Mitochondrial Sirtuin 3 and Sirtuin 5 in granulosa and cumulus cells and their contribution to the altered follicular environment in women with either reduced ovarian reserve or advanced maternal age

Abstract

Women with reduced ovarian reserve or advanced maternal age are known to have poorer IVF outcomes compared to younger women with normal ovarian reserve. While previous studies in humans have correlated metabolite concentrations in follicular fluid to IVF outcome, the impact of maternal age and reduced ovarian reserve have yet to be determined. Oocytes are dependent on mitochondrial metabolism for viability and disruptions to mitochondrial activity can reduce oocyte viability. It has been suggested that oocytes from women with either reduced ovarian reserve or advanced maternal age have a reduction in metabolic function, however, the exact mechanisms behind this reduction remain largely unknown. Interestingly, a family of proteins, the Sirtuins, are able to sense cellular metabolic state and post-translationally alter protein function, thus implicating these proteins in metabolic function. Sirtuin 3 (SIRT3) and Sirtuin 5 (SIRT5) are two proteins that are specifically located to mitochondria, thus may be important in understanding metabolic control in the pre-ovulatory follicle. Thus the aim of this thesis was to determine if a difference exists in the follicular environment in women with reduced ovarian reserve or advanced maternal age and if so does SIRT3 or SIRT5 play role in ovarian follicular cells. Women (n=111) undergoing routine IVF treatment were recruited to participate in this study. They were allocated to one of three cohorts based on maternal age (young maternal age [≤35]; advanced maternal age ≥40) and ovarian reserve for age, as measured by serum anti-mullerian hormone (AMH) levels. Surplus follicular fluid, granulosa and cumulus cells were collected, de-identified and randomly allocated to experimental protocols. Follicular fluid concentrations of carbohydrates (glucose, lactate and pyruvate), hormones FSH, LH, progesterone, estrogen and AMH), and selected ions were determined. Metabolic analysis of granulosa and cumulus cells was performed. Granulosa and cumulus gene expression of phosphofructokinase platelet (PFKP) and lactate dehydrogenase A (LDHA) was determined. SIRT3 and SIRT5 gene expression and protein activity was confirmed in granulosa and cumulus cells via qPCR, immunohistochemistry, western blotting and deacetylation/desuccinylation activity. Granulosa and cumulus cell carbamoyl phosphate synthase I (CPS1) protein, a SIRT5 target, was confirmed using immunohistochemistry. Follicular fluid ammonium concentration and granulosa and cumulus cell glutamate dehydrogenase (GDH) activity, a SIRT3 target, were assessed using microfluorometry. Granulosa and cumulus cell acetylated mitochondrial proteins were separated by immunoprecipitation and acetylation of GDH was assessed via western blotting. Data from young women with normal ovarian reserve were compared with those from young women with reduced ovarian reserve and those of advanced maternal age. Young women with normal ovarian reserve had significantly lower starting FSH doses and fewer previous cycles compared to the remaining two groups. Young women with normal ovarian reserve had significantly more oocytes collected compared to young women with reduced ovarian reserve and the advanced maternal age women. Fertilisation rate was significantly higher in the young women with normal ovarian reserve compared with the advanced maternal age group. Women of young maternal age with normal ovarian reserve had significantly less embryos transferred compared to the advanced maternal age group. The clinical pregnancy rate in young women with normal ovarian reserve was significantly increased compared to both the reduced ovarian reserve and advanced maternal age groups. No differences were found in clinical pregnancy rate between the reduced ovarian reserve and advanced maternal age groups. Follicular fluid glucose concentrations were significantly decreased, whereas lactate and progesterone concentrations, granulosa and cumulus cell glucose uptake, lactate production, and phosphofructokinase platelet gene expression were significantly increased in women with reduced ovarian reserve and in women of advanced maternal age. SIRT3 and SIRT5 mRNA and active protein were present in granulosa and cumulus cells and co-localized to the mitochondria. Women with reduced ovarian reserve or advanced maternal age had decreased granulosa and cumulus cell SIRT5 mRNA, protein, desuccinylation activity and an accumulation of follicular-fluid ammonium. CPS1 protein was present in granulosa and cumulus cells. Compared to young women with normal ovarian reserve granulosa cell SIRT3 mRNA was decreased in young women with reduced ovarian reserve and advanced maternal age whereas cumulus cell SIRT3 mRNA was decreased in women of advanced maternal age only. Granulosa cell GDH activity was decreased in young women with reduced ovarian reserve and in women of advanced maternal age, whereas cumulus cell GDH activity was reduced in the advanced maternal age group only. Granulosa and cumulus cell acetylated mitochondrial GDH was increased in women of advanced maternal age while young women with reduced ovarian reserve had increased granulosa cell GDH acetylation only. The data presented within this thesis suggest that in women with either reduced ovarian reserve or advanced maternal age both SIRT3 and SIRT5 may regulate granulosa and cumulus cell GDH and CPS1 activity, therefore altering the microenvironment surrounding the oocyte, as reflected by the altered follicular environment. This perturbed microenvironment may be responsible for impaired oocyte developmental competence, subsequent embryo development and reduced clinical pregnancy rates, also reported in this study. Considering the association between the decline in pregnancy rates in women with reduced ovarian reserve and in women of advanced maternal age and the knowledge of perturbed granulosa and cumulus cell SIRT3 and SIRT5 function this may lead to novel therapies to improve mitochondrial metabolism in the oocyte and follicular cells in women undergoing IVF treatment.