(294) ATAC-seq Reveals Age- and IVF-Response-related Changes in Genomic Binding Regions in Human Granulosa Cells

Dipanwita Das^1,2, Navya P. Basrur², Jagadesan Sankarasubramanian³, Ailenn Castillo², Chittibabu Guda3, John S. Davis^1,2,4

¹Department of Biochemistry and Molecular Biology, University of Nebraska Medical Centre, Omaha, Nebraska

²Department of Obstetrics and Gynecology, University of Nebraska Medical Centre, Omaha, Nebraska

³Department of Genetics, Cell Biology & Anatomy, University of Nebraska Medical Centre, Omaha, Nebraska

⁴Veterans Affairs Nebraska-Western Iowa Health Care System, Omaha, Nebraska

Abstract Text:

Human female fertility starts declining after 30 years of age, and rapidly declines after the age of 38. During these years the ovaries undergo a dynamic remodeling both structurally (stiffening of ovary) and functionally (decreased follicular function and reduced oocyte quality). Ovarian follicular granulosa cells are crucial for steroid production and maturation of the oocyte. With the advancement of age, the oocyte quality decreases, however, age-related alterations and varied response to gonadotropins are poorly understood. This is particularly relevant for infertile patients undergoing ovarian stimulation for  in vitro fertilization (IVF). A gap exists in understanding of the chromatin landscape and genomic binding regions in granulosa cells of patients undergoing ovarian stimulation treatments for infertility. To address this gap, we employed ATAC seq, an unbiased approach to identify regions of open chromatin, to predict accessibility of transcriptions factors to DNA regions in granulosa cells of patients undergoing IVF. We evaluated human granulosa cells retrieved from four groups of IVF patients: (1) good responders ( >17 oocytes retrieved), (2) poor responders (< 8 oocytes retrieved), (3) young responders (< 31 years old) and (4) old responders ( >38 years old). The granulosa cells were washed and processed on Percoll gradients to remove contaminating blood cells. Immediately thereafter, ATAC-sequencing was performed on granulosa cells to profile the genome wide chromatin accessibility landscape. Initial alignment to the human reference genome identified increased chromatin accessibility (~50%) in intergenic and intronic regions. Principle component analysis (PCA) showed that the young good responders and old good responders clustered well whereas the old poor responders clustered separately, suggesting that the good responders respond in a similar manner to ovarian stimulation. Differential foot printing analysis predicted group-specific increases in transcription factor binding activity in each group: young good responders - ASLC1, TBX5, KLF9, KLF15, MYC, CTCF, and PAX2; old good responders – KLF9, KLF15, MYC, TP73, HIF1A, TBX2/3; old poor responders – FOS/FOSL2, JUN, ATF3, GATA6, and POU4F1. Bioinformatics analysis revealed pathways (FDR < 0.05) associated with the cell cycle, Hippo pathway, MAPK pathway, cAMP signaling, and PI3K/AKT signaling in all groups. Interestingly, enhanced ECM receptor interaction was predicted in the old poor responder group, but not HIF-1 and TGF beta signaling pathways, which were found in good responders irrespective of age. Furthermore, motif enrichment analysis in regions associated with chromatin accessibility in each group was performed: GATA, FOS, and ETS1 motifs were highly enriched in young good responders, whereas AP1, RUNX, ATF3, NRF1/2, CTCF, and KLF5, were highly enriched in old poor responders. YAP/TAZ interacting with the transcription factors TEAD1, and TEAD4 were predicted to be enriched in young responders compared to TEAD3 which was enriched in old poor responders. These insights suggest distinct role of multiple factors that dictate response to IVF. Thus, ATAC seq reveals unique chromatin landscapes in granulosa cells of patients grouped according to age and response to ovarian stimulation. These finding provide new insight into the understanding of chromatin remodeling in granulosa cells in human patients and may lead to the development of a pipeline of targets to better understand the molecular alterations associated with aging and poor response in patients undergoing IVF.