【摘要】：目的:卵巢過(guò)度刺激綜合征(ovarian hyperstimulation syndrome,OHSS)是輔助生殖技術(shù)(ART)中使用促排卵藥物后引起的一種醫(yī)源性疾病,自然周期妊娠者較罕見(jiàn)。卵巢過(guò)度刺激綜合征是由于卵巢對(duì)促性腺激素的刺激產(chǎn)生過(guò)度反應(yīng),造成多個(gè)卵泡發(fā)育并分泌過(guò)多的雌激素。隨著無(wú)排卵性不孕癥發(fā)病率的逐年升高,控制性促排卵作為在輔助生殖技術(shù)中的主要步驟,其導(dǎo)致的卵巢過(guò)度刺激綜合征已成為輔助生殖技術(shù)中高風(fēng)險(xiǎn)的并發(fā)癥之一,OHSS不但影響胚胎的發(fā)育著床,嚴(yán)重者甚至?xí)＜被颊呱�。多囊卵巢綜合征(polycystic ovarian syndrome,PCOS)是一種常見(jiàn)的多病因,多系統(tǒng)的內(nèi)分泌紊亂疾病,好發(fā)于生育年齡女性,發(fā)病率約為5-10%,其中75%女性出現(xiàn)無(wú)排卵性不孕,是引起不排卵性不孕的主要原因。符合以下三點(diǎn)中的兩點(diǎn)即可診斷為多囊卵巢綜合征,即稀發(fā)排卵或無(wú)排卵,臨床上或生化上的高雄激素血癥,及卵巢PCO樣改變。針對(duì)多囊卵巢綜合征(PCOS)的不排卵或稀發(fā)排卵,對(duì)患者進(jìn)行促排卵治療,后期指導(dǎo)同房或人工授精(IUI)是目前解決不孕的主要方法。而對(duì)于難治性PCOS患者或PCOS并發(fā)輸卵管梗阻、盆腔粘連、子宮內(nèi)膜異位癥及男方精液異常等原因時(shí),體外受精-胚胎移植(IVF-ET)是一種非常有效的治療方法。PCOS患者在控制性促排卵時(shí)易出現(xiàn)對(duì)促排卵藥物不敏感或過(guò)度敏感,甚至發(fā)生OHSS威脅患者生命。促排卵后PCOS患者卵巢反應(yīng)的難以控制性,尤其是OHSS的發(fā)生以及卵子質(zhì)量問(wèn)題是當(dāng)今輔助生育技術(shù)領(lǐng)域的難點(diǎn)和熱點(diǎn)。MicroRNA(miRNA)是一類(lèi)短鏈非編碼RNAs,主要在轉(zhuǎn)錄后水平對(duì)基因表達(dá)進(jìn)行調(diào)節(jié),對(duì)細(xì)胞的增殖、分化、凋亡起著重要調(diào)節(jié)作用。近年來(lái)研究表明miRNA水平的改變與疾病的發(fā)生密切相關(guān),如糖尿病,胰島素抵抗,炎癥及多種腫瘤的發(fā)生。因?yàn)閙iRNA在人類(lèi)血液中大量存在,而且具有相對(duì)穩(wěn)定性、易于檢測(cè)及疾病的特異性等特性,miRNA已經(jīng)作為多種疾病的預(yù)測(cè)因子。本課題旨在探索血清中mirna是否可以作為pcos患者在進(jìn)行控制性促排卵前預(yù)測(cè)ohss發(fā)生的生物學(xué)指標(biāo),并進(jìn)行驗(yàn)證。方法和結(jié)果:(1)選取2010年-2013年于南京醫(yī)科大學(xué)附屬南京市婦幼保健院行人工授精-胚胎移植或者單精子卵泡漿內(nèi)注射(ivf/icsi)的pcos患者為研究對(duì)象,根據(jù)其發(fā)生ohss的嚴(yán)重程度分為2組,ohss組:發(fā)生中、重度ohss的患者;pcos對(duì)照組:未發(fā)生或發(fā)生輕度ohss的患者。收集兩組患者血清,采用定量低密度芯片技術(shù)(taqmanlowdensityarraychips,tlda)來(lái)篩選兩組患者血清中差異表達(dá)的的mirnas。根據(jù)我們的篩選標(biāo)準(zhǔn),結(jié)果表明有8個(gè)mirnas出現(xiàn)了較明顯的差異表達(dá),其中三個(gè)(包括mir-146a、mir-30c、mir-191)在ohss組中明顯升高,五個(gè)(包括mir-16、mir-223、mir-212、mir-451、mir-92a)在ohss組中明顯下降(p0.05)。(2)擴(kuò)大樣本量,對(duì)篩選出的8個(gè)mirnas采用實(shí)時(shí)定量聚合酶鏈?zhǔn)椒磻?yīng)(qrt-pcr)的方法進(jìn)行進(jìn)一步驗(yàn)證,結(jié)果表明:其中兩個(gè)mirnas即:mir-16和mir-223在ohss組的表達(dá)明顯低于對(duì)照組(p值分別為0.000和0.012),剩余mirnas在兩組之間的表達(dá)無(wú)明顯統(tǒng)計(jì)學(xué)差異(p0.05)。(3)通過(guò)建立roc曲線并計(jì)算曲線下面積的方法,評(píng)估臨床特征包括基礎(chǔ)lh值、基礎(chǔ)lh/fsh比值及體重指數(shù)在預(yù)測(cè)ohss發(fā)生上的靈敏性及特異性。結(jié)果表明基礎(chǔ)lh值、基礎(chǔ)lh/fsh比值及體重指數(shù)的曲線下面積(auc)分別是0.712、0.772及0.702,提示基礎(chǔ)lh/fsh比值具有更高的準(zhǔn)確性。(4)我們采用了計(jì)算roc曲線下面積分析的方法,進(jìn)一步驗(yàn)證篩選所得的mirnas對(duì)中重度ohss的預(yù)測(cè)的靈敏性及特異性,計(jì)算mir-16、mir-223及mir-16/mir-223的面積值分別是0.848、0.801及0.836,結(jié)果提示mir-16的曲線下面積最大,可以作為一個(gè)更準(zhǔn)確的預(yù)測(cè)ohss發(fā)生風(fēng)險(xiǎn)的指標(biāo)。(5)通過(guò)david生物信息學(xué)(davidbioinformaticsresources)軟件,對(duì)差異mirnas的靶基因進(jìn)行g(shù)o(geneontology)分析及biocarta通路(biocartapathway)分析。go分析表明mir-16主要參與了基因表達(dá)的調(diào)節(jié),氨基酸的磷酸化等;miR-223主要參與基因表達(dá)的調(diào)節(jié)劑細(xì)胞的黏附等。Biocarta通路分析表明miR-16主要參與了調(diào)節(jié)VEGF的生成及血管生成的調(diào)節(jié)。miR-223主要參與胰島素受體信號(hào)通路并調(diào)節(jié)體重及葡萄糖的代謝等。(6)采用多元邏輯回歸分析的方法,分析血清miR-223水平與體重指數(shù)(BMI)相關(guān)性。結(jié)果表明miR-223與BMI呈正相關(guān)。結(jié)論:本研究采用了2012-2013年南京醫(yī)科大學(xué)附屬南京市婦幼保健院收集的PCOS患者血清樣本,分析了其控制性促排卵過(guò)程中發(fā)生中重度OHSS的患者血清mi RNAs表達(dá)譜,發(fā)現(xiàn)OHSS患者具有自身特異的miRNAs表達(dá),即與對(duì)照組相比實(shí)驗(yàn)組患者血清miR-16和miR-223表達(dá)明顯下降,并且通過(guò)ROC分析表明miR-16和mi R-223有可能作為評(píng)估中重度OHSS發(fā)生風(fēng)險(xiǎn)的臨床診斷指標(biāo)。進(jìn)一步生物信息學(xué)分析表明這些差異表達(dá)的miRNAs參與了OHSS的病理生理機(jī)制的調(diào)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.
【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類(lèi)號(hào)】：R711.75

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