OBESITY-RELATED ALTERATIONS IN PROTEIN EXPRESSION IN HUMAN FOLLICULAR FLUID

Abstract

Obesity has a complex and significant impact on female reproduction. Prior research demonstrates that obesity is associated with decreased chances of conception, longer time-to-pregnancy, decreased response to medications in patients undergoing in vitro fertilization (IVF), and poorer IVF outcomes. In addition, obesity has been shown to directly alter the follicular microenvironment resulting in abnormal hormonal and metabolomic profiles. While proteomics has previously been employed to investigate follicular fluid in women with PCOS, there is a paucity of data investigating the impact of obesity in normo-ovulatory women. This study aimed to compare the proteomic composition of follicular fluid from normal vs. obese women without a history of PCOS or other ovulatory dysfunction undergoing IVF. Follicular fluid from 8 normal weight women (NW) and 8 women with obesity (OW) undergoing IVF was analyzed for proteomic content. Women undergoing IVF and without a history of PCOS, ovulatory dysfunction, endometriosis and diminished ovarian reserve were eligible for study inclusion. At the time of oocyte retrieval, follicular fluid from the 2nd and/or 3rd follicle from each ovary was collected and cryopreserved. Follicular fluid was subsequently thawed, and the most abundant proteins depleted using the ProteoMinerTM depletion kit (Biorad). Individual samples were then proteoylsed with Trypsin and labeled with a TMT 10-plex kit (ThermoFisher). Liquid chromatography mass-spectrometry (LC-MS) analysis was then performed through the University of Michigan proteomics core. Proteome Discoverer (v2.4) was used for data analysis. The ratio of protein abundance from obese/normal weight samples was calculated and the error rate corrected by utilization of the Benjamini-Hochberg method. Statistical significance was determined by a p<0.05. Western blotting was performed for validation of a subset of proteins. The mean age of NW vs OW was similar (32.88 vs 32.63 yrs). The mean BMI of NW was 21.1 kg/m2 compared with a BMI of 37.05kg/m2 in OW. A total of 1174 proteins were identified with >= 2 peptides present. 25 proteins were found to be significantly altered in the follicular fluid from women with obesity. 19 of these proteins were upregulated while 6 were downregulated. Notably, C-reactive protein was 11-fold higher in the follicular fluid from OW compared with NW (p<0.0001). Confirmatory western blots were consistent with the LC-MS findings. In this study we utilized high-resolution LC-MS to identify 1174 proteins in human follicular fluid. Further we found significant alterations in the proteomic composition of follicular fluid from women with obesity vs normal weight women without a history of ovulatory dysfunction. Future studies are needed to validate these findings in larger populations of patients and to further explore the mechanisms of these altered proteins in the pathophysiology of obesity.