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Expression of Connexin43in Human Ovarian Granulosa Cells and Its Relationship with the Outcome of IVF-ET

Author: SongLin
Tutor: SunYingZuo
School: Zhengzhou University
Course: Reproductive Medicine
Keywords: Connexin43mural granulosa cells cumulus cells in vitro fertilizationand embryo transfer pregnant
CLC: R714
Type: Master's thesis
Year: 2013
Downloads: 9
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ObjectiveWith the gradually increased incidence of infertility, more and more people are able to have their babies through assisted reproductive technology. Since in vitro fertilization and embryo transfer (IVF-ET) program carried out, clinicians and researchers in this field are continuously improving technologies and adjusting the therapeutic regimens through a large amount of scientific experiments and clinical work. Although the success rate of IVF-ET has been significantly enhanced, there are always some patients who cannot get satisfactory results after the assisted reproduction, with less retrieved oocytes, less qualified embryos and low pregnancy rate. How to obtain the high-quality oocytes in a safe way is still a challenge.Follicular development and oocyte maturation is affected by a variety of different factors. The nutrients and metabolites transmission and signal transduction between oocytes and its surrounding granulosa cells and the granulosa cells themselves are mediated by connexins. Connexin43(Cx43) is considered as the most important gap junction protein in folliculogenesis. At present, most studies of the role of Cx43during folliculogenesis are carried out in animals, while only a few in human. In assisted reproduction, the relationship between the connexin expression levels in cumulus cells and the IVF outcomes is still controversial. This study aims to compare the difference of Cx43expression between mural granulosa cells and the cumulus cells and to investigate the relationship between Cx43and IVF-ET outcomes. Moreover, its predictive value of oocyte and embryo developmental competence is also discussed.Methods32patients’mural granulosa cells were collected from the IVF-ET programs while25patients’cumulus cells were collected from the ICSI-ET programs in Reproductive Medical Center of the First Affiliated Hospital of Zhengzhou University during the October2012to January2013. All the patients in this study were treated with the standard long protocol in COH. Pituitary down-regulation was achieved with GnRH-a in midluteal phase. During the COH process, the serum estradiol, progesterone, luteinizing hormone levels and follicle diameters were dynamically monitored and the doses of Gn were adjusted in time. HCG was administrated at a proper time and after36~37h the ultrasound-guided transvaginal oocytes retrieval would be taken.72h after in vitro fertilization, the cultured embryos were selected to transfer. Progesterone and Duphaston were given to luteal support at the same time. Serum β-HCG was examined on14th and18th day after embryo-transfer. Clinical pregnancy was confirmed by observation of gestational sac with ultrasonography35days after embryo-transfer. Patients were divided into2groups:pregnant and non-pregnant group.Cx43mRNA expression in mural granulosa cells and cumulus cells was tested by quantitative real-time PCR and the protein level of Cx43in mural granulosa cells was determined by immunocytochemistry. Different expression between the two groups was compared respectively. Immunofluorescent staining was used to examine the localization of Cx43in mural granulosa cells.Results1. Compared to the pregnant group, the level of mRNA and protein of Cx43in mural granulosa cells was significantly higher in the non-pregnant group (1.00(0.50, 2.08)vs.2.30(1.77,6.90), P<0.05;114.72±12.40vs.147.54±18.91, P<0.05)2. There was no significant difference in the expression of Cx43mRNA in cumulus cells between the two groups (1.68(0.99,3.13) vs.2.34(1.01,3.95),P>0.05)3. The expression of Cx43mRNA in mural granulosa cells was negatively correlated with fertilization rate and cleavage rate (r=-0.482, P<0.05;r=-0.453, P<0.05)ConclusionsCx43is expressed in ovarian mural granulosa cells and it is mainly located in membrane. To some extent, its expression level can reflect the quality of oocytes and embryos. It also can help us to predict the outcome of IVF-ET.

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