The Mechanism Of Cyclophosphamide Exposure-inducedl Long-term Effects On Oocyte Developmental Competence

With advancements in diagnosis and treatment,the survival rate of patients with malignant cancer has increased.Cyclophosphamide(CTX)is widely used in various cancer therapies and in immunosuppression.CTX directly causes primordial follicle loss with overactivation and DNA damage-induced apoptosis,with ovary reserve loss.However,patients can still have offsprings after CTX chemotherapy.Previous studies have shown that maternal exposure to CTX before conception increases the incidence of birth abnormalities and alters the methylation of genes in the oocytes of offspring.This study is designed to study the mechanism and potential protection of long-term detrimental effect with CTX exposure on oocytes surviving from primordial follicles during chemotherapy in mice.We constructed a pre-pubertal CTX exposure mouse model for this study.Mice were treated with a single dose of CTX(100 mg/kg)at post-natal day 21 and sacrificed 47 days later when primordial follicles surviving chemotherapy developed to the antral stage.We primed pregnant mare serum gonadotropin(PMSG)and human chronic gonadotropin(h CG)injection to collect metaphase II(MII)-stage oocytes to analyze oocyte quality,and embryos were cultured for further development.We collected fully-grown germinal vesicle(GV)oocytes for in vitro maturation or directly subjected to single-cell sequencing and immunostaining to detect the mechanism of impact on oocyte developmental competency after CTX exposures.Lastly,we used lysophosphatidic acid(LPA)to rescue the oocyte quality and embryonic development after CTX injury.We found that acute DNA damage and acceleration of the activation of primordial follicles after CTX treatment were repaired within several days,but the remaining follicle numbers remarkably decrease.Although partial surviving primordial follicle were developed to mature oocyte,oocyte quality hemostasis was impaired exhibiting aberrant meiosis progression,abnormal spindle and aneuploidy,mitochondrial dysfunction and increased endoplasmic reticulum stress.Thereafter,embryo development competency significantly decreased with fewer blastocyst formation after CTX exposure.CTX treatment resulted in alteration of DNA methylations and histone modifications in fullygrown GV oocytes.It revealed that H3K4me3 level was down-regulated while H3K9me3 was opposite,and demethylation of DNA was impaired.Single-cell RNA-seq revealed CTX treatment suppressed transcription of multiple maternal genes and methyltransferases,which caused the impairment of oocyte function and quality.Yap1 is enriched in RNA-seq and the expression of Yap1 was decreased significantly,which probably accounts for loss function of CTX-impaired oocyte.In vitro addition of lysophosphatidic acid(LPA)to embryo culture media to promote YAP1 nuclear localization improved CTX-impaired embryo developmental competence,and LPA supplement could also improve the meiotic progress and mitochondrial function of oocytes surviving from CTX exposure.In conclusion,the toxic effect of CTX on oocytes developed from primordial follicles following pre-pubertal exposure lasting consistently.The transcription and methylation of genes are also affected by CTX,which induced the decrease of oocyte function and quality.LPA supplement could partially improve the embryonic development competency after CTX insult.This study provides evidence for the consistent toxic effect of CTX exposure during follicle development,and provide a new insight for fertility preservation.