Endometrial stromal cell proteome mapping in repeated implantation failure and recurrent pregnancy loss cases and fertile women

Abstract

Research question

Are there proteomic differences between endometrial stromal cells of repeated implantation failure (RIF), recurrent pregnancy loss (RPL) and normal fertile women, and is there differential protein expression upon decidualization?

Design

This exploratory study investigated the proteome of in-vitro cultured endometrial stromal cells of women with RIF (n = 4), women with RPL (n = 3) and normal fertile women (n = 4), comparing day 0 with 5 days of decidualization. Total proteins extracted from cell lysates were analysed by high-definition mass spectrometry. Data analysis was performed using significance analysis of microarray in R (P < 0.05; false discovery rate [FDR] 10%).

Results

In the RIF group, ANXA6, PSMC5 and FSCN1 were up-regulated (1.9-fold, 2.5-fold and 1.9-fold, respectively), whereas PBXIP1 was down-regulated (7.7-fold) upon decidualization. In the RPL group, RPS25 and ACADVL were down-regulated (1.9-fold and 2.4-fold, respectively; FDR 10%) between the non-decidualized and the decidualized samples. In the normal fertile group VIM and RPL23A were down-regulated (1.9-fold and 2.4-fold, respectively). Comparing ratios of expression of decidualized over non-decidualized samples in the different groups revealed six differentially expressed proteins: DUX4L2, CNPY4, PDE7A, CTSK, PCBP2 and PSMD4. Comparison of RPL versus normal fertile in the decidualized condition revealed serotransferrin to be differentially expressed. The changes in expression levels for serotransferrin, ANX6, ACDVL and VIM were confirmed by western blot.

Conclusions

Results show a varying response of endometrial stromal cells in distinct clinical groups (RIF, RPL and normal fertile) upon in-vitro decidualization. Serotransferrin could serve as a marker for the aberrant decidualization process in RPL.

Introduction

Endometrial receptivity is an important concept in the biology of implantation, as it permits a framework in which temporal and spatial interactions occur between the endometrium and the embryo (Giudice, 1999). Decidualization denotes the transformation of endometrial stromal fibroblasts into specialized decidual secretory cells, and is considered to be the key process that accounts for the establishment of a receptive endometrial microenvironment to enable and sustain pregnancy (Gellersen and Brosens, 2014, Gibson et al., 2016). According to the embryo biosensoring and selection hypothesis, the process of decidualization is not only critical for placental development but also signals the end of the implantation window and enables the endometrium to recognize, respond to and eliminate implanting compromised embryos (Macklon and Brosens, 2014, Teklenburg et al., 2010). However, the mechanisms behind this vast cellular reprogramming of endometrial stromal cells upon decidualization are not yet fully understood. In-vitro decidualization of primary cell cultures provides a powerful tool to study these molecular mechanisms. Not only do these cells maintain their in-vivo characteristics in low passages, they also allow for a more controlled manipulation of cellular functions and processes (Irwin et al., 1989, Krikun et al., 2004).

Growing evidence suggests that failure of the endometrium to express a receptive phenotype is thought to be a major cause of repeated implantation failure (RIF), and considered to be linked to recurrent pregnancy loss (RPL).

Several studies have investigated gene expression in endometrial cells from RIF patients as compared with fertile patients. These studies showed a deregulation of the receptive endometrial gene expression profile in RIF patients. The altered gene expression involved a multitude of diverse biological processes and pathways such as cell adhesion, cytoskeleton formation, cell motility, ECM–receptor interaction, signal transduction, Wnt signalling, LIF signalling, JAK-STAT signalling, PAR signalling, hedgehog signalling, cytokine–cytokine receptor interaction, transcription, xenobiotic metabolism, complement and coagulation cascade, regulation of cell cycle and apoptosis (Altmae et al., 2016, Choi et al., 2016, Herington et al., 2016, Huang et al., 2017, Koler et al., 2009, Koot et al., 2016, Shi et al., 2017).

Likewise, evidence suggests that distorted endometrial receptivity might account for RPL. The altered gene expression thus far investigated in RPL involves several biological processes and pathways such as cell adhesion, cell migration, cell differentiation, cell signalling and communication, angiogenesis, immune response, complement and coagulation cascade, Wnt signalling and organ/system development (Huang et al., 2017, Kosova et al., 2015, Othman et al., 2012).

Proteomics, unlike genomics/transcriptomics, studies the final effectors directly, thus showing a more representative reflection of the functional significance in the biological system. Moreover, the proteome is not limited to the translated complement of the protein-encoding genome. While a few studies have attempted to profile the endometrial proteome, current innovative high-throughput mass spectrometry approaches allow the analysis of a substantially higher number of proteins, not only predominantly highly abundant proteins (Altmae et al., 2014).

This study used a state-of-the-art proteomic approach to screen for differences in endometrial stromal cell proteome of patients with repeated implantation failure (RIF; ≥3 transfers of high-quality embryos or transfer of ≥10 embryos in multiple transfers), patients with recurrent pregnancy loss (RPL; ≥3 consecutive miscarriages) and normal fertile women (≥1 live birth after spontaneous conception or insemination with donor spermatozoa), and the differential expression upon decidualization.