P-448 Changes in gene transcription induced by cyclophosphamide treatment in an experimental ovarian culture model
Authors: Hao X , Reyes Palomares A , Rodriguez-Wallberg K 
Published: June 2022

Abstract

Study question
How does cyclophosphamide (CPA) treatment impact at transcriptional level on mouse ovarian tissue?

Summary answer
Cultured murine ovarian tissue with CPA versus control showed up-regulated intrinsic and extrinsic apoptotic signaling pathways, associated with DNA damage, DNA repair and oxidative response.

What is known already
Alkylating chemotherapeutic treatment depletes the ovarian pool and induces infertility in women. The suggested mechanisms behind these adverse effects include apoptosis and/or over-activation of the dormant primordial follicle pool. However, there is a lack of knowledge about the pathways that lead to these outcomes and previous researches have been inconclusive. The investigation of changes in the ovarian transcriptomic profiling following the alkylating drug CPA treatment can be useful to identify new potential targets for fertility preservation in women treated for cancer.

Study design, size, duration
Controlled experimental study using 20 female B6CBA/F1 4-day-old mice. Ovaries were collected and randomly assigned to CPA (4-hydroperoxycyclophosphamide) treated group (n = 20) or control group (n = 20). Five ovaries/group were collected at 8, 12, 24 and 36 h to investigate the dynamic of the changes. RNA extraction and RNA sequencing analysis were carried out.

Participants/materials, setting, methods
Ovaries were cultured on Millicell cell culture inserts floating on 0.25 mL culture medium in a 24-well plate. Freshly prepared 4-hydroperoxycyclophosphamide solution was added to the wells of CPA group (final concentration = 5 µM). Equal amount of solvent was added to the wells of control group. Culture medium was refreshed at 48 h with culture medium only. RNA sequencing data were processed for subsequent differentially expressed genes (DEGs) and gene set enrichment analysis (GSEA).

Main results and the role of chance
At 8 h, CPA treatment induced the up-regulation of biological processes related to hypoxia, cell growth and embryonic organ development. At 12 h, DNA damage and the ovarian cell responses were evidenced by an increased activity of DNA damage response, DNA damage checkpoint, DNA repair (double-strand break, mismatch, single strand binding), stress-activated MAPK cascade, antioxidant activity and intrinsic apoptotic signaling pathway. The representative genes of these processes there were Bbc3, Bax, Trp73, Cdkn1a, Trp53inp1 and Mdm2. A dramatic increase in the number of DEGs was found at 24 h (8 h, n = 209; 12 h, n = 239; 24 h, n = 2013). Also at 24 h DNA repair, intrinsic and extrinsic apoptotic signaling pathways were the most representative processes evidenced by the addition of Rad9a, H2afx, Casp3, Bak1 and Casp8 genes to the above mentioned. Whereas, germ cell related genes Ybx2, Nobox and Ddx4 were all down-regulated. At 36 h, the number of DEGs (n = 3804) still increased, the up-regulated pathways were similar to 24 h, while meiosis and microtubule-based movements pathways were observed in the down-regulated set too.

Limitations, reasons for caution
Although the age of the mice chosen for the experiment ensured a high and representative content of primordial follicles in the ovary, whole ovaries were used for RNA sequencing analysis containing a heterogeneous composition of cells other than follicles.

Wider implications of the findings
Our results provide evidence of dynamic sequential changes in transcriptional level where apoptosis was involved in CPA-induced ovarian follicle depletion. Our research indicates a time frame before the occurrence of DNA definitive damage following CPA-treatment, where application of possible treatments in order to prevent the following apoptosis would be possible.

Trial registration number
Not Applicable