The tumour suppressor gene maspin is differentially regulated in cytotrophoblasts during human placental development

Identification of factors that play a role in regulating the highly invasive ability of human placental cells throughout gestation will contribute to a better understanding of this unique developmental process. The aims of this study were to determine whether the tumour suppressor gene maspin is present in the human placenta and plays a putative role in the regulation of cytotrophoblast invasion during placental development. The data showed that the expression of maspin mRNA was maximum in term placentae compared to the first and second trimester tissues, and absent in the HTR-SVneo (immortalized extravillous cytotrophoblast), JEG-3 and JAR (choriocarcinoma) cell lines. Maspin protein, detected by Western blot analysis, was twofold higher in the second trimester and 4.4-fold higher in the third trimester compared to the first trimester. Maspin immunohistochemical staining was localized in cytotrophoblasts with increased and more diffuse staining in the second and third trimesters. Corresponding to the period of maximum maspin expression, cytotrophoblasts isolated from term placentae had significantly lower invasive ability as compared to first and second trimester cytotrophoblasts (P< 0.03). Further, addition of recombinant maspin significantly decreased cytotrophoblast invasion in vitro by 40-50 per cent in all three trimesters of gestation. This study provides the first evidence of the temporal expression of maspin during human gestation and suggests a putative role for maspin in regulating the invasive activity of cytotrophoblasts at term. The down-regulation of maspin expression may be critical at the time of implantation and early placental development, whereas upregulation of maspin may serve as a signal for the end of cytotrophoblast invasion and gestation.     

Immunocytochemical localization of chorionic gonadotropin, placental lactogen, and placental alkaline phosphatase in the diagnosis of complete and partial hydatidiform moles

Complete hydatidiform moles (CHMs) and partial hydatidiform moles (PHMs) represent different clinicopathologic entities with characteristic morphologic and cytogenetic findings. In the absence of cytogenetic data, the histologic distinction between these lesions and abortuses showing hydropic swelling (AHS) may be difficult. An immunocytochemical study analyzing the distribution of human chorionic gonadotropin (hCG), human placental lactogen (hPL), and placental alkaline phosphatase (PlAP) in CHMs, PHMs, and AHS was undertaken to determine whether the expression of these trophoblastic proteins might assist in the differential diagnosis. A total of 24 CHMs, 22 PHMs, and 13 AHS were selected on the basis of established morphologic criteria. Thirty-four specimens of abortuses without hydropic swelling and normal placentas, ranging from 6 to 24 weeks gestational age, were similarly analyzed. The immunocytochemical localization of the three trophoblastic proteins, predominantly in syncytiotrophoblast (ST), was scored using a semiquantitative scoring system. In CHMs hCG is widely distributed and PlAP is patchily distributed in ST regardless of the gestational age, whereas hPL tends to increase with increasing gestational age. In contrast, in PHMs hPL is more widely distributed in ST compared with CHMs regardless of gestational age, while PlAP increases with increasing gestational age; in PHMs the distribution of hCG is markedly less than in CHMs except early in the first trimester when the staining patterns are similar. The different patterns of distribution of hCG, hPL, and PlAP may reflect differences in the pathobiology of trophoblast in CHMs and PHMs and appear to be useful in the differential diagnosis of these conditions.     

Expression of CD44, E-cadherin, and antimetastatic protein nm23-H1 in complete hydatidiform moles

INTRODUCTION: There is scant information about the expression of CD44 and E-cadherin, two cell adhesion molecules, and the antimetastatic protein nm23-H1, in complete hydatidiform moles. We measured the expression of these markers to determine their usefulness in predicting the development of invasive disease. MATERIALS AND METHODS: We performed a retrospective study of 27 patients with complete hydatidiform moles, collecting clinical information including the patient's age, pre-evacuation hCG level, pathology, hCG monitoring, and the development of gestational trophoblastic neoplasia. Immunohistochemical staining for CD44, E-cadherin, and nm23-H1 was performed. CD44 expression was classified as positive or negative. For E-cadherin and nm23-H1, the intensity of expression was graded on a 0 to 3 scale. Chi-square or Fisher's exact testing was used to evaluate the relationship between these markers and the development of invasive disease. RESULTS: CD44 was expressed in 26% of cases. E-cadherin expression was 1+, 2+, and 3+in 8%, 33%, and 59% of cases, respectively. Nm23-H1 expression was 1+, 2+, and 3+in 4%, 11%, and 85% of cases. The risk of developing invasive disease did not correlate with the expression of CD44, E-cadherin, or nm23-H1. CONCLUSION: In this preliminary study, there is no relationship between CD44, E-cadherin, and nm23-H1 expression in complete hydatidiform moles and the risk of invasive disease. Other molecular markers predictive of invasive disease should be sought to limit hCG surveillance to those at risk.     

Fatty Acid Binding Protein-4 is expressed in the mouse placental labyrinth,yet is dispensable for placental triglyceride accumulation and fetal growth

Introduction

Fatty Acid Binding Protein-4 (FABP4) is a member of a family of FABP proteins that regulate intracellular lipid trafficking in diverse tissues. We recently showed that FABP4 regulates triglyceride accumulation in primary human trophoblasts. To assess the function of placental FABP4 in vivo, we tested the hypothesis that FABP4 is expressed in the murine placenta, and regulates placenta triglyceride accumulation.

Methods

C57Bl/6 wild type or Fabp4-null mice were time-bred, and fetuses and placentas harvested at different time points during pregnancy. Placental FABP4 expression was assessed at different gestational ages, using quantitative PCR, immunohistochemistry, immunofluorescence and western immunoblotting. FABPs expression was examined by RT-qPCR. Placental lipids were extracted using the Folch method and triglyceride levels determined using a colorimetric quantification kit.

Results

Using immunohistochemistry, we found that FABP4 was expressed in the placental labyrinthine layer, predominantly in endothelial cells in association with CD31 positive fetal capillaries. The level of placental FABP4 mRNA and protein increased from E12.5 to E16.5 and slightly decreased at E18.5. Breeding of Fabp4 heterozygous mice resulted in embryonic genotypes that followed a Mendelian distribution and exhibited normal weight and morphology, triglyceride content, and expression of other FABP family members. Exposure to hypoxia (O₂ = 12%) between E12.5–E18.5 did not uncover a difference between wild type and Fabp4-null mice.

Conclusions

FABP4 is expressed in the mouse placental labyrinth, with highest expression at E16.5. FABP4 is dispensable for feto-placental growth and placental lipid accumulation.     

Angiogenesis during implantation, and placental and early embryonic development

Angiogenesis, the development of new capillaries from pre-existing vessels, is induced by inflammation, wound healing, immune reactions and neoplasia, and is required for tumour growth and progression. Angiogenesis participates in a wide range of ovulatory-related and non-ovulatory-related reproductive processes. We present a review of current data pertaining to angiogenesis of pregnancy, with specific emphasis on implantation and placental and embryonic development in both normal physiology processes and various pathological conditions. To this goal, MEDLINE, Current Contents and Index Medicus were searched for studies published between 1966 and August 1999. Pertinent studies (including human and animal models) pertaining to angiogenesis of implantation and placental and embryonic development were reviewed. Current literature supports that angiogenesis is an essential physiological component of implantation, and placental and embryonic development. Angiogenesis also actively participates in abnormal implantation, and various pathological processes of the placenta including those observed in association with pre-eclampsia, growth restriction, maternal anaemia in the first-trimester and other hypoxia-related conditions during pregnancy. Finally, administration of an angiogenesis inhibitor (AGM-1470) in mice has been shown to result in complete failure of embryonic growth due to interference with decidualization, placental and yolk sac formation, and embryonic vascular development.     

Localization of telomerase hTERT protein and survivin in placenta: relation to placental development and hydatidiform mole

OBJECTIVE: To find if a difference in telomerase or survivin expression exists between non-neoplastic tissues and hydatidiform moles, and explore expression of those proteins in normal placental development, post-term gestation, and preeclampsia. METHODS: Formalin-fixed placental tissues were selected from collections of the Department of Pathology at the University of Colorado. Five specimens of each trimester, five each of preeclamptic and post-term placentas, and 23 molar pregnancies were selected. The telomerase catalytic protein hTERT was localized in placental tissues using the catalyzed signal amplification system, and survivin was localized by conventional immunoperoxidase method. Staining was graded on a scale of zero to 4. RESULTS: hTERT staining was detected in sections of 42 of 48 specimens (23 of 23 hydatidiform moles, 19 of 25 non-neoplastic placental tissues). The intensity of staining for hTERT was higher in hydatidiform moles (mean 3.3, median 3) compared with levels in non-neoplastic placental tissues (mean 0.92, median 1) (P <.001). Survivin was detected in 39 of 48 specimens (22 of 23 hydatidiform moles, 17 of 25 non-neoplastic placental tissues). Compared with non-neoplastic tissues (mean 0.88, median 1), survivin levels were elevated in hydatidiform moles (mean 1.35, median 1) (P =.031). CONCLUSION: Survivin and telomerase were increased in hydatidiform moles, suggesting that regulation of apoptosis and stabilization of telomere length might be involved in neoplastic transformation of the placenta. The patterns of expression observed for survivin and telomerase in non-neoplastic placental tissues suggest that the control of apoptosis and stabilization of telomeric DNA might also be involved in normal gestational development.     

Increased expression of interleukin-1 beta is associated with persistence of the disease and invasion in complete hydatidiform moles (CHM)

Complete hydatidiform moles (CHM), a post-conceptual pathologic condition of the placenta, have a high prevalence rate (12/1,000 deliveries) in Kerala, India. This study addresses the expression of IL-1 alpha and beta by immunohistochemistry in relation to persistence and invasion of the disease. Mild to moderate expression of IL-1 alpha in the villous cytotrophoblasts, syncytiotrophoblasts and decidua of the first trimester in the normal placenta and all gestational ages in the molar placenta were observed. IL-1 beta expression was observed in the extravillous trophoblasts, syncytiotrophoblasts and decidua in both the normal and molar placentae and also in the villous cytotrophoblasts and the stromal Haufbaur cells in molar placentae. Strong expression of IL-1 beta in the placenta suggests its involvement in placental physiology supporting earlier reports. Higher expression of IL-1 beta correlated well with the invasive and persistent nature of the tumour and holds potential as a marker of persistence and invasion in CHM.     

Microarray analysis of differentially expressed fetal genes in placental tissue derived from early and late onset severe pre-eclampsia

Although it has been well documented that pre-eclampsia is caused by a combination of maternal and fetal susceptibility genes, little is known about the precise etiology of this complicated disorder. To investigate how the expression of fetal genes contributes to the mechanisms underlying the progression of this disease, we have analyzed differentially expressed genes using placentas from 13 normal pregnancies and 14 pregnancies with severe pre-eclampsia. We performed genome-wide expression profiling using high-density oligonucleotide microarrays, followed by validation using real-time PCR. Among the 47,000 genes that were screened in the microarray, 137 genes were found to be differentially expressed between normal and pre-eclamptic tissues. Among these candidates, 70 were up-regulated and 67 were down-regulated. The up-regulated genes included leptin and inhibin A, which are well-known biological markers for pre-eclampsia, as well as FLT1, which was recently proved to be tightly linked with the etiology of this disease. Gene ontology analysis further revealed several biological processes that could be associated with the development of pre-eclampsia, including response to stress, host-pathogen interactions, lipid metabolism, and carbohydrate metabolism. Analyses of biological mechanisms highlighted some important pathways that may be involved in this disorder, such as the TGF-beta and CEBPA-related pathways. Furthermore, when our present subjects were classified as either severe cases of early onset or late onset pre-eclampsia, the expression of 11 genes could be correlated with the severity of this disorder. These genes may therefore prove to be novel biological markers by which the severity of this condition could be predicted. Our data are likely to be a useful future resource in the elucidation of the disease-process and in the identification of novel markers for pre-eclampsia.     

A distinct pattern in the DNA ploidy histograms of hydatidiform moles and nonmolar abortuses is caused by accumulation of trophoblasts in the late s-phase.

DNA ploidy analysis is a useful tool to distinguish the partial hydatidiform moles (PMs) from complete hydatidiform moles (CMs) and nonmolar abortuses (NAs). DNA ploidy histograms of hydatidiform moles are sometimes difficult to interpret because of the uneven distribution of nuclei in the S-phase, simulating aneuploid peaks. In this study, we analyzed DNA ploidy histograms of 25 CMs, 16 PMs, and 28 NAs, with special reference to the accumulation of cells in the late S-phase using a high-resolution DNA image cytometry. All the gestational products demonstrated the accumulation of cells in the late part of the S-phase fraction. To objectify the observation, we compared the percentage of cells in the second quarter with that of the third quarter of the S-phase fraction. All the gestational products had significantly lower (P < 0.001) percentage of cells in the second compared with that of the third quarter of the S-phase. The mean ratios of the third quarter to the second quarter in CMs, PMs, and NAs were 1.9, 1.7, and 2.5, respectively. This was significantly different from that of highly proliferative endometrial carcinomas. The knowledge of this specific S-phase fraction distribution in molar and nonmolar pregnancy material is important when interpreting the DNA histograms. The possibility of hypoxia being the cause of this phenomenon is also discussed.     

Plasma homocysteine,vitamin B<Subscript>12</Subscript> and folate levels in hydatidiform moles and histopathological subtypes

Objective  To investigate the association of mole hydatidiform with plasma homocysteine, vitamin B₁₂, and folate levels. Methods  Sixty-eight patients diagnosed with mole hydatidiform at our clinic between February and October 2007 were assessed in a case-control study. Plasma homocysteine, vitamin B₁₂, and folate levels, taken before evacuation of patients with hydatidiform mole, were compared with the results of 100 healthy normal pregnants of first trimester; and also plasma homocysteine, vitamin B₁₂, and folate levels were compared according to histopathological types of mole hydatidiforms. SPSS 14.0 package program was used to analyze the data. Logarithmic transformation was applied for variables. Parameters were expressed as mean ± standard deviation. Results  The mean of plasma homocysteine levels was higher in molar group (0.8 ± 0.13) than in normal pregnant group (0.7 ± 0.13) and the difference was statistically significant (P < 0.001). The mean of plasma vitamin B₁₂ levels was found to be similar both in normal pregnant women (2.4 ± 0.17) and in the molars (2.4 ± 0.15) (P = 0.272). The mean of plasma folate levels was lower in molar group (1.0 ± 0.15) than in normal pregnant women (1.2 ± 0.17) and the difference was statistically significant (P < 0.001). The hydatidiform moles of 68 patients were divided into two groups according to histopathological examination: 36 patients were partial moles and the others were complete. The difference of plasma mean homocysteine, vitamin B₁₂, and folate levels in these two groups was not statistically significant. There were statistically significant differences of plasma mean homocysteine and folate levels one by one in complete and in partial moles when compared with the normal pregnants. The mean of plasma folate levels were lower (1.0 ± 0.17 for partials, 1.0 ± 0.13 for completes) and the homocysteine levels were higher (0.9 ± 0.14 for partials, 0.8 ± 0.12 for completes) than the levels of normal group. Conclusion  This study suggests that there may be an association between plasma folate and homocysteine levels with hydatidiform moles. Folate may play a protective role in preventing molar pregnancy. Further controlled prospective studies are needed to investigate the possible effect of homocysteine, vitamin B₁₂, and folate in molar pregnancies.     

The genetics of recurrent hydatidiform moles in Mexico: further evidence of a strong founder effect for one mutation in NLRP7 and its widespread

Journal of Assisted Reproduction and Genetics - To investigate the frequency of a founder mutation in NLRP7, L750V, in independent cohorts of Mexican patients with recurrent hydatidiform moles...