O-038 Role of apoptosis and autophagy in ovarian follicle pool decline from birth to late reproductive age and after gonadotoxic chemotherapeutic treatments

Abstract

Abstract Study question Which biological mechanisms are responsible for physiological age-dependent and non-physiological ovarian reserve decline throughout a woman’s life? Summary answer Ovarian follicle pool size is modulated by both apoptosis and autophagy, the latter playing a major role in its decline before puberty. What is known already Among different pathways of controlled cell death, apoptosis and autophagy are both involved in follicle death. Apoptosis participtes in eliminating damaged follicles, like those affected by chemotherapy (CHT)-induced DNA damage, but its role in physiological age-related follicle decline is less understood. Autophagy has proved crucial in follicle quiescence maintenance in murine models, but its contribution to human follicle pool modulation is still unclear. Study design, size, duration Eighty-four patients aged between 1 and 35 years, with ovarian tissue stored for histological analyses at the time of cryopreservation (between 2012 and 2021) at the Université Catholique de Louvain (Belgium) were included in retrospective evaluations. Participants/materials, setting, methods Ovarian fragments were used for the following analyses: hematoxylin and eosin staining for follicle count and classification; caspase-3 immunostaining to identify follicle apoptosis; and microtubule-associated proteins 1A/1B light chain 3B (LC3B) immunolabeling to detect follicle autophagy. Ovarian fragments from 9 patients were assigned for transmission electron microscopy to investigate ultrastructural features of oocytes and granulosa cells. All analyses were conducted stratifying patients by age, menarchal status (premenarchal=32; postmenarchal=52), and potentially gonadotoxic CHT before cryopreservation (n = 14). Main results and the role of chance Premenarchal patients had a larger follicle pool in terms of total follicle density (4979.98±4952.16 vs 918.8±903.86, p < 0.0001), but higher rates of morphologically abnormal (8.52±6.48% vs 3.54±4.54%, p < 0.0001) and atretic follicles (15.8±9.14% vs 10.6±9.43%, p = 0.01) than postmenarchal subjects. Apoptosis rates did not change with increasing age (27.94±28.78% in prepubertal subjects and 29.5±33.58% in postpubertal subjects). Autophagic follicles were around 10 times more common in premenarchal compared to postmenarchal subjects (10.21±16.% vs 1.34±4.02%, p < 0.0001), playing a crucial role in age-related follicle decline and elimination of ‘abnormal follicles’, that are rarely seen after menarche. The impact of previous CHT varied according to age. In premenarchal patients with previous CHT, significantly more apoptotic (40.22±40.44% vs 26.79±25.35%, p = 0.04) and fewer abnormal (3.84±3.91% vs 9.83±6.5%, p = 0.02) follicles were detected than in those with no CHT prior to ovarian tissue cryopreservation, suggesting a direct effect on follicle elimination, especially of those with abnormalities. In postmenarchal subjects with previous CHT, lower quiescent follicle rates were observed compared to patients with no CHT before tissue freezing (70.1±36.36% vs 85.89±12.61%, p = 0.04), suggesting accelerated follicle activation and growth. Limitations, reasons for caution The present study could not investigate the impact of specific CHT protocols, since the group of patients with previous CHT was highly heterogeneous. However, these patients could not be excluded a priori, since the effect of follicle pool depletion has not been fully elucidated. Wider implications of the findings This study yields a deeper understanding of follicle pool decline regulation, evidencing for the first time that (i) different cell death pathways are involved in physiological and non-physiological follicle depletion, and (ii) the follicle pool responds differently to gonadotoxic damage according to age and ovarian reserve size. Trial registration number N/A