P-192 ATP5G3 and VEGFC expression in cumulus cells are non-invasive markers of fertilization rate

Abstract

Abstract Study question Could expression of aging- and hypoxia-related genes in cumulus cells be used as non-invasive markers of fertilization rate in ART? Summary answer The expression levels of specific aging- (ATP5G3) and hypoxia-related genes (VEGFC) in human cumulus cells correlate with oocyte ability to be fertilized. What is known already Throughout a woman reproductive life, cumulus cells (CC) protect the dormant oocyte from damage, act as sensors of the follicular microenvironment and act as gatekeeper for the oocyte developmental potential. One such mechanism relays on the hypoxia-dependent response, which controls the cell metabolic and oxidative state. With age, hypoxia tolerance decreases in the whole organism, including the ovary. Recently, alterations in CC response to the follicular microenvironment have been described in advanced maternal age (AMA) women. We investigate whether expression levels of hypoxia-responsive genes in CC could be used as non-invasive marker of fertilization rate in human oocytes. Study design, size, duration Prospective study of 94 women (41 oocyte donors and 53 patients) recruited from 2018 to 2021. The overall age was 32±8.0 (range 18-46); 25±4.3 (range 18-34) for oocyte donors and 38±4.6 (range 26-46) for patients. The mean antral follicular count (AFC) was 18±10.9 (range 1-48) and the mean MII rate at ovum pick-up (OPU) was 75±17.8% (range 20-100%). Participants/materials, setting, methods Pooled CC were collected from each woman after OPU; total RNA was extracted, reverse transcribed to cDNA using random hexamers and expression of aging- (LYZ, FGF2, ATP5G3, AMH, ANXA5) and hypoxia-related genes (HIF-1A, NOS2, NOS3, HMOX1, TXNIP, CLU, FABP3, TGFBR3, VEGFC) was detected by qPCR and normalized against TBP. Expression levels were plotted against woman age, AFC, MII rate at OPU (MR), and fertilization rates (FR) and non-parametric Spearman’s correlation was applied. Main results and the role of chance Overall, MII and FR rates did not decrease with increasing woman’s age (rs= -0.2, p = 0.06 and rs= -0.1, p = 0.39, respectively). When analyzing expression levels of aging markers (LYZ, FGF2, ATP5G3, AMH, ANXA5), none of them correlate with age, AFC, or MR (rs<│0.1│, p > 0.05) in CCs. Moreover, although abundant, the expression of HIF1A, the main hypoxia-related gene, was not correlated with age, AFC, MR or FR. However, the expression of several genes regulated by HIF1A did show correlation with age, MR or FR. CLU (a secreted chaperone involved in cell death), TXNIP (a tumor suppressor gene involved in redox regulation) and NOS2  (an inducible and calcium-independent isoform of nitric oxide synthase) correlated positively with age (rs=0.25, p = 0.014; rs=0.31, p = 0.0027; and rs=0.24, p = 0.03, respectively). We further analyzed CC gene expression against FR and we found that ATP5G3 and VEGFC levels decreased with increasing FR, independently of age (rs=-0.26, p = 0.044 and rs=-0.31, p = 0.014; respectively). Limitations, reasons for caution The analysis of pooled CC allows for cohort interpretation of results, and they cannot be extrapolated at this time to single oocyte’s reproductive outcome prediction. Wider implications of the findings We found that the fertilization potential of a cohort of oocytes is related to the ability of CC to respond to oxidative stress and hypoxia, pointing at ATP5G3 and VEGFC expression as markers for fertilization success. Further research is needed to assess their association with embryo quality and pregnancy outcomes. Trial registration number NA