P-216 Low meiotic progression rates in oocytes from older women are due to impaired mitochondrial metabolism
Authors: Zambelli F , Pietroforte S , Ibañez E , Vassena R 
Published: June 2022

Abstract

Study question
Could a decreased mitochondrial metabolism explain the lower oocyte meiotic progression competence observed in advanced maternal age (AMA) oocytes?

Summary answer
Inhibition of mitochondrial activity in young oocytes mirrors the phenotype of AMA oocytes, pointing to a causative link between effective mitochondrial metabolism and meiotic competence

What is known already
Oocyte meiotic maturation is a fundamental and finely regulated process required to form haploid gametes, and errors during this process invariably cause loss of oocyte competence. Similarly, mitochondria metabolism is required for the acquisition of oocyte competence and support of embryo development. In oocytes from AMA women, both meiotic competence and mitochondrial activity are impaired; in mice, it has been suggested the two processes might be functionally linked. However, functional studies in human oocytes evaluating whether alterations in mitochondrial metabolism cause reduced meiotic competence are lacking

Study design, size, duration
A total of 216 oocytes from young (age ≤30) and AMA (age &gt;37) women were included. Oocytes at the germinal vesicle (GV) stage were matured in vitro (in G2plus medium for 30 h; maturation was assessed by polar body (PB) extrusion. For loss of function studies on mitochondrial metabolism, 64 GVs from young women were treated with 1uM of the mitochondria inhibitor Trifluoromethoxy-carbonylcyanide-phenylhydrazone (FCCP) for 30 min and cultured for 30 h in G2plus

Participants/materials, setting, methods
The proteins Dihydrolipoamide S-Acetyltransferase (D-LAT) and Translocase of outer mitochondrial membrane (TOMM20) were analyzed by immunofluorescence in young and AMA oocytes to assess mitochondrial activity and localization, respectively. Fluorescent mean intensities were quantified with ImageJ and compared with t-test. For D-LAT, we assessed an area of 30 um approx. inward from the oocyte cortex, whereas for TOMM20, the whole oocyte diameter was used. Maturation rates were compared between groups by Chi-squared test

Main results and the role of chance
Mitochondrial staining of the two proteins showed a common pattern in the two age groups, with a uniform localization of mitochondria in the whole ooplasm observed by TOMM20, and a subcortical localization of active organelles detected by D-LAT. Quantification of the fluorescent signal showed no differences in term of total mitochondrial presence (intensity of 61674±24322 AU in young, 32186±33414 AU in AMA, p = 0.195), whereas a significant decrease in activity (D-LAT) was measured in the AMA group (intensity of 78614±58534 AU in young, 12517±10187 AU in AMA, p = 0.003). Young oocytes also matured at a higher rate (86.3%; 63/73) than AMA oocytes (62.3%; 38/61; p = 0.002). Young GV oocytes treated with FCCP presented a phenotype similar to AMA oocytes, with a significant decrease in of DLAT (intensity of 78614±58534 AU in non-treated, 11554±16131 AU in treated, p = 0.019), indicating a decrease in mitochondria activity, and a concomitant drop in maturation rate, with only 39.5% (17/43) of the treated oocytes able to extrude the PB

Limitations, reasons for caution
Maturation rates have been assessed by PB extrusion and some variability in spindle assembly may have been overlooked. We assessed one mitochondria activity inhibitor, FCCP, so indirect effects of this molecule on the observed phenotype cannot be ruled out

Wider implications of the findings
We show a functional link between decreased mitochondrial metabolism and impaired meiotic progression. The metabolic inhibition with FCCP in young oocytes might serve as tool to further investigate the molecular mechanisms driving the loss of quality observed in AMA oocytes

Trial registration number
NA