fg12 and TNF-alpha mRNA and protein, and increased fibrin deposition was detected in placental trophoblast., Conclusions: Activation of fg12 prothrombinase by Th1-type cytokines in pregnancy may lead to spontaneous abortion, or in ongoing pregnancy, to pre-eclampsia and/or IUGR.

Objective: To investigate the selenium glutathione and glutathione peroxidase (glutathione-Px) levels in blood in women who experience miscarriage., Design: An observational study., Participants: Forty women with miscarriage occurring in the first and second trimesters., Methods: Concentrations were measured in whole blood and plasma. Glutathione was measured in red cells and glutathione-Px activity was measured in the red cells and plasma. The results were compared with 36 women in the same period of viable pregnancy and 28 age-matched, healthy, non-pregnant controls., Results: The selenium concentrations in whole blood and plasma of women following abortion were the same as in viable pregnancy, but were significantly lower compared with controls. The glutathione levels were significantly higher in women with miscarriage compared with viable pregnancies and with non-pregnant women. Red cell and plasma glutathione-Px activities of women who had had a miscarriage were significantly lower than in both normal pregnancies and the control group., Conclusion: The decreased activities of the antioxidant enzymes, red cell and plasma glutathione-Px, may play an important role in the aetrology of spontaneous abortion.

Background: Endocannabinoids such as anandamide are thought to have adverse effects on pregnancy and embryonic development. The activity of the degradative enzyme anandamide hydrolase may therefore be crucial for prevention of excessive concentrations of anandamide in the uterus, and thus prevention of pregnancy failure or female infertility. We tested this hypothesis in a preliminary study, and then used the results to find out whether anandamide hydrolase activity could predict miscarriage in a group of pregnant women., Methods: We assessed anandamide hydrolase activity in peripheral lymphocytes from 50 healthy, pregnant women at weeks 6-11 of gestation by a specific radiochromatographic method. The expression of the enzyme at the protein level was measured by ELISA with specific polyclonal antibodies. In a further study, we measured anandamide hydrolase concentration in 120 women who were 7-8 weeks pregnant and compared these findings with subsequent pregnancy outcome., Findings: In the first study, seven of the 50 women had a miscarriage. Anandamide hydrolase activity was lower in the seven women who miscarried than in the 43 who did not (60.43 pmol/min per mg protein [SD 29.34] vs 169.60 pmol/min per mg protein [30.20]; difference 109.17 pmol/min per mg protein [95% CI 26.64-191.70]; p<0.0001 by the Mann-Whitney test). Enzyme activity correlated with enzyme concentration, and a threshold concentration represented by an optical density (after ELISA) of 0.15 absorbance units at 450 nm separated the women who had miscarriages from those who did not. In the second study, 15 women had anandamide hydrolase concentrations below the threshold, and 105 had concentrations at or above the threshold. All 15 women in the low anandamide hydrolase group had miscarriages, compared with one of the 105 women with high concentrations (p<0.0001 by Fisher's exact test)., Interpretation: Decreased anandamide hydrolase activity and expression in peripheral lymphocytes is an early (<8 weeks of gestation) marker of spontaneous abortion, and may prove useful as a diagnostic tool for large-scale, routine monitoring of gestation. Our results also suggest that endocannabinoids might be critical in regulating the lymphocyte-dependent cytokine network associated with human fertility and successful pregnancy.

To investigate the relationship between inducible nitric oxide synthase (iNOS) and the early spontaneous abortion., in situ hybridization and immunohistochemistry were used to detect the expression of iNOS in trophoblasts in the early pregnancy with and without spontaneous abortion (group I and group II). By light microscopy and computer color magic image analysis system (CMIAS), light density (D) and the positive cell number per statistic square (N/S) in situ hybridization were used to analyze the positive cell index, while total positive cells (N) and the positive unit (Pu) were used in immunohistochemistry. By in situ hybridization, D and N/S in trophoblasts were 0.35 +/- 0.028, 0.07 +/- 0.011 respectively in group I and 0.18 +/- 0.016, 0.015 +/- 0.003 in group II. In terms of immunohistochemical staining, N and Pu were 0.058 +/- 0.007, 11.94 +/- 2.01 in group I and 0.013 +/- 0.009, 1.08 +/- 0.35 in group II in trophoblasts. Significant differences existed between two groups. It is concluded that the higher nitric oxide produced by the higher expression of iNOS in trophoblasts might play an important role in the early spontaneous abortion.

We conducted a nested case-control study to examine the association of microsomal epoxide hydrolase polymorphism with spontaneous abortion. The analysis included 127 cases and 107 controls from a rural community in China. The prevalence of the homozygous wild-type (AA), the heterozygous variant (Aa), and the homozygous variant (aa) in exon 3 of epoxide hydrolase was 13.4%, 34.6%, and 52.0% in cases and 27.1%, 30.8%, and 42.1% in controls, respectively. In contrast, the variant genotypes in exon 4 of epoxide hydrolase were less frequent in cases than controls. Using women with genotype AA as the referent, the adjusted odds ratio of spontaneous abortion was 2.69 [95% confidence interval (CI) = 1.33-5.42] for those with genotype Aa or aa in exon 3; but it was 0.45 (95% CI = 0.22-0.94) for those with genotype Aa or aa in exon 4, indicating that the two variants have opposite associations with spontaneous abortion. The findings persisted after adjustment for age, education, parity, smoking, alcohol use, occupation, and pesticide exposure, as well as in subgroup analysis. Moreover, for the variant genotypes Aa or aa in exon 3, the odds ratio was twice as great in those cases with three or more spontaneous abortions than in those with fewer spontaneous abortions.