P-362 Cellular senescence in mouse ovaries as an explanatory mechanism of cyclophosphamide detrimental effect to female fertility
Authors: Hao X , Zhao J , Rodriguez-Wallberg K 
Published: July 2024

Abstract

Study question
Is cellular senescence a causative mechanism of cyclophosphamide’s detrimental effect to female fertility?

Summary answer
Cyclophosphamide induced up-regulation of genes related to cellular senescence and associated pathways, oocyte organelles alterations and increased beta-galactosidase (b-Gal) positive cells in mouse ovaries.

What is known already
Cyclophosphamide (CPA) is frequently used for treatment of cancer and chronic benign diseases; however, secondary effects include infertility and POI. The mechanisms of CPA-induced POI have not been fully elucidated. Research in possible causative mechanisms include apoptosis and over-activation with follicle burnout, however, new hypothesis including affecting cellular senescence has been proposed. Cellular senescence is a complex cellular reaction to stress and the cells enter an irreversible proliferative arrest state. It is associated with alterations in cell secretory profile (secretion of pro-inflammatory factors) and morphology (vacuolization and granularity in cytoplasm, abnormal organelles).

Study design, size, duration
Controlled experimental study. Intact ovaries from B6CBA/F1 4-day-old mice were randomly assigned to 4-hydroperoxycyclophosphamide (n = 22) or control group (n = 22). Five ovaries per group were collected at 8, 12, 24, 36 h and processed for RNA sequencing. Two ovaries per group were collected at 24 h for transmission electron microscopy analysis. B6CBA/F1 12-day-old mice were either intraperitoneally injected with 100 mg/kg CPA (n = 2) or no treatment (n = 2). Ovaries were collected for histological analysis 2 weeks later.

Participants/materials, setting, methods
Ovaries from 4-day-old mice were cultured on Millicell cell culture inserts floating on 0.25 mL culture medium in a 24-well plate, maximum 5 ovaries per insert. Freshly prepared 4-hydroperoxycyclophosphamide solution was added to the wells of CPA group (final concentration is 5 µM). 12-day-old mice were intraperitoneally injected with 100 mg/kg CPA or no treatment, 2 weeks later, ovaries were collected and fixed, then processed for b-Gal staining.

Main results and the role of chance
Transcriptomic analysis of 4-day-old ovaries cultured with 4-hydroperoxicyclophosphamide (5 µM) presenting up-regulation of genes related to cellular senescence and its associated signaling pathways, e.g. TNF signaling pathway, P53 signaling pathway, cell cycle, cytokine-cytokine receptor interaction. Literature reported cellular senescence related genes Cdkn1a, Hdac1, E2f3, Ccne1, Jun, Pcna, Smad3, Mapk1, Smad1, Cdkn2cc, Ccne2, Rbl1, Tp53, Myc, Cdk2, Cdk1 were found up-regulated by CPA treatment at 36 h.
CPA treatment also caused organelles alterations in ovarian follicles revealed by transmission electron microscopy, e.g. endoplasmic reticulum became elongated and attached to mitochondria and forming a necklace-like structure, vacuolization could be observed in some oocytes’ cytoplasm, especially in some growing follicles. And these changes were not observed in the control group.
Young (12-day-old) mice that received CPA (100 mg/kg) showed increased trend of staining of cellular senescence markers including b-Gal two weeks after the treatment comparing to control ovaries, suggesting an involvement of cellular senescence in CPA induced ovarian damage.

Limitations, reasons for caution
We used an experimental model and it is not known if similar results could be found in human ovarian tissue.

Wider implications of the findings
Our results using in vitro and in vivo models suggest that cellular senescence may be induced by CPA at transcriptomic, cellular ultrastructural and histological levels. This might partially explain the occurrence of infertility following treatment with alkylating drugs since some ovarian cells are arrested and influence follicle development.

Trial registration number
Not Applicable