| Background:Since the birth of the first ’test tube baby’ in1978through out the world, human assisted reproductive technology (ART) has made considerable progress and brought hope to the infertile couples. However, the pregnancy rate and live birth rate in in vitro-fertilization and embryo-transfer (IVF-ET) and intracytoplasmic sperm injection-embryo transfer (ICSI-ET) are still far below people’s expectation. Selection of embryos with high developmental potential for transfer is the key process to improve the pregnancy outcomes and the’fate’of embryo is lagely decided by the oocyte quality.In ART, how to select qualified embryos is a challenge to embryologists. Nowadays, noninvasive dynamic morphological grading system is widely used in clinical work. It includes the evaluation of pronuclear, early cleavage status, the number and the uniformity of the blastomeres on Day2and Day3, the percentage of fragments, the grade of blastocyst on Day5, and so on. However, these subjective morphological criteria cannot accurately reflect the embryo developmental competence. In order to acquire stable implantation rate and clinical pregnancy rate,2-3embryos with good morphology or1-2blastocysts are transferred. This may enhance the risk rate of multiple pregnancy and the complications of mothers and babies during pregnancy. At the same time, this will also bring economical burden to both family and the society.There is a close relationship between ovarian granulosa cells and oocytes. The former can affect the development, maturation and fertilization of the latter via autocrine, paracrine, nutrients flow, signal transduction and so on. It can even affect the early developmental status of embryos which come from the corresponding oocytes. In the process of IVF/ICSI, study on discarded ovarian granulosa cells may offer us objective and noninvasive methods to select embryos. So, the pregnancy rates can be increased, the elective single embryo/blastocyst transfer protocol can be promoted, the incidence rates of high-order multiple pregnancy and the complications it brings can be reduced.The proliferation of ovarian somatic cells is the pivotal process for ovarian reproductive function (oogenesis) and endocrine function. Previous studies have demonstrated that the proliferation of ovarian granulosa cells is closely related to the oocyte quality. Oogenesis accompanies with the proliferation of granulosa cells and it is regulated by a spatio-temporal way. The maintenance of telomere length is one of the main genetic mechanisms of cellular proliferation. Telomerase catalytic subunit or telomerase reverse transcriptase (TERT) is the major rate-limiting enzyme capable of lengthening telomric DNA. TERT is only expressed in tissues and cells with high proliferative ability, such as marrow stem cells, germ cells, endometrium, liver cells and epidermics and so on. Although there have been some studies on the relationship between telomerase activity and telomere length in human granulosa cells and the IVF outcomes, they did not analyze the TERT expression quantitatively in granulosa cells from mRNA levels and its relationship with in vitro fertilization-embryo transfer outcomes.Objective:The objective of this study was to locate the TERT expression in ovarian granulosa cells by immunofluorescence and immunohistochemical staining and determine the TERT mRNA expression quantitatively in mural granulosa cells and cumulus cells by quantitative real time PCR and study the relationship between TERT expression in granulosa cells and in vitro fertilization-embryo transfer outcomes, so that it can offer us an objective and quantitative criteria for oocyte quality evaluation and embryos selection.Methods:1.80patients’discarded follicular fluid and26patients’cumulus cells were collected from the IVF/ICSI-ET programs in Reproductive Medical Center of the First Affiliated Hospital of Zhengzhou University. All the patients were suffered from infertility due to tubal factor and male factor.2. Mural granulosa cells were purified from follicular fluid by Ficoll gradient cen-trifugation method and the red cell lysis buffer was used to eliminate the interference from red cells. The granulosa cells were separated to3different parts: one part was to be stained with immunofluorescence after a short-term culture in vitro and the cells’pictures were taken by laser scanning confocal microscope; one part was stored at-80℃until subsequent RNA isolation, reverse transcription and qRT-PCR to quantitatively determine the TERT mRNA; the last part was immobilized by4%formaldehyde after a short-term culture in vitro and stored at-80℃until subsequent immunohistochemical staining.3. SPSS12.0software was used to analyze the data. α=0.05was set as the inspection level and P＜0.05was considered significant.Results:1. The protein TERT was expressed in ovarian mural granulosa cells and it was located in cytoplasma and nucleus. TERT mRNA was expressed in both mural granulosa cells and cumulus cells.2. In mural granulosa cells, compared to the elderly group, TERT and its mRNA expression in younger group were significantly higher (145.13±28.63vs.123.82±24.34,.P＜0.05;1.27(1.00,2.2) vs.0.51(0.23,0.97), P＜0.05). 3. In mural granulosa cells of younger group, compared to the non-pregnant group, TERT expression of pregnant group was significantly higher (155.73±29.36vs.132.06±22.37, P＜0.05).4. In mural granulosa cells, TERT and its mRNA expression were negatively correlated with patient’s age (r=-0.401, P＜0.05; r=-0.386, P＜0.05).5. In cumulus cells, compared to the elderly group, TERT mRNA expression in younger group was significantly higher (0.45(0.18,0.83) vs.0.13(0.03,0.34), P＜0.05).6. In cumulus cells, compared to the non-pregnant group in both younger group and elderly group, TERT mRNA expression of pregnant group were significantly higher (0.77(0.44,1.00) vs.0.21(0.12,0.47), P＜0.05;0.22(0.09,0.40) vs.0.017(0.005,0.028), P＜0.05).7. In cumulus cells, TERT mRNA expression was negatively correlated with patient’s age (r=-0.654, P＜0.05).Conclusions:1. Telomerase reverse transcriptase and its mRNA are expressed in ovarian mural granulosa cells, the level of TERT and TERT mRNA expression are negatively correlated with patient’s age. To some extent, the expression of TERT may serve as a predictor of pregnancy outcome.2. In cumulus cells, the level of TERT mRNA expression is negatively correlated with patient’s age. It can predict pregnancy outcome. |
PDF Full Text Request