发生OHSS的PCOS患者血清miRNA的表达谱的特征及诊断性分子标志物的筛选
发布时间：2018-07-26 17:40
【摘要】：目的:卵巢过度刺激综合征(ovarian hyperstimulation syndrome,OHSS)是辅助生殖技术(ART)中使用促排卵药物后引起的一种医源性疾病,自然周期妊娠者较罕见。卵巢过度刺激综合征是由于卵巢对促性腺激素的刺激产生过度反应,造成多个卵泡发育并分泌过多的雌激素。随着无排卵性不孕症发病率的逐年升高,控制性促排卵作为在辅助生殖技术中的主要步骤,其导致的卵巢过度刺激综合征已成为辅助生殖技术中高风险的并发症之一,OHSS不但影响胚胎的发育着床,严重者甚至会危急患者生命。多囊卵巢综合征(polycystic ovarian syndrome,PCOS)是一种常见的多病因,多系统的内分泌紊乱疾病,好发于生育年龄女性,发病率约为5-10%,其中75%女性出现无排卵性不孕,是引起不排卵性不孕的主要原因。符合以下三点中的两点即可诊断为多囊卵巢综合征,即稀发排卵或无排卵,临床上或生化上的高雄激素血症,及卵巢PCO样改变。针对多囊卵巢综合征(PCOS)的不排卵或稀发排卵,对患者进行促排卵治疗,后期指导同房或人工授精(IUI)是目前解决不孕的主要方法。而对于难治性PCOS患者或PCOS并发输卵管梗阻、盆腔粘连、子宫内膜异位症及男方精液异常等原因时,体外受精-胚胎移植(IVF-ET)是一种非常有效的治疗方法。PCOS患者在控制性促排卵时易出现对促排卵药物不敏感或过度敏感,甚至发生OHSS威胁患者生命。促排卵后PCOS患者卵巢反应的难以控制性,尤其是OHSS的发生以及卵子质量问题是当今辅助生育技术领域的难点和热点。MicroRNA(miRNA)是一类短链非编码RNAs,主要在转录后水平对基因表达进行调节,对细胞的增殖、分化、凋亡起着重要调节作用。近年来研究表明miRNA水平的改变与疾病的发生密切相关,如糖尿病,胰岛素抵抗,炎症及多种肿瘤的发生。因为miRNA在人类血液中大量存在,而且具有相对稳定性、易于检测及疾病的特异性等特性,miRNA已经作为多种疾病的预测因子。本课题旨在探索血清中mirna是否可以作为pcos患者在进行控制性促排卵前预测ohss发生的生物学指标,并进行验证。方法和结果:(1)选取2010年-2013年于南京医科大学附属南京市妇幼保健院行人工授精-胚胎移植或者单精子卵泡浆内注射(ivf/icsi)的pcos患者为研究对象,根据其发生ohss的严重程度分为2组,ohss组:发生中、重度ohss的患者;pcos对照组:未发生或发生轻度ohss的患者。收集两组患者血清,采用定量低密度芯片技术(taqmanlowdensityarraychips,tlda)来筛选两组患者血清中差异表达的的mirnas。根据我们的筛选标准,结果表明有8个mirnas出现了较明显的差异表达,其中三个(包括mir-146a、mir-30c、mir-191)在ohss组中明显升高,五个(包括mir-16、mir-223、mir-212、mir-451、mir-92a)在ohss组中明显下降(p0.05)。(2)扩大样本量,对筛选出的8个mirnas采用实时定量聚合酶链式反应(qrt-pcr)的方法进行进一步验证,结果表明:其中两个mirnas即:mir-16和mir-223在ohss组的表达明显低于对照组(p值分别为0.000和0.012),剩余mirnas在两组之间的表达无明显统计学差异(p0.05)。(3)通过建立roc曲线并计算曲线下面积的方法,评估临床特征包括基础lh值、基础lh/fsh比值及体重指数在预测ohss发生上的灵敏性及特异性。结果表明基础lh值、基础lh/fsh比值及体重指数的曲线下面积(auc)分别是0.712、0.772及0.702,提示基础lh/fsh比值具有更高的准确性。(4)我们采用了计算roc曲线下面积分析的方法,进一步验证筛选所得的mirnas对中重度ohss的预测的灵敏性及特异性,计算mir-16、mir-223及mir-16/mir-223的面积值分别是0.848、0.801及0.836,结果提示mir-16的曲线下面积最大,可以作为一个更准确的预测ohss发生风险的指标。(5)通过david生物信息学(davidbioinformaticsresources)软件,对差异mirnas的靶基因进行go(geneontology)分析及biocarta通路(biocartapathway)分析。go分析表明mir-16主要参与了基因表达的调节,氨基酸的磷酸化等;miR-223主要参与基因表达的调节剂细胞的黏附等。Biocarta通路分析表明miR-16主要参与了调节VEGF的生成及血管生成的调节。miR-223主要参与胰岛素受体信号通路并调节体重及葡萄糖的代谢等。(6)采用多元逻辑回归分析的方法,分析血清miR-223水平与体重指数(BMI)相关性。结果表明miR-223与BMI呈正相关。结论:本研究采用了2012-2013年南京医科大学附属南京市妇幼保健院收集的PCOS患者血清样本,分析了其控制性促排卵过程中发生中重度OHSS的患者血清mi RNAs表达谱,发现OHSS患者具有自身特异的miRNAs表达,即与对照组相比实验组患者血清miR-16和miR-223表达明显下降,并且通过ROC分析表明miR-16和mi R-223有可能作为评估中重度OHSS发生风险的临床诊断指标。进一步生物信息学分析表明这些差异表达的miRNAs参与了OHSS的病理生理机制的调控。
[Abstract]:Objective: ovarian hyperstimulation syndrome (ovarian hyperstimulation syndrome, OHSS) is a iatrogenic disease caused by the use of oviposit drugs in assisted reproductive technology (ART). The patients with natural cycle pregnancy are rare. Ovarian hyperstimulation syndrome is due to ovarian hyperstimulation of gonadotropin stimulation, resulting in multiple follicles. As the incidence of Anovulatory Infertility increases year by year, controlling ovulation is the main step in assisted reproductive technology. The ovarian hyperstimulation syndrome has become one of the high risk complications in assisted reproductive technology. OHSS not only affects the development of embryo, but also affects the development of the embryo. Polycystic ovarian syndrome (PCOS) is a common multiple cause, multiple system of endocrine disorders, good hair in the reproductive age of women, the incidence of about 5-10%, of which 75% female anovulatory infertility is the main cause of anovulatory infertility. It is in line with two of the following three points. Points can be diagnosed as polycystic ovary syndrome, that is, dilute ovulation or ovulatory, clinical or biochemical Kaohsiung steroids, and ovarian PCO like changes. For polycystic ovary syndrome (PCOS), ovulating or dilute ovulation, promoting ovulation treatment for patients, and guiding the same room or artificial insemination (IUI) at the later stage are the main methods to solve infertility. In the case of refractory PCOS patients or PCOS complicated with tubal obstruction, pelvic adhesions, endometriosis, and male semen abnormalities, in vitro fertilization and embryo transfer (IVF-ET) is a very effective treatment for.PCOS patients who are susceptible to ovulatory or oversensitive, or even OHSS, during controlled ovulation. The difficult control of ovarian response in PCOS patients after ovulation, especially the occurrence of OHSS and the quality of egg is a difficult and hot spot in the field of assisted reproductive technology,.MicroRNA (miRNA) is a class of short chain non coding RNAs, which mainly regulates the expression of the gene at the post transcriptional level, and the proliferation, differentiation and apoptosis of the cells. In recent years, studies have shown that changes in miRNA levels are closely related to the occurrence of disease, such as diabetes, insulin resistance, inflammation and the occurrence of a variety of tumors. Because miRNA exists in human blood, and is relatively stable, easy to detect and the specificity of disease, miRNA has been used as a variety of diseases. The purpose of this study is to explore whether serum miRNA can be used as a biological indicator for predicting the occurrence of OHSS in PCOS patients before controlling ovulation. Methods and results: (1) an artificial insemination embryo transfer or single sperm egg in the Nanjing Maternity and Child Care Center affiliated to Nanjing Medical University in 2010 was selected. PCOS patients with intratural injection (ivf/icsi) were divided into 2 groups according to the severity of the occurrence of OHSS. Group OHSS: Patients with severe OHSS; PCOS control group: patients who did not have or have mild OHSS. Collect two groups of patients' serum, use quantitative low density chip technology (taqmanlowdensityarraychips, tlda) to screen two groups of patients. The differential expression of mirnas. in serum was based on our screening criteria, and the results showed that 8 miRNAs showed significant differential expression, of which three (including miR-146a, mir-30c, mir-191) were significantly increased in the OHSS group, and five (including mir-16, mir-223, miR-212, mir-451, mir-92a) decreased significantly in the OHSS group. (2) expanded sample size, 8 selected miRNAs were further verified by real-time quantitative polymerase chain reaction (qRT-PCR). The results showed that the expression of two miRNAs, mir-16 and mir-223 in the OHSS group was significantly lower than that of the control group (P value was 0 and 0.012 respectively), and the expression of the remaining miRNAs in the two groups was not statistically significant (P0.05). (3) After establishing the ROC curve and calculating the area under the curve, the sensitivity and specificity of the clinical features including basic LH value, basic lh/fsh ratio and body mass index in predicting the occurrence of OHSS were evaluated. The results showed that the basal LH value, the basal lh/fsh ratio and the body mass index curve area (AUC) were 0.712,0.772 and 0.702 respectively, suggesting the basis lh/fsh ratio. The value has higher accuracy. (4) we use the method of calculating the area analysis under the ROC curve to further verify the sensitivity and specificity of the selected miRNAs for the prediction of moderate and severe OHSS. The area values of mir-16, mir-223 and mir-16/mir-223 are 0.848,0.801 and 0.836 respectively. The results suggest that the area under the mir-16 is the largest. As a more accurate index for predicting the risk of OHSS occurrence. (5) go (geneontology) analysis and biocarta pathway (biocartapathway) analysis of the target genes of differential miRNAs are carried out by David bioinformatics (davidbioinformaticsresources) software and.Go analysis shows that mir-16 is mainly involved in the regulation of gene expression, phosphorylation of amino acids, and so on. The.Biocarta pathway analysis of miR-223 mainly involved in the gene expression of regulator cells showed that miR-16 was mainly involved in regulating the formation of VEGF and regulating the angiogenesis of.MiR-223 mainly involved in insulin receptor signaling pathway and regulating body weight and glucose metabolism. (6) multivariate logistic regression analysis was used to analyze serum miR- The 223 level was associated with body mass index (BMI). The results showed that miR-223 was positively correlated with BMI. Conclusion: This study adopted the serum samples of PCOS patients collected by Nanjing Maternity and Child Care Center of Nanjing Medical University for 2012-2013 years, and analyzed the serum mi RNAs expression profiles of patients with moderate OHSS in the process of controlling ovulation, and found OHSS patients. The expression of miR-16 and miR-223 in the sera of the experimental group was significantly lower than that of the control group, and the ROC analysis showed that miR-16 and MI R-223 could be used as a clinical diagnostic indicator to assess the risk of moderate severe OHSS. Further bioinformatics analysis showed that the miRNAs of these differentially expressed miRNAs participated in O. The regulation of the pathophysiological mechanism of HSS.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R711.75
[Abstract]:Objective: ovarian hyperstimulation syndrome (ovarian hyperstimulation syndrome, OHSS) is a iatrogenic disease caused by the use of oviposit drugs in assisted reproductive technology (ART). The patients with natural cycle pregnancy are rare. Ovarian hyperstimulation syndrome is due to ovarian hyperstimulation of gonadotropin stimulation, resulting in multiple follicles. As the incidence of Anovulatory Infertility increases year by year, controlling ovulation is the main step in assisted reproductive technology. The ovarian hyperstimulation syndrome has become one of the high risk complications in assisted reproductive technology. OHSS not only affects the development of embryo, but also affects the development of the embryo. Polycystic ovarian syndrome (PCOS) is a common multiple cause, multiple system of endocrine disorders, good hair in the reproductive age of women, the incidence of about 5-10%, of which 75% female anovulatory infertility is the main cause of anovulatory infertility. It is in line with two of the following three points. Points can be diagnosed as polycystic ovary syndrome, that is, dilute ovulation or ovulatory, clinical or biochemical Kaohsiung steroids, and ovarian PCO like changes. For polycystic ovary syndrome (PCOS), ovulating or dilute ovulation, promoting ovulation treatment for patients, and guiding the same room or artificial insemination (IUI) at the later stage are the main methods to solve infertility. In the case of refractory PCOS patients or PCOS complicated with tubal obstruction, pelvic adhesions, endometriosis, and male semen abnormalities, in vitro fertilization and embryo transfer (IVF-ET) is a very effective treatment for.PCOS patients who are susceptible to ovulatory or oversensitive, or even OHSS, during controlled ovulation. The difficult control of ovarian response in PCOS patients after ovulation, especially the occurrence of OHSS and the quality of egg is a difficult and hot spot in the field of assisted reproductive technology,.MicroRNA (miRNA) is a class of short chain non coding RNAs, which mainly regulates the expression of the gene at the post transcriptional level, and the proliferation, differentiation and apoptosis of the cells. In recent years, studies have shown that changes in miRNA levels are closely related to the occurrence of disease, such as diabetes, insulin resistance, inflammation and the occurrence of a variety of tumors. Because miRNA exists in human blood, and is relatively stable, easy to detect and the specificity of disease, miRNA has been used as a variety of diseases. The purpose of this study is to explore whether serum miRNA can be used as a biological indicator for predicting the occurrence of OHSS in PCOS patients before controlling ovulation. Methods and results: (1) an artificial insemination embryo transfer or single sperm egg in the Nanjing Maternity and Child Care Center affiliated to Nanjing Medical University in 2010 was selected. PCOS patients with intratural injection (ivf/icsi) were divided into 2 groups according to the severity of the occurrence of OHSS. Group OHSS: Patients with severe OHSS; PCOS control group: patients who did not have or have mild OHSS. Collect two groups of patients' serum, use quantitative low density chip technology (taqmanlowdensityarraychips, tlda) to screen two groups of patients. The differential expression of mirnas. in serum was based on our screening criteria, and the results showed that 8 miRNAs showed significant differential expression, of which three (including miR-146a, mir-30c, mir-191) were significantly increased in the OHSS group, and five (including mir-16, mir-223, miR-212, mir-451, mir-92a) decreased significantly in the OHSS group. (2) expanded sample size, 8 selected miRNAs were further verified by real-time quantitative polymerase chain reaction (qRT-PCR). The results showed that the expression of two miRNAs, mir-16 and mir-223 in the OHSS group was significantly lower than that of the control group (P value was 0 and 0.012 respectively), and the expression of the remaining miRNAs in the two groups was not statistically significant (P0.05). (3) After establishing the ROC curve and calculating the area under the curve, the sensitivity and specificity of the clinical features including basic LH value, basic lh/fsh ratio and body mass index in predicting the occurrence of OHSS were evaluated. The results showed that the basal LH value, the basal lh/fsh ratio and the body mass index curve area (AUC) were 0.712,0.772 and 0.702 respectively, suggesting the basis lh/fsh ratio. The value has higher accuracy. (4) we use the method of calculating the area analysis under the ROC curve to further verify the sensitivity and specificity of the selected miRNAs for the prediction of moderate and severe OHSS. The area values of mir-16, mir-223 and mir-16/mir-223 are 0.848,0.801 and 0.836 respectively. The results suggest that the area under the mir-16 is the largest. As a more accurate index for predicting the risk of OHSS occurrence. (5) go (geneontology) analysis and biocarta pathway (biocartapathway) analysis of the target genes of differential miRNAs are carried out by David bioinformatics (davidbioinformaticsresources) software and.Go analysis shows that mir-16 is mainly involved in the regulation of gene expression, phosphorylation of amino acids, and so on. The.Biocarta pathway analysis of miR-223 mainly involved in the gene expression of regulator cells showed that miR-16 was mainly involved in regulating the formation of VEGF and regulating the angiogenesis of.MiR-223 mainly involved in insulin receptor signaling pathway and regulating body weight and glucose metabolism. (6) multivariate logistic regression analysis was used to analyze serum miR- The 223 level was associated with body mass index (BMI). The results showed that miR-223 was positively correlated with BMI. Conclusion: This study adopted the serum samples of PCOS patients collected by Nanjing Maternity and Child Care Center of Nanjing Medical University for 2012-2013 years, and analyzed the serum mi RNAs expression profiles of patients with moderate OHSS in the process of controlling ovulation, and found OHSS patients. The expression of miR-16 and miR-223 in the sera of the experimental group was significantly lower than that of the control group, and the ROC analysis showed that miR-16 and MI R-223 could be used as a clinical diagnostic indicator to assess the risk of moderate severe OHSS. Further bioinformatics analysis showed that the miRNAs of these differentially expressed miRNAs participated in O. The regulation of the pathophysiological mechanism of HSS.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R711.75
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