Elsevier

Placenta

Volume 61, January 2018, Pages 1-10
Placenta

Proteomics and bioinformatics analysis of altered protein expression in the placental villous tissue from early recurrent miscarriage patients

https://doi.org/10.1016/j.placenta.2017.11.001Get rights and content

Highlights

  • Identified 314 differentially expressed proteins in placental villous tissue from recurrent miscarriage patients.

  • These 314 proteins were found to play important roles in the growth of embryo.

  • Network analysis show that Angiotensinogen (AGT), MAPK14 and Prothrombin (F2) are core factors in early embryonic development.

Abstract

Introduction

Recurrent miscarriage (RM) affects 5% of women, it has an adverse emotional impact on women. Because of the complexities of early development, the mechanism of recurrent miscarriage is still unclear. We hypothesized that abnormal placenta leads to early recurrent miscarriage (ERM). The aim of this study was to identify ERM associated factors in human placenta villous tissue using proteomics. Investigation of these differences in protein expression in parallel profiling is essential to understand the comprehensive pathophysiological mechanism underlying recurrent miscarriage (RM).

Methods

To gain more insight into mechanisms of recurrent miscarriage (RM), a comparative proteome profile of the human placenta villous tissue in normal and RM pregnancies was analyzed using iTRAQ technology and bioinformatics analysis used by Ingenuity Pathway Analysis (IPA) software.

Results

In this study, we employed an iTRAQ based proteomics analysis of four placental villous tissues from patients with early recurrent miscarriage (ERM) and four from normal pregnant women. Finally, we identified 2805 proteins and 79,998 peptides between patients with RM and normal matched group. Further analysis identified 314 differentially expressed proteins in placental villous tissue (≥1.3-fold, Student's t-test, p < 0.05); 209 proteins showed the increased expression while 105 proteins showed decreased expression. These 314 proteins were analyzed by Ingenuity Pathway Analysis (IPA) and were found to play important roles in the growth of embryo. Furthermore, network analysis show that Angiotensinogen (AGT), MAPK14 and Prothrombin (F2) are core factors in early embryonic development. We used another 8 independent samples (4 cases and 4 controls) to cross validation of the proteomic data.

Discussion

This study has identified several proteins that are associated with early development, these results may supply new insight into mechanisms behind recurrent miscarriage.

Introduction

Recurrent miscarriage (RM), the loss of two or more consecutive pregnancies prior to the 20th week of gestation; it is one of the most common clinical problems in reproduction, affects 5% of couples trying to conceive [1], [2]. The known causes of RM include genetic or chromosomal abnormalities, endocrinological disorders, anatomic anomalies, and other such factors [3], [4], [5]. However, these related molecular mechanisms of recurrent miscarriage are still unexplained.

The placenta is the organ that transports nutrients, respiratory gases, and wastes between the maternal and fetal systems [6]. In the early embryonic development, the placental barrier facilitates the embryonic growth and effectively avoiding radical damage; once embryogenesis is complete, the maternal intervillous circulation becomes fully established [7]. There is speculation that an abnormal placenta leads to early recurrent miscarriage (ERM), but the unclear relationship between the placenta and ERM remains requiring further elucidation.

In contrast to conventional biochemical approaches that monitor one or only a few specific proteins at a time, proteomics, particularly the combination of iTRAQ and 2D-LC-MS/MS, is a useful method to recognize altered protein expression as well as proteins involved in disease pathogenesis. In 2006, Kim, Y.S. et al. have identified for the first time recurrent miscarriage (RM)-associated proteins in follicular fluid of RM patients using 2D-gel-based proteomic tools [8]. They think that coagulation factors (fibrinogen γ and antithrombin) play an important role in maintaining the normal pregnancy. Later in 2011, they analyzed blood samples from normal and RM patients to conduct a comparative proteomic study, these results suggest that ITI-H4 expression may be used as a biomarker [9]. In 2014, Metwally, M. performed a proteomic analysis of the endometrium in obese and overweight women with recurrent miscarriage. These findings provide preliminary evidence for an alteration in the endometrial protein profile in overweight/obese women with recurrent miscarriage mainly in the form of increased haptoglobin, an inflammatory marker associated with obesity [10].

Nevertheless, up to date, there have been no reported attempts to screen the proteins associated with RM involved in placenta villous tissue, using large-scale proteomic analysis. Therefore, in this study, we aim to establish a comparative proteome profile of the human placenta villous tissue in normal and RM pregnancies using the iTRAQ technology coupled with 2D nano LC-MS/MS. In addition, we used Ingenuity Pathway Analysis (IPA) software to analysis these differentially expressed proteins to determined molecular function, downstream effects, and upstream regulator, which are useful for providing molecular insights into early recurrent miscarriage (ERM).

Section snippets

Clinical specimen collection and preparation

For this study, a total number of eight placental villous samples, four from ERM patients and four from elective abortions, were collected from Shaoxing women and children hospital in Zhejiang, China. Placental villous tissues were taken through the cervix during dilatation and aspiration according to strict clinical procedures. Informed consents were provided by all participants. This study was approved by the Research and Ethics Committee of the Shaoxing women and children hospital in

Protein profiles of placental villous tissue

In this work, we used high accuracy LC-MS/MS to quantitatively detect and map proteins in human placenta villous specimens of the ERM and control groups. Using iTRAQ technology, we identified 2805 non-redundant proteins (Additional file 1: Table S1) in human placenta villous with high confidence (one or more unique peptides with an FDR less than 1%). The detailed information of the identified peptides is shown in Table S2 (Additional file 2). Among them, 314 proteins were found to be

Discussion

Proteins that perform biological functions directly are rich in information that has been extremely valuable for the description of biological processes. The correlation between mRNA/DNA and protein levels is insufficient to predict protein expression levels [13]. Proteomics has many advantages compared with other technology. Mass spectrometry (MS) allows the multivariate analysis of complex patterns of new biomarkers without knowledge of the human proteome could help fill the gap between

Authors' roles

H-G.D, M.F, B.Y, Q-Q.F, B.L and H-G.C prepared samples. H-T.P and Y.C performed LC-MS/MS analysis. H-T.P, Y-J.T, X. J and X-Q.X analyzed the data, Z.T, H-G.D, M.F and H-T.P designed and conceived the study. All authors read and approved the final manuscript.

Conflicts of interest

None declared.

Acknowledgements

We thank the Shanghai Bioprofile Technology Co., Ltd., for technological assistance. This work was supported by the National Natural Science Foundation of China (No. 81701522); the Science Technology Department of Zhejiang Province, China (No.2015C33280) and the Health and Family Planning Commission of Zhejiang Province, China (No.2014KYA276, No.2017KY669 and 2018KY845); the Natural Science Foundation of Zhejiang Province, China (No.LY14H040003, No.LY14H040004, No.LQ15H040001 and No.LY15H040001

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