Effects of Gonadotropin-Releasing Hormone Analogues on Ovarian Function and Embryogenesis: A Cyclophosphamide-Induced Mouse Model Study.

Abstract

Objective: To clarify the protective effects of gonadotropin-releasing hormone analogues (GnRHas) on cyclophosphamide (CTX)-induced oocyte number loss and development of potential damage.

Design: Mice model study.

Setting: Laboratory-based animal study conducted in controlled research facilities.

Population: Female C57/BL6 mice subjected to CTX-induced ovarian damage.

Methods: The effects of GnRHa on CTX mice were evaluated in terms of hormones, oocyte count on slices, oocyte count in established three-dimensional-constructed ovaries, in vitro fertilisation, RNA sequencing and microinjection.

Main outcome measures: The main outcome measures were the number of oocytes in intact mouse ovaries and oocyte quality, evaluated using three-dimensional (3D) tissue-clearing methods, oxidative stress markers (reactive oxygen species [ROS] and malondialdehyde [MDT]), mitochondrial function (ATP levels), and embryogenesis rates at the two-cell, four-cell and blastocyst stages.

Results: In CTX mice, GnRHa pretreatment did not protect endocrine hormone changes, but protected loss of oocyte number on slice counting. A tissue-clearing technique, CUBIC (Clear, Unobstructed Body Imaging Cocktails), was a suitable method for ovaries clearing, and a 3D method for oocyte counting was validated with accuracy of 105.22% ± 3.48%. By this method, GnRHa was also found to protect the loss of oocyte number (597 ± 28 vs. 222 ± 15, p < 0.0001), which may be mediated by upregulated anti-Müllerian hormone (AMH) levels inhibiting primordial follicle development approved by in vitro culture of ovaries. GnRHa also increased the number of retrieved oocytes in CTX mice (19.4 ± 2.1 vs. 15.0 ± 1.6, p < 0.0001) and developmental ability of oocytes (65.0 ± 4.6 vs. 48.1 ± 4.2 for blastocyst, p < 0.0001). RNA sequencing revealed GnRHa pretreatment downregulated pathways of exogenous drug metabolism, oxidative stress and cytochrome P450, validated by detection of adenosine triphosphate (ATP), MDA and ROS levels. The up-expression of Cox17 (cytochrome c oxidase copper chaperone 17) after GnRHa pretreatment was confirmed by PCR and microinjection of siCox17 increased the embryogenesis from CTX mice.

Conclusions: GnRHa was associated with reduced oocyte loss and improved embryogenesis, likely mediated by AMH and Cox17 upregulation.