妊娠期肝内胆汁淤积症胎盘OATP1A2和OATP1B3的表达及其多态性研究

目的:探讨胎盘OATP1A2和OATP1B3的表达水平及OATP1B3基因多态性与妊娠期肝内胆汁淤积症(ICP)的关系。方法:收集42例ICP胎盘和42例正常妊娠胎盘标本及母体静脉血,RT-q PCR和Western blot法分析OATP1A2和OATP1B3的mRNA及蛋白水平,测序法分析OATP1B3 c.334TG和c.699GA多态性位点基因型。结果:ICP胎盘中OATP1A2和OATP1B3的mRNA及蛋白表达水平均显著低于正常妊娠组(P0.05)。ICP组和正常妊娠组中,OATP1B3 c.334CT多态性纯合突变频率分别为50.0%和47.6%(P0.05),c.699GA多态性纯合突变频率分别为54.8%和45.2%(P0.05);两组的两种多态性的突变等位基因频率比较,差异均无统计学意义(70.2%vs 70.2%和73.8%vs 67.9%,P0.05)。ICP组OATP1B3不同基因型对其mRNA及蛋白的表达无显著影响(P0.05)。结论:ICP胎盘中OATP1A2和OATP1B3表达下调可能与ICP高胆汁酸的发生有关,而OATP1B3 c.334TG和c.699GA多态性不影响OATP1B3表达。     

妊娠期肝内胆汁淤积症孕妇胎盘合体滋养细胞OATP8基因表达及意义的研究

目的:探讨有机阴离子转运多肽8(OATP8)在妊娠期肝内胆汁淤积症(ICP)孕妇胎盘组织中的表达及其意义。方法:研究对象分为ICP轻度组(8例)、ICP重度组(8例)和正常对照组(8例)。采用免疫组化链霉亲和素-生物素-过氧化物酶复合物(SABC)法检测胎盘组织中OATP8蛋白定位表达;采用逆转录(RT)-PCR技术检测OATP8mRNA表达;蛋白印迹法检测OATP8蛋白表达水平。结果:①OATP8蛋白定位在胎盘合体滋养细胞膜上。②3组孕妇胎盘组织中OATP8mRNA的表达:ICP重度组明显高于对照组和ICP轻度组,差异均有统计学意义(P<0.05),ICP轻度组高于对照组,差异有统计学意义(P<0.05);③3组孕妇胎盘组织中OATP8蛋白的表达:ICP重度组高于轻度组,差异有统计学意义(P<0.05),ICP轻度组高于对照组,差异有统计学意义(P<0.05)。结论:ICP孕妇胎盘合体滋养细胞OATP8基因表达水平上调,可能有助于胎盘对胆汁酸或胆红素的清除作用。     

Effect of maternal cholestasis on TGR5 expression in human and rat placenta at term

Background & aims

TGR5 (Gpbar-1) is a plasma membrane-bound bile acid receptor expressed in several tissues, including liver, intestine and brain. High levels of TGR5 mRNA have been detected in human and rodent placenta, however, localization of the TGR5 protein has not been studied in this tissue. We aimed at characterizing TGR5 expression in placental tissue and investigated the effect of bile acids and progesterone metabolites, which accumulate during intrahepatic cholestasis of pregnancy (ICP), on receptor expression and localization.

Methods

TGR5 mRNA levels and cell-specific localization were determined by quantitative PCR and immunofluorescence, respectively.

Results

In human term placentas, TGR5 was mainly localized in fetal macrophages and to a lower extent in trophoblasts. In placentas from ICP patients and pregnant rats with obstructive cholestasis a marked down-regulation of TGR5 mRNA expression was observed. However, the cell-specific distribution of the TGR5 protein was unaffected. Besides bile acids, progesterone and its metabolites (5α-pregnan-3α-ol-20-one/5α-pregnan-3β-ol-20-one), which increase in serum during ICP, were able to dose-dependently activate TGR5. In addition, progesterone metabolites but not their sulfated derivatives nor taurolithocholic acid, significantly down-regulated TGR5 mRNA and protein expression in isolated human macrophages and a macrophage-derived cell line.

Conclusion

Since fetal macrophages and trophoblast cells are exposed to changes in the flux of compounds across the placental barrier, the expression of TGR5 in these cells together with its sensitivity to bile acids and progesterone metabolites regarding receptor activity and mRNA expression suggest that TGR5 may play a role in the effect of maternal cholestasis on the placenta.     

Down-regulation of soluble fms-like tyrosine kinase 1 expression in invasive placentation

Purpose

To confirm reduced expression of soluble fms-like tyrosine kinase 1 (sFlt-1) in accreta/increta.

Methods

Formalin-fixed tissue sections from 11 peripartum hysterectomies with invasive placentation and 5 controls were stained for sFlt-1. Stain intensity was scored in selected 100× microscopic fields. We compared sFlt-1 expression in invasive areas among cases, non-invasive areas among cases and areas from control placentas.

Results

Chorionic villi displayed significantly decreased sFlt-1 expression in invasive areas of cases compared to control placentas (p = 0.003), as well as in non-invasive areas of cases compared to control placentas (p = 0.01). There was no difference in sFlt-1 expression between invasive and non-invasive areas among cases.

Conclusions

Expression of sFlt-1 is diminished in villous trophoblasts from patients with placenta increta or percreta. Local depth of invasion was not associated with sFlt-1 expression, suggesting a more global abnormality across the implantation site rather than localized to areas of histologic invasion.

     

Cytokine-related genes and oxidation-related genes detected in preeclamptic placentas

Purpose

To investigate cytokine- and oxidation-related genes for preeclampsia using DNA microarray analysis.

Methods

Placentas were collected from 13 normal pregnancies and 13 patients with preeclampsia. Gene expression was studied using DNA microarray. Among significantly expressed genes, we focused on genes associated with cytokines and oxidation, and the results were confirmed using quantitative real time-polymerase chain reaction (QRT-PCR).

Results

415 genes out of 30,940 genes were altered by ≥2-fold in the microarray analysis. 121 up-regulated genes and 294 down-regulated genes were found to be in preeclamptic placenta. Six cytokine-related genes and 5 oxidation-related genes were found from among the 121 up-regulated genes. The cytokine-related genes studied included oncostatin M (OSM), fms-related tyrosine kinase (FLT1) and vascular endothelial growth factor A (VEGFA), and the oxidation-related genes studied included spermine oxidase (SMOX), l cytochrome P450, family 26, subfamily A, polypeptide 1 (CYP26A1), acetate dehydrogenase A (LDHA). These six genes were also significantly higher in placentas from patients with preeclampsia than in those from women with normal pregnancies. The placental tissue of patients with preeclampsia showed significantly higher mRNA expression of these six genes than the normal group, using QRT-PCR.

Conclusion

DNA microarray analysis is one of the great methods for simultaneously detecting the functionally associated genes of preeclampsia. The cytokine-related genes such as OSM, FLT1 and VEGFA, and the oxidation-related genes such as LDHA, CYP26A1 and SMOX might prove to be the starting point in the elucidation of the pathogenesis of preeclampsia.     

MiR133b is involved in endogenous hydrogen sulfide suppression of sFlt-1 production in human placenta

Increased production of soluble fms-like tyrosine kinase-1 (sFlt-1) from placenta is one of the major contributors to the development of preeclampsia. Our previous study has shown that hydrogen sulfide (H₂S) inhibits sFlt-1 release in placenta. In the present study, we sought to investigate whether endogenous H₂S affects sFlt-1 production and elucidate which H₂S-producing enzyme is responsible for its effect in placenta. It was found that, besides cystathionine β-synthase (CBS) and cystathionine γ-lyase (CSE), 3-mercaptopyruvatesulfurtransferase (3-MST) was identified in human placenta and mainly localized in syncytiotrophoblasts. There was no significant difference in expression level of 3-MST among preeclamptic and normal placentas. Treatment of cultured syncytiotrophoblasts with NaHS and l-cysteine suppressed sFlt-1 mRNA expression and caused a decrease in sFlt-1 protein content in culture media of the cells. Transfection of syncytiotrophoblasts with CBS siRNA and CSE siRNA reversed the above effects of l-cysteine. Furthermore, NaHS and l-cysteine treatment decreased the half-life of sFlt-1 mRNA and increased the expression of miR-133b targeting sFlt-1. MiR-133b expression was downregulated in preeclamptic placentas and correlated with the level of CBS and CSE. These results indicate that H₂S is an important regulatory factor in sFlt-1 production in placenta. Reduced H₂S generation in placenta contributes to development of preeclampsia by enhancing sFlt-1 production.     

Polymorphisms in genes HSD17B1 and HSD17B2 and uterine leiomyoma risk in Chinese women

Purpose

To evaluate the association of HSD17B1 and HSD17B2 gene polymorphisms with uterine leiomyoma in Chinese women.

Methods

121 Chinese women with clinically diagnosed uterine leiomyoma and 217 healthy normal Chinese women were investigated to compare three single nucleotide polymorphisms (SNPs) (rs605059 and rs676387 of HSD17B1 gene and rs8191246 of HSD17B2 gene) by polymerase chain reaction?Crestriction fragment length polymorphism and DNA sequencing method.

Results

All the SNPs were polymorphisms in Chinese women. Frequencies of rs605059 AA genotype and A allele were significantly increased in patients with uterine leiomyoma compared to healthy controls (GG vs. AA, OR 0.40, 95?% CI 0.20?C0.82; G vs. A, OR 0.68, 95?% CI 0.50?C0.94).

Conclusion

The results suggest that the genotype of HSD17B1 rs605059 may play a role in the tumourgenesis of uterine leiomyoma.     

Gene and protein expression of progesterone receptor isoforms A and B, p53 and p21 in myometrium and uterine leiomyoma

Purpose

To assess gene and protein expression of progesterone receptor isoforms A and B, cell cycle regulators p53 and p21 in leiomyoma and myometrium.

Methods

Samples were collected from 14 patients in reproductive age who underwent abdominal hysterectomy. Gene expression of PRA, PRB, p53 and p21 was analyzed by real-time PCR. Protein expression was assessed by Western blots.

Results

There was no change in gene and protein expression of PRA and PRB in both tissues. The ratio between isoforms (PRA:PRB) was not different between tissues and showed a strong correlation (r?=?0.767, P?=?0.004). The analysis of gene expression and protein showed increased levels of mRNA and protein p53 in leiomyoma compared to myometrium (P?=?0.030 and P?=?0.002, respectively). The same increase was observed in p21 mRNA levels (P?=?0.016) and protein p21 levels (P?=?0.026) in samples of uterine leiomyoma.

Conclusions

PRA:PRB ratio is similar in normal myometrium and leiomyomas. p53 and p21 mRNA and protein levels are increased in leiomyomas.     

Histone demethylase KDM4A and KDM4B expression in granulosa cells from women undergoing in vitro fertilization

Purpose

To assess expression of the histone demethylases KDM4A and KDM4B in granulosa collected from women undergoing oocyte retrieval and to determine if expression was related to pregnancy outcome.

Methods

Cumulus and mural granulosa cells were obtained from women undergoing oocyte retrieval. KDM4A and KDM4B mRNA expression was determined by qRT-PCR. KDM4A and KDM4B proteins were immunohistochemically localized in ovarian tissue sections obtained from archival specimens.

Results

KDM4A and KDM4B protein was localized to oocytes, granulosa cells, and theca and luteal cells in ovaries from reproductive-aged women. KDM4A and KDM4B mRNA expression was overall higher in cumulus compared to mural granulosa. When comparing granulosa demethylase gene expression, KDM4A and KDM4B mRNA expression was higher in both cumulus and mural granulosa from not pregnant patients compared to patients in the pregnant-live birth group.

Conclusions

Histone demethylases KDM4A and KDM4B mRNA are differentially expressed in cumulus and mural granulosa. Expression of both KDM4A and KDM4B mRNA was lower in cumulus granulosa and mural granulosa from pregnant compared to not pregnant patients. These findings suggest that altered expression of histone demethylases may impact epigenetic changes in granulosa cells associated with pregnancy.

     

Periconceptional alcohol consumption causes fetal growth restriction and increases glycogen accumulation in the late gestation rat placenta

Introduction

Alcohol consumption is a common social practice among women of childbearing age. With 50% of pregnancies being unplanned, many embryos are exposed to alcohol prior to pregnancy recognition and formation of the placenta. The effects of periconceptional (PC) alcohol exposure on the placenta are unknown.

Methods

Sprague-Dawley rats were exposed to alcohol (12.5% v/v ad libitum) from 4 days prior to 4 days after conception and effects on placental growth, morphology and gene/protein expression examined at embryonic day (E) 20.

Results

PC ethanol (EtOH)-exposed fetuses were growth restricted and their placental/body weight ratio and placental cross-sectional area were increased. This was associated with an increase in cross-sectional area of the junctional zone and glycogen cells, especially in PC EtOH-exposed placentas from female fetuses. Junctional Glut1 and Igf2 mRNA levels were increased. Labyrinth Igf1 mRNA levels were decreased in placentas from both sexes, but protein IGF1R levels were decreased in placentas from male fetuses only. Labyrinth mRNA levels of Slc38a2 were decreased and Vegfa were increased in placentas following PC EtOH-exposure but only placentas from female fetuses exhibited increased Kdr expression. Augmented expression of the protective enzyme 11βHsd2 was found in PC EtOH-exposed labyrinth.

Discussion

These observations are consistent with a stress response, apparent well beyond the period of EtOH-exposure and demonstrate that PC EtOH alters placental development in a sex specific manner.

Conclusion

Public awareness should be increased to educate women about how excessive drinking even before falling pregnant may impact on placental development and fetal health.     

Nitrofurantoin transport by placental choriocarcinoma JAr cells: involvement of BCRP, OATP2B1 and other MDR transporters

Objective  

To determine the role of BCRP in nitrofurantoin (NF) transport in JAr cells and the possible contribution of OATP2B1, P-gp and MRPs to this transport.     

Decreased expression of complement 3a receptor (C3aR) in human placentas from severe preeclamptic pregnancies

Objectives

The aim of this study was to determine the expression of the anaphylatoxin receptors complement C3a receptor (C3aR) and C5a receptor (C5aR) in the placentas of pregnancies complicated by severe early onset preeclampsia.

Study design

We recruited women with pregnancies complicated by severe early-onset preeclampsia (n = 19, 11 of which were further complicated with IUGR) and women with preterm pregnancies not affected by preeclampsia (n = 8). Gene and protein expression of C3aR and C5aR was analysed by quantitative RT-PCR and Western blotting, respectively.

Results

C3aR was detected in the Hofbauer cells in the villous stroma of the placenta. C5aR staining was detected in the syncytiotrophoblast and endothelial cells. We found significantly decreased expression of C3aR mRNA and protein expression in placentas with preeclampsia compared to controls. However, C5aR expression was not significantly different between preeclamptic and control placentas at either the mRNA or protein level.

Conclusions

Decreased C3aR expression indicates a dysregulation of the complement system in the placentas of preeclamptic women. Further studies would elucidate the exact mechanisms that complement has in preeclampsia.     

KISS1 expression in human female adipose tissue

Objective

The current focus of kisspeptin research is an exploration of its key role in the hypothalamic control of human and animal fertility. Notwithstanding the importance of these studies, strong evidence exists that the gene encoding human kisspeptin, KISS1, is present in several peripheral sites, including the placenta. We also provided evidence that kiss1 is also expressed and regulated in rodent adipose tissue. This study describes a pilot investigation into the possibility that human female adipose tissue might also express the KISS1 gene.

Methods

Samples of fat were taken from women undergoing open abdominal surgery, for example, during caesarian section. Two small samples of fat were obtained, one from subcutaneous tissue (n = 35) and one from the omentum (n = 32). RNA was isolated from all fat samples and KISS1 mRNA was detected by realtime RT-PCR.

Results

KISS1 gene expression was detected at varying levels in all samples of fat tissue but levels were significantly higher in subcutaneous fat. There was no significant correlation between KISS1 gene expression and body mass index (BMI) in subcutaneous fat (P = 0.43), but there was a significant positive correlation (P = 0.01) between KISS1 mRNA levels and BMI in omental adipose tissue.

Conclusion

We have shown for the first time that human female adipose tissue may be a source of kisspeptins. Further studies are required to establish whether kisspeptins of adipose tissue origin might be correlated with some aspects of infertility.     

HMGB1-RAGE signaling pathway in pPROM

Objective

Increased inflammation of the placenta is considered as a risk factor and a promoter of preterm premature rupture of the membranes (pPROM). High-mobility group box 1 (HMGB1) is a recently identified inflammatory cytokine, and HMGB1-RAGE signaling pathway has been associated with many pathophysiological processes. This study aims to reveal the mechanisms of HMGB1-RAGE signaling pathway in pPROM.

Thrombospondin-1 serum levels do not correlate with pelvic pain in patients with ovarian endometriosis

Objetive

Thrombospondin-1 serum levels is correlate with pelvic pain in patients with ovarian endometriosis.

Patients

Thrombospondin-1 serum levels were prospectively analysed in 51 patients (group A asymptomatic patients or patients presenting mild dysmenorrhea and women comprised group B severe dysmenorrhea and/or chronic pelvic pain and/or dyspareunia) who underwent surgery for cystic ovarian endometriosis to asses whether a correlation exists among thrombospondin-1 serum levels and pelvic pain.

Results

From 56 patients, five cases were ultimateley excluded, because the histological diagnosis was other than cystic ovarian endometriosis (2 teratomas and 3 haemorragic cysts). The mean thrombospondin-1 serum levels in group A was 256,69 pg/ml_+37,07 and in group B was 291,41 pg/ml + 35,59.

Conclusion

Pain symptoms in ovarian endometriosis is not correlated with thrombospondin-1 serum levels.     

Intercellular adhesion molecule-1 expression in massive chronic intervillositis: Implications for the invasion of maternal cells into fetal tissues

Introduction

Massive chronic intervillositis (MCI), also known as chronic intervillositis of unknown etiology, is a placental lesion associated with massive infiltration of mononuclear cells in the intervillous space, poor perinatal outcome, and high rate of recurrence. Our previous demonstration of increased syncytiotrophoblast (st) intercellular adhesion molecule-1 (ICAM-1) expression in villitis lesions and the finding of extensive monocyte/macrophagic cells in the maternal intervillous space in MCI, led us to further investigate stICAM-1 in MCI.

Materials and methods

A cross-sectional study of placentas from the third trimester of pregnancy (34–41 weeks gestation) was conducted to determine stICAM-1 in MCI (n = 7). MCI stICAM-1 expression was compared to stICAM-1 in villitis (n = 7) and in normal villi from placentas with (n = 7) and without (n = 7) villitis. Maternal cells within villi in MCI were identified in placentas mismatched for maternal/fetal human leukocyte antigen (HLA)-DRw52. Villitis was diagnosed with hematoxylin and eosin staining and antibody to CD3 in serial sections, and ICAM-1 in syncytiotrophoblasts was confirmed with antibodies to ICAM-1 and cytokeratin.

Results

Placentas with MCI had higher stICAM-1 (79.8%) than placentas with villitis (27.1%), normal villi from placentas with villitis (11.5%), and normal villi from placentas without villitis (0.3%). Maternal cells were identified within villi of placentas (n = 5) mismatched (mothers positive, fetuses negative) for HLA-DRw52.

Conclusions

Placentas with MCI have more stICAM-1 than placentas with or without villitis lacking MCI. The finding that MCI and villitis have prominent stICAM-1 and maternal cells in the villi suggests that MCI and villitis could have a similar pathophysiologic mechanism.