Altered decidual and placental catabolism of vitamin D may contribute to the aetiology of spontaneous miscarriage.

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 CoCL2 (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 CoCL2 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.

Early pregnancy loss in 15-hydroxyprostaglandin dehydrogenase knockout (15-HPGD-/-) mice due to requirement for embryo 15-HPGD activity.

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 (PGE2) and prostaglandin F2α (PGF2α), 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 PGF2∝ 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.

poFUT1 promotes uterine angiogenesis and vascular remodeling via enhancing the O-fucosylation on uPA.

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.

Why AMPK agonists not known to be stressors may surprisingly contribute to miscarriage or hinder IVF/ART.

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.

Potential roles of metalloproteinases of endometrium-derived exosomes in embryo-maternal crosstalk during implantation.

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.

Association of Plasminogen Activator Inhibitor-1 Gene Polymorphisms and Methylene Tetrahydrofolate Reductase Polymorphisms with Spontaneous Miscarriages.

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.

Infertility and recurrent miscarriage with complex II deficiency-dependent mitochondrial oxidative stress in animal models.

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.

[Association of gene polymorphisms of matrix metalloproteinases with reproductive losses in the first trimester of pregnancy].

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 MMP1­1607insG, MMP9 A­8202G, and TIMP1 С536T, were reported. The frequency of homozygotes for allele MMP9 A­8202 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.

Triclosan causes spontaneous abortion accompanied by decline of estrogen sulfotransferase activity in humans and mice.

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.

Polymorphisms in the methylene tetrahydrofolate reductase and methionine synthase reductase genes and their correlation with unexplained recurrent spontaneous abortion susceptibility.

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.

Altered ß1,6-GlcNAc and bisecting GlcNAc-branched N-glycan on integrin ß1 are associated with early spontaneous miscarriage in humans.

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 ß1,6-N-acetylglucosamine (ß1,6-GlcNAc) and bisecting N-acetylglucosamine (bis-GlcNAc) branched N-glycans on integrin ß1. 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 ß1,6-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 α5ß1 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 ß1,6-GlcNAc and bis-GlcNAc structures in integrin α5ß1 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, ß1,6-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 ß1,6-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. ß1,6-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 ß1,6-GlcNAc-branched N-glycans and increased bis-GlcNAc-branched N-glycans on integrin ß1 (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.

[Involvement of matrix metalloproteinases in obstetrics: from implantation to delivery]. / Implication des métalloprotéases matricielles en obstétrique: de la nidation à l'accouchement.

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.

Polymorphisms of human placental alkaline phosphatase are associated with in vitro fertilization success and recurrent pregnancy loss.

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.

Hyaluronan up-regulates growth and invasion of trophoblasts in an autocrine manner via PI3K/AKT and MAPK/ERK1/2 pathways in early human pregnancy.

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.

Reduced serum selenium concentration in miscarriage incidence of Indonesian subjects.

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 activity in normal pregnancy and pregnancy associated disorders.

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.

Circulating matrix metalloproteinase MMP-9 and MMP-2/TIMP-2 complex are associated with spontaneous early pregnancy failure.

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.

Paradoxical role of C1561T glutamate carboxypeptidase II (GCPII) genetic polymorphism in altering disease susceptibility.

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.

Protective role of heme oxygenase-1 in Listeria monocytogenes-induced abortion.

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.

Study of methyl transferase (G9aMT) and methylated histone (H3-K9) expressions in unexplained recurrent spontaneous abortion (URSA) and normal early pregnancy.

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.