High throughput, cell type-specific analysis of key proteins in human endometrial biopsies links infertility to leukemia inhbitory factor, progesterone receptor-B and integrin αIIb dysregulation

Abstract

OBJECTIVE: We have examined the cyclical and cell-specific expression of implantation-associated proteins in the human endometrium and their dysregulation in infertile patients.DESIGN: Endometrial biopsies from 173 patients obtained by the Reproductive Medicine Network were evaluated for expression of progesterone receptor-B (PR-B), leukemia inhibitory factor (LIF), glycodelin (Gd), HOXA10, osteopontin (OPN), heparin-binding EGF-like growth factor (HBEGF), calcitonin (CT), and integrin αIIb. Based on histological dating, the specimens were grouped into early (days 14-19), mid (days 20-24) or late (days 25-28) secretory phase. Antigen levels were evaluated in stromal cells, glandular epithelia, and luminal epithelia. Protein expression profiles were compared between fertile and infertile women.MATERIALS AND METHODS: Endometrial biopsies were obtained on cycle days 21-22 and 26-27, based upon the time of LH surge. Fertile and infertile women were distributed similarly between the two biopsy intervals, with 44 fertile and 43 infertile on days 21-22, and 42 fertile and 44 infertile on days 26-27. Paraffin embedded specimens were serially sectioned for immunohistochemistry with antibodies against the respective proteins using a robotic DAKO Autostainer. Digital images of the stained tissues were analyzed using a morphometry program (Simple PCI, Compix) to separately quantify the grey level of circumscribed stroma, glandular epithelium and luminal epithelium. Results of the antibody staining intensities were analyzed using ANOVA and the Student-Newman-Keuls post hoc test, with statistical significance set at p<0.05.RESULTS: All proteins examined displayed significant cyclical regulation and were differentially expressed in the three tissue compartments. PR-B was over expressed during early secretory phase within all cell types of infertile patients. LIF stromal expression was significantly higher in fertile than infertile patients throughout the cycle. Integrin αIIb was significantly elevated in the luminal epithelium of infertile patients during the mid-secretory phase.CONCLUSIONS: This high throughput method for protein profiling in the human endometrium is an effective means of evaluating expression of implantation-associated gene products. Previously reported LIF dysregulation in infertile tissues was confirmed, and the identification of altered PR-B and integrin αIIb expression patterns in infertile patients was reported for the first time. OBJECTIVE: We have examined the cyclical and cell-specific expression of implantation-associated proteins in the human endometrium and their dysregulation in infertile patients. DESIGN: Endometrial biopsies from 173 patients obtained by the Reproductive Medicine Network were evaluated for expression of progesterone receptor-B (PR-B), leukemia inhibitory factor (LIF), glycodelin (Gd), HOXA10, osteopontin (OPN), heparin-binding EGF-like growth factor (HBEGF), calcitonin (CT), and integrin αIIb. Based on histological dating, the specimens were grouped into early (days 14-19), mid (days 20-24) or late (days 25-28) secretory phase. Antigen levels were evaluated in stromal cells, glandular epithelia, and luminal epithelia. Protein expression profiles were compared between fertile and infertile women. MATERIALS AND METHODS: Endometrial biopsies were obtained on cycle days 21-22 and 26-27, based upon the time of LH surge. Fertile and infertile women were distributed similarly between the two biopsy intervals, with 44 fertile and 43 infertile on days 21-22, and 42 fertile and 44 infertile on days 26-27. Paraffin embedded specimens were serially sectioned for immunohistochemistry with antibodies against the respective proteins using a robotic DAKO Autostainer. Digital images of the stained tissues were analyzed using a morphometry program (Simple PCI, Compix) to separately quantify the grey level of circumscribed stroma, glandular epithelium and luminal epithelium. Results of the antibody staining intensities were analyzed using ANOVA and the Student-Newman-Keuls post hoc test, with statistical significance set at p<0.05. RESULTS: All proteins examined displayed significant cyclical regulation and were differentially expressed in the three tissue compartments. PR-B was over expressed during early secretory phase within all cell types of infertile patients. LIF stromal expression was significantly higher in fertile than infertile patients throughout the cycle. Integrin αIIb was significantly elevated in the luminal epithelium of infertile patients during the mid-secretory phase. CONCLUSIONS: This high throughput method for protein profiling in the human endometrium is an effective means of evaluating expression of implantation-associated gene products. Previously reported LIF dysregulation in infertile tissues was confirmed, and the identification of altered PR-B and integrin αIIb expression patterns in infertile patients was reported for the first time.