| BackgroundThe world’s first test-tube baby was born in 1978, and since then human assisted reproductive technology(ART) has developed rapidly and brought hope to many infertile couples. However, the pregnancy rate and live birth rate in in vitro-fertilization and embryo-transfer(IVF-ET) are still far away form our expectations.Oocyte and emhryo quality are the main factors, and they can determine the outcome of ART. How to select qualified embryos is a challenge to clinical reproductive physicians and how to accurately assess the quality of oocytes and embryos before transplantation, has always been the focal problem with troubled embryologists.Available assessment methods for the oocytes and embryos quality rely mainly on morphological observation, but there are certain limitations of morphological assessment and it can’t objectively reflect the developmental potention of embryo. Clinicians transplante 2-3 quality embryos to ensure good pregnancy outcomes, which leads to the incidence of multiple pregnancies and the risk of a variety of adverse complications greatly increased. And it usually brings the double burden of physical, psychological and economic to the patient and family. Follicular fluid is microenvironment to the growth of oocytes, and it offers a variety of hormones and cytokines for the development and mature of oocytes, and even affects the egg fertilization and embryo developmental potential. The relationship between oocyte and granulosa cells in follicles is close. Cumulus-oocyte complexes is made up of cumulus granulosa cells and oocytes,with wide gap junctions between cells. Granulosa cells may exchange informations with oocytes through these connections, develop and aging synchronously. Granulosa cells can also regulate oocyte maturation via the way of endocrine, what’s more, it may affect fertilization and the potential development of embryo. By studying the follicular fluid and granulosa cells, it is expected to provide a more objective selection of oocytes and embryos. With this accurate and noninvasive method, the pregnancy rates can be increased, the incidence rates of high-order multiple pregnancy and the complications would be reduced, which will ensure good pregnancy outcomes.Previous studies have demonstrated that the Cytokines level in follicle and the granulosa cells are closely related to the Egg maturation, fertilization and embryo quality. During the process of oocyte development, the type and level of cytokines is changed. At the same time, granular cells in ovarian could multiply. AMH is secreted by the granular cell, and it can regulate granulosa cells and follicular function via autocrine and paracrine mechanisms. The balance of cell proliferation and apoptosis is regulated by telomere and telomerase. There is rare research about human cytokines AMH in follicular fluid and the telomere length of ovarian granulosa cell with oocyte maturation and the pregnant outcome in IVF-ET. Study on the relationship between AMH and granular cell telomeres may offer us some method to delay shorten telomere and promote follicle development. ObjectiveThe objective of this study investigate the relationship between AMH in follicular fluid together with the telomere length of ovarian granulosa cell and oocytes at different mature stages, the outcome in in vitro fertilization and embryo transfer(IVF-ET). Moreover, it is also discussed whether AMH and telomere length of granulosa ceils can be a objective clinical indicator for evaluation of oocytes quality, embryo developmental capacity. Methods92 patients’ follicular fluids were collected from the IVF-ET programs while 81 patients’ cumulus cells were collected in Reproductive Medical Center of the Third Affiliated Hospital of Zhengzhou University during the October 2014 to March 2015.All patients were treated with the standard long protocol in COH in this study. Follicular fluids were obtained from 92 women undergoing IVF-ET. Oocyte–cumulus complex samples were collected from 81 patients and Cumulus granular cells(CCs) were manually separated from the oocyte-cumulus complex under microscope. Oocyte maturation of all the samples were recorded. On the basis of age was divided into the young age group(<35 years), the older age group(≥35 years). The follicular fluids AMH basal levels were evaluated by ELISA. Cumulus GC were assayed by quantitative RealtimePCR for telomere length. Results1.AMH concentrations in the immature follicles group were higher than in the mature follicles group, it failed to achieve statistical significance in the young age group(P=0.18) while it achieved statistical significance in the older age group(P=0.01). There was no statistically significant AMH in the pregnant subgroup than in the non-pregnant subgroup among the young and older age group(P>0.05)2. There was a negative correlation between AMH concentrations in follicular fluids with the patients’ age(r=-0.637, P=0.01).3. The relative telomere length of CCs was higher in mature follicles than immature follicles in both the young and older age group and it achieved statistical significance(P<0.05). There was a statistically significantly higher telomere length of CCs in the pregnant subgroup than in the non-pregnant subgroup among the older age group(P=0.01). It was a similar trend in the young women but it failed to achieve statistical significance(P=0.08).4. There was a negative correlation between relative telomere length of CCs with the patients’ age(r=-0.367, P=0.02), it also has the same trend between AMH and relative telomere length in cumulus granular cells(r=-0.241, P=0.04). Conclusions1. AMH may inhibite the maturation of oocyte, AMH level in follicular fluids may be used as a diagnostic criterion of oocyte maturation. It is negatively correlated with patient’s age.2. The telomere length of CCs is negatively correlated with patient’s age. The telomere length of CCs could reflect the degree of oocyte maturation and telomere may be associated with pregnancy outcome in IVF-ET.3. There was a correlation between telomere length of CCs with AMH, AMH may affect telomeres through some mechanism. |
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