DOI: 10.14457/tu.the.2016.1450
title: Phosphoproteomics and glycoproteomics of endometrial secretion in assisted reproductive technology cycles with implantation and non-implantation

description: Embryo implantation represents a crucial step to success in the assisted reproductive technology treatment (ART) of infertility. The endometrial receptivity assessment during the window of implantation (WOI) has been an important challenge. The analysis of endometrial receptivity using endometrial secretion has been of particular interest as they represent a non-invasive approach and the number of mediators contained may involve in the preparation for embryo to implant and by modulation of endometrial receptivity.This study aims firstly, to investigate the endometrial secretome based on the phosphoproteomics and glycoproteomics study, and secondly, to identify possible potential phosphoprotein and glycoprotein biomarkers to represent for the phases of human endometrium and receptivity during the WOI of stimulated ART treatment cycle.The cross-sectional study performed in infertile couples underwent assisted reproductive technology treatment at Thammasat Fertility Center, Thailand, during Dec 2015 – Dec 2016. This study included total of 60 infertile women of which 30 were patients with implantation and another 30 were patients with non-implantation.  Endometrial secretions were also collected from embryo transfer catheter tip at the oocyte retrieval day (day 0/ D0) of 15 implantation plus 15 non-implantation patients designated as group I (baseline or control) and of 30 implantation patients and 30 non-implantation patients at embryo transfer day (day 5/ D5) designated as group II and group III (study groups) respectively. The sample from each group was analysed for phosphoproteins and glycoproteins.To study phosphoproteins and glycoproteins in endometrial secretion, immobilized metal affinity chromatography (IMAC) and lectin concanavalin A (ConA) were used to enrich phosphorylated proteins and N-glycosylated proteins respectively. After that the proteins were digested into short peptide by trypsin, and analysed by liquid chromatography with tandem mass spectrometry (LC-MS/MS) approaches. Bioinformatic tools were used to compare phosphoproteins and glycoproteins found unique between the groups, searching for gene ontology and category classifications. In order to validate the predicted differential phosphoproteins and glycoproteins expression of endometrium receptivity biomarkers based on LC-MS/MS data, the western blot analysis using the commercial antibodies against total protein was performed in sample collected at D5 comparing between implantation and non-implantation groups. The results of these western blot analysis were then compared to the corresponding phosphopeptide and glycopeptide obtained from mass spectrometry results.From all of the endometrial secretion samples in this study, LC-MS/MS analysis resulted in the identification of total 267 phosphoproteins and 110 glycoproteins. In order to identify phosphoproteins and glycoproteins those represent for the phase of endometrium, this study compared the differential phosphoproteins and glycoproteins expression between samples collected at oocyte retrieval day (D0 as pre-receptive phase) and samples collected at embryo transfer day (D5 as receptive phase or window period of implantation). It was found in this study that 267 phosphoproteins and 105 glycoproteins expressed at D0, whereas 220 phosphoproteins and 110 glycoproteins expressed at D5. Moreover, 47 phosphoproteins were found unique at D0 and 5 glycoproteins were found unique at D5. In order to identify phosphoprotein and glycoprotein those represent for the endometrium receptivity, this study also compared the differential phosphoproteins and glycoproteins expressions at D5 between implantation and non-implantation groups. It was found that 4 phosphoproteins and 1 glycoprotein were found unique in the implantation group, whereas 36 phosphoproteins and 2 glycoproteins were found unique in the non-implantation group. Regarding validation of the LC-MS/MS results, western blot analysis was performed. This study analysed the total protein expressions in two selected phosphoproteins and one selected glycoprotein: ATP synthase subunit gamma, mitochondrial (ATPG), katanin p60 ATPase containing subunit A1 (KATNA1), and thioredoxin domain-containing protein 2 (thioredoxin2), respectively. The results of western blot of phosphoprotein and glycoprotein validation showed that there were total proteins expressed in samples of implantation and non-implantation groups. These confirmed the results of protein expression by LC-MS/MS, however, there was not quite similar in pattern of expression in which LC-MS/MS showed of the identified proteins were uniquely expressed. This study also analysed the relative quantity of total protein expression. For phosphoprotein validation, there was no significantly different in protein abundances between implantation and non-implantation groups. Whereas, for glycoprotein validation, there was significantly different in protein abundances between implantation and non-implantation groups with similar trend to those of LC-MS/MS result. The results obtained both by western blot analysis in correlation with LC-MS/MS, it is proposed that there was the same parent proteins found in both implantation and non-implantation groups as measured by western blot analysis while apparent post-translational modification forms especially phosphorylated and glycosylated were differentially found in implantation and non-implantation groups as measured by LC-MS/MS.Based on the findings, it was postulated that 267 phosphoproteins and 105 glycoproteins expressed at D0 may represent pre-receptive phase of endometrium, while 220 phosphoproteins and 110 glycoproteins expressed at D5 may represent receptive phase of endometrium. Moreover, in this study 47 phosphoproteins were found unique at D0 and 5 glycoproteins were found unique at D5. This finding may help especially to improve more sensitivity and specificity values of the ability to discriminate between pre-receptive and receptive phases of endometrium. Based on their sequential temporal expression with respect to the implantation window, it was considered that the number of phosphoproteins and glycoproteins identified may regulate the functions of human endometrium in preparation for the implantation process during early and mid-luteal phase of stimulated endometrium. It was also postulated that 4 phosphoproteins and 1 glycoprotein found unique in implantation group may represent the activity for promotion of the endometrium receptivity and may be regarded as potential biomarkers for positive regulation of endometrium receptivity. Whereas 36 phosphoproteins and 2 glycoproteins found unique in non-implantation group may represent barrier activity of the endometrium receptivity and might be regarded as potential biomarkers for negative regulation of endometrium receptivity. All of these functional proteins might reflect important physiological activities and supposedly characteristics of endometrium receptivity. In conclusion, these preliminary data apply the comprehensive phosphoproteomics and glycoproteomics study of endometrial secretion to evaluate of significance protein profiles. This study showed that a panel of proteins, rather than a single one can improve sensitivity and specificity values of the ability to discriminate between pre-receptive and receptive phases. Also, these receptivity biomarkers may help to increase sensitivity and specificity determining predictive value as definite indicator for endometrium receptivity. Finally, based on the findings in this study, it is suggested that study of biomarkers in endometrium related to implantation process may not be pointed to the difference observed by total proteomics study, but should be considered with more unique insights. Therefore, from results of this study we proposed that regulation of endometrium with implantation might exert at post-translational levels. The limitation of this study was the endometrial secretion collected during WOI using non-invasive clinical routine procedure elicited the sample containing very low abundance of proteins. Therefore, it is quite difficult for analysis since generally more protein concentration is required and then more demanding processes are needed to perform analysis, especially analysis of phosphoproteins and glycoproteins included in this study. Furthermore, antiphospho- and antiglyco-specific antibodies for western blot analysis are not always available. Further study is needed in the larger population or by way of working with other reproductive samples. Other confirmatory methods will be needed to validate these findings. Tests in the other biological fluids would be more easily and relatively non-invasive to collect sample i.e. plasma or urine that are preferred in clinical applications. The continuous dynamic analysis of identified biomarkers starting from pre-receptive to receptive period (time series) is certainly required for further study. To correlate the unique phosphoproteins and glycoproteins found in this study with other approaches e.g. histological, ultrasound, and pinopods for assessing the endometrium receptivity are of interest for future research, and certainly, other PTMs study, such as ubiquitination, acetylation, etc. are also interested.