【作者】 龚斐；

【导师】 卢光琇；

【作者基本信息】 中南大学 ， 基础医学， 2013， 博士

【摘要】 多囊卵巢综合征(Polycystic Ovary Syndrome, PCOS)是一种常见的内分泌紊乱综合征,在生育年龄的妇女人群中发病率约为5%,是引起育龄女性无排卵性不孕的主要原因[1]。约80%的PCOS患者可通过药物干预或腹腔镜下卵巢打孔术诱发排卵而自然受孕,但仍有20%的PCOS妇女需要在辅助生殖技术的帮助下才能妊娠。体外受精-胚胎移植技术(In vitro fertilization and embryo transfer, IVF-ET)是分别将卵子与精子从人体内取出后体外受精,发育成胚胎后再移植回母体子宫以帮助不孕夫妇建立妊娠的辅助生殖技术。该项技术的一个关键是控制性促排卵,用药物在可控制的范围内诱发多个卵泡的发育和成熟,得到高质量的卵子,形成多个可供筛选的胚胎进行移植,从而提高了每周期试管婴儿的成功率。对于PCOS患者,临床上常规使用长效长方案进行促排卵。但是因PCOS患者往往伴有FSH分泌不足、LH分泌异常增高、高胰岛素血症、高雄激素血症等内分泌和代谢的异常,导致其IVF妊娠率低,促排后卵巢过度刺激综合征(Ovarian hyperstimulation syndrome, OHSS)等并发症发生率高[2]。对PCOS这一特殊人群,我们想找到一种更适合的促排卵方案,以获得更高的妊娠率、并减少促排卵并发症发生的风险。针对PCOS患者特殊的病理特征,我们前期发明了—种改良的超长方案。为了回答改良的超长方案是否可以提高PCOS患者的IVF妊娠率这一问题,在本论文的第一部分,我们分别通过回顾性和前瞻性的对照研究,比较了改良超长方案和长效长方案用于PCOS患者IVF治疗的各项临床指标和临床结局。质量优良的胚胎在母体子宫内通过着床、分化,最终发育成一个成熟的胎儿。着床是建立成功妊娠的重要一步,而子宫内膜的容受性即子宫内膜接受胚胎的能力是着床的关键因素之一[3]。目前的观点认为,调节子宫内膜的容受性比较明确的指标有：子宫内膜的厚度、形态、血流,体内性激素水平,子宫内膜着床相关因子(如同源框基因相关蛋白产物和白细胞抑制因子)等。各个因素相互调节、作用,增加子宫内膜的容受性,使胚胎能够成功的植入。在人类着床窗口期,如子宫内膜厚度增加(>9mm)、内膜下血流指数上升、胞饮突出现、LIF、HOXA10基因等表达增加,则意味着良好的子宫内膜容受性[4、5]。因此,在本文的第二部分,为了探讨两种促排方案对子宫内膜容受性的影响,我们通过电镜检测了改良超长方案组和长效长方案组中PCOS患者着床窗口期胞饮突情况,用免疫组化、Real time-PCR, Western blot对比研究了两组着床窗口期HOXA10、LPAR3和COX2的表达情况,以探讨改良超长方案是否能通过改善PCOS患者的子宫内膜的容受性来提高IVF妊娠率。由于PCOS患者体内FSH水平低,导致2-8mm小卵泡的堆积[2],并且FSH的阈值窄,这部分患者接受常规IVF助孕治疗,较其他患者更易发生OHSS(高达14%)[6]。卵母细胞体外成熟培养(In vitro maturation, IVM)是指从不孕妇女卵巢中获取未成熟卵子并体外培养成熟,再结合卵胞浆内精子注射治疗不孕症的辅助生殖技术。IVM技术可以在不使用或仅少量使用促性腺激素(Gn)的情况下获得卵子进行IVM,由于未用Gn或Gn用量小,即使注射人类绒毛膜促性腺激素(hCG),在治疗过程中患者体内的雌激素水平较低,不会达到诱发OHSS发生的水平而使OHSS发生率大大降低,同时因为使用药物少可以大幅度降低治疗费用。由于采用IVM技术治疗PCOS并发症少、费用低,部分中心使用IVM作为PCOS患者IVF治疗的一个替代途径。但是常规的IVM技术成功率较低,仅10%-30%,严重地限制了该技术在临床的广泛开展。在论文的第三部分我们探讨对IVM技术进行改良,以提高IVM治疗的成功率,以期为PCOS患者提供更安全有效的治疗方案。第一部分改良超长方案在PCOS不孕患者IVF/ICSI-ET中的疗效目的：分别回顾性和前瞻性比较改良超长方案降调节结合人绝经期促性腺激素(HMG)和常规长效长方案降调节在多囊卵巢综合征(PCOS)不孕患者行体外受精/胞浆内单精子注射-胚胎移植术(IVF/ICSI-ET)助孕中的疗效。方法：(1)回顾分析我院2008.1-2012.12采用改良超长方案的1989例和长效长方案的1370例PCOS患者,对两组受精率、卵裂率、临床妊娠率、种植率、抱婴率、流产率、治疗费用等重要指标进行统计和对比。(2)将我院2011.9～2012.4行IVF/ICSI-ET的PCOS患者随机分为两组,分别实施改良超长方案降调后促排卵(n=75)和标准的长效长方案促排(n=72)；观察两种促排方案的平均获卵数、受精率、优胚率、取消率、重度OHSS发生率、移植妊娠率、种植率、流产率、宫外孕率及其他相关指标,同时评价HCG日E2、LH、P、P/E2的水平,阴道超声检测两组患者HCG日子宫内膜(EM)的厚度、形态、血流情况,并将两组的结果进行对比分析。结果：(1)在回顾性研究中,改良超长方案组和长效长方案组在年龄、基础体重指数(BMI)、不孕年限、基础内分泌水平、平均获卵数、优胚率、移植的胚胎数、周期取消率、OHSS取消率、冻胚妊娠率、重度OHSS发生率差异均无明显差异(P>0.05)。但是改良超长方案组妊娠率为74.56%,胚胎种植率为52.51%,抱婴率60.43%,显著高于长效长方案组(分别妊娠率为56.02%,胚胎种植率为37.83%,抱婴率42.88%,P<0.001)。改良超长方案注射hCG日的P水平0.62±0.29ng/ml,明显低于长效长方案组(0.81±0.39ng/ml,P<0.05),而hCG日内膜厚度则明显厚于长效长方案组(12.26±2.42mm和11.36±2.43mm,P<0.001)。改良超长方案的早期流产率较长效长方案组明显下降(6.02%和8.59%,P<0.05)。此外,改良超长方案组的整体治疗费用较长效长方案组亦有显著下降(989.58±418.59元和5831.73±2313.13元,P<0.001)。(2)在前瞻性研究中,两组患者共入选147例,无一例患者脱失。其中改良超长方案组75例,长效长方案组72例。两组患者相比其Gn用量(IU)、用药时间(天)、hCG日E2水平和LH水平、平均获卵数(个)、受精率、优胚率、取消率、移植胚胎数、重度OHSS发生率、流产率、宫外孕发生率均无统计学差异。改良超长方案组种植率及移植妊娠率(59.6%和77.9%)均高于长效长方案组(44.1%和61.3%,P<0.05)。与长效长方案相比,运用改良的超长方案促排后患者HCG日P水平及P/E2明显降低,子宫内膜(EM)的厚度、形态、血流情况明显改善。结论：对PCOS不孕患者实施IVF/ICSI-ET助孕过程中,采用改良的GnRH-a超长降调节+hMG超排方案可通过优化患者内膜形态及血流分布,增加子宫内膜厚度,抑制子宫内膜过早的向分泌期转化等显著改善PCOS患者的子宫内膜的容受性,提高患者的着床率和妊娠率,并节约临床治疗费用。第二部分改良超长方案对PCOS不孕患者着床期子宫内膜容受性的影响目的：改良超长方案和长效长方案治疗PCOS患者着床期子宫内膜胞饮突、HOXA10、LPAR3、COX2表达的对比研究。方法：分别收集改良超长方案组和长效长方案组治疗PCOS患者因OHSS原因取消移植后,取卵后第6天的子宫内膜组织,用电镜、Real time-RCR和Western blot、免疫组化方法观察两组的内膜组织胞饮突分布以及HOXA10、LPAR3、COX2基因的表达变化。结果：(1)改良超长方案组在取卵后第6天子宫内膜上出现大量呈蘑菇状、表面光滑、边界清楚的完全发育胞饮突形态。长效长方案组在取卵后6天出现胞饮突的数量少于改良超长方案组,很多呈局灶样分布,表面不光滑,多为微绒毛所覆盖,可见少量退化的胞饮突。(2)在改良超长方案组和长效长方案组中所采取的子宫内膜上均有LPAR3、COX2、HOXA10的表达。但在着床期改良超长方案组子宫内膜LPAR3、COX2、HOXA10的表达水平显著高于长效长方案组。结论：改良超长方案通过增加着床期子宫内膜上的胞饮突的出现,增强着床相关因子LPAR3、COX2、HOXA10的表达,改善了子宫内膜的容受性。第三部分不同的卵子体外成熟方案治疗PCOS患者的研究目的：比较多囊卵巢综合征(PCOS)患者三种助孕方案：自然周期卵子体外成熟培养、降调节后短暂促性腺激素(Gn)刺激再IVM、长方案降调节后常规体外受精胚胎移植(IVF-ET)助孕治疗效果。方法：前瞻性地将2010年1月至2012年12月在我院接受IVF助孕治疗的PCOS患者分别分入以下三组：A组(n=140),自然周期卵子体外成熟培养(IVM)；B组(n=152),降调节后短暂促性腺激素(Gn)刺激再IVM；C组(n=505),长方案降调节后常规体外受精胚胎移植(IVF-ET)助孕。比较三种助孕方案治疗效果。结果：(1)B组和C组临床妊娠率无明显统计学差异(B组为52.59%,C组为58.35%,P>0.05),但均明显高于A组(32.23%,P<0.05)。(2)hCG日内膜厚度A组为5.99±1.96mm,B组为7.87+2.14mm,C组为11.81±2.20mm,三组比较均有统计学差异,P<0.05。优胚率A组为47.23±29.17,B组为59.95-30.43,C组为65.66-23.35,三组比较均有统计学差异,P<0.05。(3)A组未使用Gn,B组Gn用量为600.08-291.21IU,费用为1868.11士1208.67元,C组Gn用量1776.36-693.76IU,费用5508.23-2193.17元,三组比较均有统计学差异(P<0.05)。(4)A、B组无卵巢过度刺激综合征(OHSS)发生,C组重度OHSS发生率为2.18%。结论：IVM助孕可以避免OHSS发生,若降调节后短暂Gn刺激再IVM助孕,优胚率更高,且由于子宫内膜发育得到改善,可以获得与长方案IVF相似的临床妊娠率,是PCOS患者的一个经济安全有效的助孕方式选择。更多还原

【Abstract】 Polycystic Ovarian Syndrome (PCOS) is a common endocrinal disorder in women with non-ovulation infertility in childbearing age, its incidence is about5%in women of childbearing age.About80%PCOS women get pregnant through drug intervention or after laparoscopic ovarian drilling to induce normal ovulation. But there are still20%of the PCOS women require assisted reproductive technology (ART) to obtain pregnancy. In-vitro fertilization and embryo transfer (IVF-ET) is currently the most commonly-used technology, which taking the oocytes and the sperm outside the body and fertilizing them in vitro, and then culturing the zygote to early preimplantaion embryos and then transferring them back into the womb. A key step of a successful IVF-ET procedure is controlled ovarian hyperstimulation (COH), which inducing multiple follicular development and maturation in a controlled stimulation with exogenous drugs to get enough high quality oocytes, and generating multiple embryos for transfer per cycle to improve the pregnancy rate of IVF per cycle.Long gonadotropin-releasing hormone-analogues (GnRH-a) down regulation is a commonly-used COH program for PCOS patients. But, PCOS patients are often accompanied with numerous endocrinal and metabolic abnormalities, such as:the insufficient secretion of FSH, increased secretion of LH, hyperinsulinemia, and hyperandrogenism, which leads to decreased IVF pregnancy rate and increased incidence of OHSS after COH. We wish to establish a more suitable COH program with higher pregnancy rate and decreased incidence of complication after COH. According to the pathological features of PCOS, we adopted a modified ultra-long down-regulation protocol. To answer whether our protocol improves the pregnancy rate in patients with PCOS, in the first part of this thesis, we retrospectively compared IVF outcomes of modified ultra-long protocol and routinely used long protocol for PCOS patients.Good quality embryo needs to implant in the uterus, differentiation and eventually develop to a mature fetus. Implantation is another important step of a successful pregnancy. The endometrial receptivity, which refers to the endometrial ability to accept the embryo is a key aspect for a successful implantation.At present, there are several factors which affect the endometrial receptivity:the endometrial thickness, morphology, blood flow of endometrial, hormone levels, cytokines and genes which regulating uterine receptivity. All the factors interact as a network to regulate the receptivity and aid a successful implantation. In the implantation window, the endometrial thickness increases to above9mm, blood flow index of endometrial increases, pinopodes appears and the up-regulated expression of endometrial receptivity related genes such as leukocyte inhibitory factor (LIF) and HOXA10gene. Therefore, in the second part of this thesis, to explore the influence of ultra-long protocol to the endometrial receptivity, we examined emdometrial pinopodes by electron microscopy in the PCOS patients during the implantation window from both long protocol group and the modified ultra-long protocol group, we examined the expression of HOXA10, LPAR3and COX2in two groups with immunohistochemistry, realtime-PCR, Western blot. We attempts to answer whether modified ultra-long protocol generates better clinical outcomes by improving the endometrial receptivity as compared to long protocol group.Because of the low FSH level in PCOS patients which leads to accumulation of small follicles with2-8mm diameter and the narrow FSH threshold, PCOS patients are much easier to suffer from ovarian hyperstimulation syndrome (OHSS) in routine COH protocols than non-PCOS patients. The incidence of OHSS in PCOS patients is up to14%. In vitro maturation (IVM) is a novel treatment for PCOS patients, which collects immature oocytes from infertile women without or with small dose of exogenous gonadotropin stimulation, the immature oocytes are then matured in vitro and fertilized by intracytoplasmic sperm injection (ICSI). Under this circumstance, no or much less exogenous gonadotropin (Gn) is needed to generate enough oocytes for late FVF-ET, the serum estrogen level is low in IVM patient which prevent the occurrence of OHSS after injection of HCG. Meanwhile, less usage of exogenous Gn dramatically decrease the cost for a fresh ET cycle. Due to the cost and less incidence of complications, IVM is adopted by some center to be alternative procedure for PCOS patients underwent IVF treatment. Due to the success rate of conventional IVM is10-30%, much lower than routine IVF, its clinical application is restricted. In part three of the thesis, we explored the possibility of improving the efficiency of IVM program in PCOS patients, trying to establish a safe but more effective strategy.Part I. Study of modified ultra-long protocol in patients with PCOS in IVF/ICSI-ETObjective:To retrospectively and prospectively compare clinical outcomes between modified ultra-long protocol down regulation in combination with human menopausal gonadotropin (hMG) and conventional long protocol down regulation in PCOS infertile women undergoing in vitro fertilization/intracytoplasmic sperm injection and embryo transfer (IVF/ICSI-ET).Methods:(1) We retrospectively analyze the clinical data of1989PCOS patients received modified ultra-long protocol treatment and1370PCOS patients received routine down-regulation long protocol during January2008to December2012. The fertilization rate, implantation rate, pregnancy rate, lives birth rate, abortion rate and treatment cost were compared between the two groups.(2) PCOS patients who underwent IVF/ICSI-ET treatment from September2011to April2012in our hospital were divided into two groups:ultra-long protocol group (n=75) and long protocol group(n=72). The average number of retrieved oocytes, fertilization rate, implantation rate, pregnancy rate, abortion rate, thickness, morphology and pulsed Doppler study of uterine artery of endometrial on the day of hCG administration and the day of ET were detected and assessed by Doppler ultrasound and compared. Otherwise, the related indexes of two groups such as serum progestogen, estradiol and luteinizing hormone level and P/E2on hCG day were compared.Results:(1) In the retrospective study, there were no significant differences in average age, body mass index, average infertility duration, basal FSH and LH level, average oocyte number, good-quality-embryo formation rate, average transferred embryos, cancellation rate, severe OHSS incidence between ultra-long protocol and long protocol. But the clinical pregnancy rate, implantation rate and take-home-baby rate in ultra-long protocol (74.56%,52.51%and60.43%, respectively) are much higher than in long protocol (56.02%,37.83%and42.88%, respectively). In ultra-long protocol, the serum P level on the day of hCG injection is lower than in long protocol (0.62±0.29ng/ml vs.0.81±0.39ng/ml, P<0.05), and the endometrial thickness is higher than in long protocol (12.26±2.42mm vs.11.36±2.43mm, P<0.001). The early abortion rate in ultra-long protocol is reduced than in long protocol (6.02%和8.59%, P<0.05). In addition, the average overall cost for ultra-long protocol is significantly reduced than for long protocol (989.58±418.59and5831.73±2313.13, P<0.001).(2) Totally147patients were included in the prospective study,75patients were in ultra-long protocol and72patients were in long protocol. The average Gn usage, duration for Gn, serum E2and LH level on day of hCG injection, average oocyte, fertilization rate, good quality embryo rate, cancellation rate, average transferred embryos, severe OHSS incidence, abortion rate showed no significant difference. But in ultra-long protocol, the clinical pregnancy rate per transfer cycle and the implantation rate were significantly higher than in long protocol (77.9%and59.6%vs.61.3%and44.1%,P<0.05). Compared to patients in long protocol, the patients in ultra-long protocol had lower serum P level on the day of hCG injection, and had better endometrial thickness, improved morphorlogy and blood flow.Conclusion:Modified ultra-long protocol combined with hMG could improve the implantation rate and the clinical pregnancy rate for PCOS patients underwent IVF-ET treatment through increasing the endometrial thickness, promoting endometrial morphology transition and blood flow. Part II Effect of modified ultra-long protocol on endometrium receptivity of PCOS patientsObjective:Comparatively study the morphology of endometrium pinopodes, the expression of HOXA10, LPAR3and COX2in PCOS patients in modified ultra-long protocol and long protocol.Methods:Separately collect day6endometrium after oocyte retrieval from PCOS patients who underwent IVF/ICSI-ET treatment in either ultra-long protocol or long protocol, and quit the fresh embryo transfer due to OHSS risk. The expression of HOXA10, LPAR3and COX2in two groups was determined by immunohistochemistry, Real time-PCR, Western blot, and pinopodes were observed and analyzed by electron microscopy.Results:We detected more uniform distribution of a fully developed pinopodes in the endometrium of patients in modified ultra-long protocol than in long protocol, which exhibited a large number mushroom like structure with smooth surface, clear boundary and membrane protrusions. And in the group of long protocol, the pinopodes distribute focally, the surface is not smooth, much of them covered with microvilli. The expression of implantation related factors LPAR3, COX2and HOXA10were higher in the endometrium from modified ultra-long protocol group than in the endometrium from long protocol group in either mRNA level and protein level during the period of implantation widow.Conclusion:Modified ultra-long protocol may improve the endometrium receptivity through increasing pinopodes generation and LPAR3, COX2and HOXA10expression. Part Ⅲ Study on different in vitro maturation treatments for PCOS patientsObjetive:To compare the clinical outcomes among IVM, low dose gonadotrophin stimulate IVM and long protocol in women with PCOS and find a safer and more effective treatment protocol for these patientsMethods:PCOS patients in our hospital from Jan.2010to.Dec.2012.were divided into three groups prospectively. Group A (n=140), routine IVM; Group B (n=152), low dose gonadotrophin stimulate IVM and Group C (n=505), long protocol group. The clinical outcomes of these three groups were compared.Results:(1) The clinical pregnancy rates of group B and group C showed no significant differences (52.59%and58.35%, respectively, P>0.05) and were significantly higher than that in group A (32.23%, P<0.05).(2) The thickness of endometrial on the day of hCG injection in group A, B and C were5.99±1.96mm,7.87±2.14mm and11.81±2.20mm, showed significantly differences, P<0.05. At the same time, there were statistical significant for high-quality embryo rate in groupA、B and C(47.23±29.17vs.59.95±30.43vs.65.66±23.35, P<0.05).(3) There were significantly differences in the usage of Gn, no usage in group A, and the dosage of Gn in group B were600.08±291.21IU and the cost were1868.11±1208.67yuan. The dosage of Gn in group C were1776.36±693.76IU and the cost were5508.23±2193.17yuan (P<0.05)(4) There were no OHSS in group A^B, the severe OHSS rate in group C were2.18%.Conclusion:IVM can avoid occurance of OHSS. The low dosage stimulation of Gn after down regulation then in vitro maturation can obtain similar clinical pregnancy rate compared to long protocol FVF due to improved endometrium. IVM is a safer and effective strategy for PCOS patients.更多还原