VARIATION IN THE PERCENTAGE OF ENDOMETRIAL p16-POSITIVE SENESCENT CELLS IN CONSECUTIVE MENSTRUAL CYCLES

Abstract

Recently, it was found that p16ink4a-positive senescent cells are involved in endometrial receptivity and participate in the acute cellular remodelling at the time of embryo implantation. Our previous research showed that the percentage of glandular p16-positive cells and luminal epithelial p16-positive cells is strongly associated with successful implantation and live births. The aim of the present study was to compare the percentage of p16-positive cells in the glandular and luminal epithelial compartments of the human endometrium during the mid-luteal phase in two consecutive menstrual cycles. We measured the percentage of p16ink4a-positive cells by immunohistochemistry in endometrial biopsy samples of 94 women in two consecutive menstrual cycles. This is a prospective observational study of 94 fertile women who had two endometrial biopsies during the mid-lutheal phase (7 days after LH surge) in two consecutive natural cycles. Patients older than 40 years, with BMI<18 kg/m2 or BMI≥30 kg/m2, endometriosis, polycystic ovary syndrome (PCOS), endometrial polyps, abnormal uterine development and hydrosalpinx were excluded from the study. Endometrial biopsies were obtained by pipelle suction and they were immediately fixed in 10% formalin. The endometrial tissue was submitted to paraffin embedding for histological determination and subsequent analysis. Immunohistochemistry (IHC) was performed on the paraffin-embedded sections by Novolink Polymer Detection System (Leica Biosystems, Wetzlar, Hesse, Germany). We used monoclonal antibody against p16ink4a (Master Diagnostica, Granada, Spain) to identify senescent endometrial epithelial cells. The percentage of p16+ cells in each tissue compartment was calculated after enumeration by two independent investigators in multiple endometrial sections. Values were expressed as mean ± SD. Paired t-test was used to compare the percentage of p16-positive cells between the two time points. P<0.05 was considered statistically significant. The percentage of p16-positive cells in the endometrial glands during the mid-luteal phase of the cycle ranged between 0.12% and 53.22%, while it varied between 1.39% and 85.29% in the luminal epithelium. The temporal change in the percentage of p16-positive cells varied at both time points between 0.55% and 15.56% in the endometrial glands, and between 1.58% and 30.74% in the endometrial luminal epithelium. In addition, for both endometrial glands and luminal epithelium, the proportion of p16+ senescent cells was not significantly different between the two time measurements (p=0.56 and p=0.93, respectively). In 80% of the patients the difference in the percentage of p16-positive cells at both time points was lower than 20% in the endometrial luminal epithelium and lower than 9% in the luminal epithelium. The percentage of endometrial epithelial p16-positive senescent cells is relatively constant in human endometrium during the mid-luteal phase in consecutive menstrual cycles.