Earlier it was believed that uterine fluid (uf) or secretions are not relevant after the embryo implantation in humans. However, recent reports suggest that uterine secretions continue to play important role till the first trimester of human pregnancy. Evidences also suggest that uf mirrors endometrial functions or dysfunctions. Considering the relevance of uf in endometrial functions, studies were undertaken 1. to develop human uf proteomes and 2. to identify uf proteins that display differential abundance during the receptive phase and also 3. to assess the functional relevance of differentially abundant proteins using an animal model.
Materials and methods
2D-PAGE and gel-free Isobaric Tag for Relative and Absolute Quantitation (iTRAQ) were employed. Samples were collected in the pre-receptive (i.e. day two postovulation, n=7) or receptive phase (i.e. day six post-ovulation, n=7) of the menstrual cycles, from regularly cycling healthy fertile women. Receptive phase samples were also collected from women with unexplained infertility. Further, to test the functional significance, rats (Rattus norvegicus) were used as an experimental model. Samples were collected in the proestrous (n=8) and metestrous (n=8) phases of estrous cycles and also from pregnant rats (n=18) during day 3-5 post-coitum (p.c.).
Our studies demonstrated higher abundance of uf alpha-1 antitrypsin precursor and apolipoprotein A-1 in the receptive phase, than in the nonreceptive phase, in regularly cycling women (Parmar et al, 2008). iTRAQ revealed identities of 127 proteins in the human uf. Of these, 27 proteins displayed differential abundance in the receptive (R) phase, compared to the pre-receptive (PR) phase. High Mobility Group Binding Protein 1(HMGB1), one of the differentially abundant proteins displayed less abundance in the R phase than in the PR phase; in secretions as well as in endometrial tissues. Interestingly rats also revealed a lesser abundance of HMGB1 in the receptive phase, compared to that in the nonreceptive phase uf (Bhutada et al. 2013). Thus, human and rat data indicated an association of endometrial receptivity with a decline in the levels of uf HMGB1. A significant decline was also observed in the expression of endometrial HMGB1 on the day of implantation in pregnant rats. Further, recombinant HMGB1 (0.25-1.5 ug/horn) was administered on day three p.c. in mated rats. The horns administered with HMGB1 showed pregnancy failure, whereas those with saline remained unaffected. This indicated the detrimental effect of an excess of extracellular HMGB1 on pregnancy in rats. Also, morphological changes in the endometrium, an increase in the expression of luminal epithelial NFκβ; and also various inflammatory molecules such as Receptor for Advanced Glycation End Products, Tumor necrosis factor-alpha and interleukin- 6;were observed in HMGB1 treated rats, when compared with untreated rats (Bhutada et al. 2014). Significantly higher expression of endometrial HMGB1 was also observed during the receptive phase in the women with unexplained infertility, compared with healthy proven fertile women.
Our study, for the first time, employed iTRAQ, a gel-free approach to characterize human uf proteome. The study also demonstrated that an excess of extracellular HMGB1 in the receptive phase induces inflammatory changes in the endometrium which interfere with pregnancy.