The Long-term Effects Of Repeated Ovulation On Ovarian Structure And Function Were Evaluated Using The Rhesus Monkey Model

During the past30years, Assisted reproductive technologies (ARTs) have been a highly successful and widely used to treat human infertility since the world’s first test tube baby Louis Brown born in1978. In clinical, the patients are always given the exogenous follicle stimulating hormone (FSH) and luteinizing hormone (LH) to increase the gonadotropin levels in vivo, to induce multiple follicular development, known as controlled ovarian hyperstimulation (COH), the collection of multiple oocytes increases the chances of embryos for transplantation and pregnancy in a given treatment cycle and allows for excess embryos to be frozen for subsequent pregnancy attempts. However, despite the improvements in the success of ART, the fact remains that the majority of patients require more than one treatment cycle to achieve pregnancy. Therefore, an increasing number of researchers are focusing on the effects of repeated ovulation on offspring and maternal. Especially, whether the treatment of COH would have an impact on the structure and function of the ovary has become a problem that can’t be ignored. Although there are some related ovulation treatment safety assessment report, but there is no definite conclusion between these reports, more importantly, many of these studies are retrospective cohort and case-control studies based on human populations, which are not suitable for the assessment of the long-term influence of repeated ovulation on the ovary. Therefore, there have an important significance to systematic evaluation of the long-term effects of repeated superovulation on the structure and function of the ovaries.As a primate animal, rhesus macaque has many similar physiological characteristics with human, especially female macaque menstrual cycle and human is very similar, therefore it has become the ideal experimental animal to investigate the reproductive regulation mechanism. In our study, we used six adult female rhesus macaques (average age,13years) that had been provided by the Kunming Primate Research Center (KPRC). Three rhesus macaques had received four cycles of ovarian stimulation five years ago (superovulation group). The remaining three monkeys had not received any treatment (normal control group). Using morphological, molecular biology, proteomics to evaluate the structure and function of ovaries that have previously received repeated COH treatment and determining whether repeated ovulation stimulations have a long-term effect on ovaries, and further investigate the potential mechanism.Firstly, morphological observation on ovarian tissues, the results show that all the ovaries in the superovulation and control groups were non-cancerous. Follicles at different developmental stages, including primordial, primary, secondary, antral, and atretic, were found in the ovaries of both the groups, and there were no discernible abnormalities in cell morphology and histology of these follicles between the two groups. There were no significant differences in the proportion of follicles of different developmental stages between the superovulation and normal groups; however, the proportion of secondary, antral, and atretic follicles in the superovulation group showed a rising trend. The ultrastructure of follicle cells was observed by transmission electron microscopy. In the superovulation group, there were many granulosa cells with abnormal mitochondria, showing degradation of mitochondrial cristae, matrix vague, and vacuolization.Further we assessed the ovarian function by detecting the proliferation and apoptosis of follicle cells, the developmental status of the oocytes, the hormone levels in serum sample and ability of the follicles to synthesize hormones. The results show that:1. Ki67immunostaining and TUNEL assay were conducted to assess the cell proliferation and apoptosis of follicles. The results show that both Ki67-and TUNEL-positive signals were mainly observed in the granulosa cells. By calculating the ratio of Ki67-positive follicles and TUNEL-positive cells, we revealed no significant differences in the proliferation and apoptotic index between the superovulation and normal groups.2. We then assessed the developmental competence of oocytes by detecting the expression of specific oocyte-derived mRNAs in the ovaries. The mRNA levels of five genes, including GDF9, ZP3, Nlrp5, Tcll and NOBOX, were examined, showing no significant differences between the superovulation and normal groups.3. On days3and10of the menstrual cycle, the serum E2, P4, and FSH concentrations showed no significant differences between the superovulation and normal groups. The expression levels of estrogen synthesis-related molecule StAR and CYP19A1were significanty lower in the superovulation group than in the normal group. All these results suggest that repeated ovulation induction might have long-term adverse effects on the development and function of follicle cells, decreasing their hormone synthesis ability.Finally, we use the TMT labeling with MS to compare the protein expression level between normal group and ovulation group, by analysing the differentially expressed proteins, to assess the possible mechanism from molecular level. The results show that:We identified79proteins that exhibited significantly differential expression in the superovulation group compared to the normal group(P<0.05). Of these,51proteins showed at least a1.5-fold significant change. Most of these differentially expressed proteins (43/51) were downregulated, and only a few (8/51) were upregulated in the superovulation group. These differentially proteins were mainly localized in mitochondrion and cytosol. This may be related to the abnormal mitochondria that we found in the superovulation group, which is consistent with the previous studies who reported that repeated ovulation could result the mitochondrial damage. Through the discovery of previously reports which is associated with the differentially expressed proteins, we found that the changes in protein levels in superovulation group mainly inhibited cell proliferation and differentiation but promoted apoptosis. Moreover, many differentially expressed proteins(56.8%)were associated with tumorigenesis.In conclusion, our study indicated that repeated COH could change the expression of many proteins in the ovaries even after several years, the changes in protein levels mainly inhibited cell proliferation and differentiation but promoted apoptosis, which showed the negative effect on the ovary. Although our study did not found the obviously abnormal changes in the ovary that had repeated COH many years ago, but on the observation through the molecular level and the ultrastructural, we still found that the ovarian granulosa cells showed low potential development trend and the expression level of the key factor and enzyme involved in hormone synthesis were decreased.