P-605 Hypoxia leads to diminished ovarian reserve in an age dependent manner
Authors: Bachar G , Gutzeit O , Nebenzahl-Sharon K , Weiner Z , Beloosesky R , Fainaru O 
Published: June 2023

Abstract

Study question
To determine whether hypoxic ovarian damage is due to increased growth and follicles “burnout” or due increased apoptosis, and whether this damage is age dependent.

Summary answer
Direct tissue hypoxia causes activation and recruitment of dormant follicles without apoptotic changes. This effect is age dependent, more pronounced in adult- versus newborn ovaries.

What is known already
In a previous study we have shown that perinatal hypoxia causes premature activation and growth initiation of dormant follicles leading to diminished ovarian reserve. Other reports have also linked intrauterine deprivation conditions, premature delivery and small for gestational age newborns with diminished ovarian reserve due to premature ovarian follicular recruitment and exhaustion. Nevertheless, an indirect mechanism influencing ovarian follicle recruitment under hypoxic conditions, such as the release of stress related hormones cannot be ruled out.

Study design, size, duration
Animal studies were carried out using adult 6-week-old (n = 8) and one-day-old newborn (n = 20) ICR (CD-1) female mice. Throughout the study, animals were housed with an alternating 12-h light/dark cycle with access to food and water ad libitum, under constant room humidity and temperature. Animals were sacrificed and ovaries harvested and immediately cultured in Leibovitz media with L-Glutamine and 10% Fetal Bovine Serum.

Participants/materials, setting, methods
Ovarian tissue from dams and pups was cultured for 3 hours at 37oC to hypoxia (1% O2 and 99% N2) or normoxia (21% O2 and 5% CO2). Afterwards tissue was embedded in 4% formaldehyde for further processing and analyses. Sections were stained with H&E for follicular counts. For immunohistochemistry, sections were stained with Ki-67 (proliferation marker), anti-Caspase 3 and anti-FOXOp (apoptosis markers).

Main results and the role of chance
Hypoxia led to a significant decrease in the percentage of primordial follicles out of total follicles as compared to normoxia, both among dams and among pups (3.17±2.75% vs. 17.89±4.4%; p = 0.004 and 40.59±14.88 vs. 81.92±31.56%, p = 0.001 respectively). This was accompanied by a concomitant increases in the percentages of growing (primary and secondary) follicles. Strikingly, this effect was more pronounced in adult dams as compared to that in newborn pups (6 fold versus 2 fold respectively). Ki67 staining indicated increased cell proliferation scores in follicular granulosa cells following hypoxia as compared to normoxia . Both Caspase 3 and FoxoP staining did not detect any changes in both markers of apoptosis either in oocytes, granulosa cells, theca cells or in stromal cells when ovaries were exposed to hypoxia as compared to normoxia.

Limitations, reasons for caution
This model was performed in mice and generalization to human ovarian physiology remains to be determined.

Wider implications of the findings
We hereby show that direct tissue hypoxia causes premature activation and growth initiation of dormant follicles without concomitant changes in apoptosis. This effect is age dependent being more pronounced in adult ovaries. Taken together it supports follicular “burn out” as a potential mechanism for hypoxia induced loss of ovarian reserve.

Trial registration number
IL-117-08-2017