【作者】 王海英；

【导师】 全松；

【作者基本信息】 南方医科大学 ， 妇产科生殖医学（专业学位）， 2013， 博士

【摘要】 控制性超促排卵(COH)是辅助生殖技术(ART)中的关键环节之一。它对于提高IVF-ET技术的成功率和现代辅助生殖技术的建立与发展发挥了重要的作用。经典的GnRH激动剂(GnRHa)长方案仍然存在药物刺激时间较长、费用贵,卵巢过度刺激发生率高的缺点。寻求性价比高、患者依从性好,安全有效的超促排卵方案一直是临床生殖医学医生追求的目标。上世纪末GnRH拮抗剂(GnRH antagonist, GnRH-ant)的出现给临床促排卵方案带来新的选择。近年来GnRH-ant方案在国内得到了越来越多的应用,在欧洲一些大型ART中心GnRH-ant方案使用比例占90%以上,很有希望成为今后超促排卵的主导方案之一。拮抗剂可以迅速高效地抑制内源性的LH水平,有效地防止早发LH峰,但同时造成的低LH水平,对卵泡发育,子宫内膜和黄体功能产生的影响,也会影响其新鲜胚胎种植的成功率。在拮抗剂方案中如何适当地补偿LH活性,已成为目前一个备受关注的活跃的研究领域。人绒毛膜促性腺激素(HCG)最近被誉为21世纪科学中的奇迹,其是人类胚胎细胞最早分泌的分子物质,参与着人类胚胎的着床,胎盘的形成,胎儿的发育,介导着从人类生殖,肿瘤的发生到人类死亡许多的功能。HCG是由胎盘滋养细胞分泌的一种促性腺激素,与LH有85%的氨基酸序列相同。因此,它们的生物学作用与免疫学特性接近。LH与HCG作用于同一个受体(LH/HCG受体),并且运用其中一个激素结合LH/HCG受体,两者均同样起到刺激作用。近期研究发现LH/HCG受体广泛地分布于生殖系统组织中,特别是在不同发育阶段的卵泡细胞、黄体细胞和子宫内膜细胞均有表达,hCG可能在各生殖环节中均起到了重要的调节作用。hCG与LH相比其半衰期更长、与受体结合的亲和力较高,其生物学效价约为LH的6倍,如何恰当地利用hCG来补充LH活性日渐受到重视。近年来,国外临床研究发现在促排卵中加入小剂量HCG,以更稳定与持续的方式对发育中卵泡起支持作用,并完成卵泡的生成。在长方案或拮抗剂方案的卵泡晚期添加小剂量HCG,都有报道可以起到减少Gn用量、提高成熟卵及优胚数,在低反应,低LH的病人还能起到提高妊娠率的作用,而在高反应的患者中又有减低OHSS等并发症发生的作用,但其可能的机理未得到进一步研究。既往研究多是关于HCG在晚卵泡期的应用,而在卵泡早期添加小剂量HCG的报道较少。前瞻性、多中心的荟萃分析已经证实运用含有HCG活性的HP-hMG促排卵比单纯用rFSH有更高的妊娠率及活产率。可见HCG在整个刺激周期中运用是安全的,可采用的。HCG是滋养层细胞和众多胚胎源性物质中最早出现的标志物,近期研究发现最早在第二天的胚胎培养液中能测定到胎源性HCG的水平,其可望成为新的无创性评估胚胎质量与种植的标志物。LH/HCG受体mRNA表达存在人类的输卵管,子宫,精液,卵子,早期胚胎,囊胚,子宫内皮,胎儿循环内皮前基因细胞中。提示HCG介导相关的生殖过程。胚胎源性的hCG通过旁、自和内分泌途径与靶细胞表面hCG/LH受体结合,在胚胎发育、早期妊娠黄体功能维持和胚胎种植与胎盘形成过程中发挥着不可缺少的生物学作用。LH和HCG的加入,可能独立于甾体类激素而促进卵子的最终成熟,胚胎发育及胚胎种植。既往有关早期胚胎LH/HCG受体的研究都是在mRNA水平上,但未见有关胚胎HCG受体蛋白水平表达的相关研究。本研究拟在拮抗剂超促排卵周期中的早卵泡期开始添加小剂量HCG,观察各项临床及实验室指标、优质胚胎数、妊娠率、着床率及并发症等；通过检测第三天胚胎培养液中微量的胚胎源性HCG水平,分析其与传统胚胎形态评分、胚胎发育、种植间的关系,比较两种不同的促排卵方案对于胚胎自分泌HCG的影响。此外利用胚胎活检技术提取废胚中单个卵裂球,用单细胞免疫组化荧光方法检测LH/HCG受体蛋白水平的表达,分析其表达水平与临床各项指标的关系。评估拮抗剂超促排卵周期中早期添加小剂量HCG对于各临床指标、胚胎发育种植,胚胎自分泌HCG的调控和HCG受体表达的影响。以便进一步理解HCG在人类促排卵及胚胎发育种植中的作用,为其广泛应用提供可靠的实验依据。第一部分拮抗剂超促排卵方案中早卵泡期添加小剂量HCG的临床应用材料与方法研究设计采用单中心、前瞻性、配对设计的临床试验研究。所有受试者均签署知情同意书。研究对象,分组及治疗方案从2012年3月至2012年11月在广东省人民医院生殖中心就诊的需要行IVF治疗的病人,选取其中拟行拮抗剂方案超促排卵的病人100例,按照同期病人年龄,不孕类型即原发性继发性不孕,不孕年限,不孕原因,基础窦卵泡数等因素配对成组,分试验组及对照组患者各50例。对照组采用标准拮抗剂方案,给予重组FSH(普利康)启动。试验组在FSH启动的同时加用小剂量HCG100IU/每天。药物为丽珠公司(HCG1000IU/支),需稀释注射1/10量。药物统一在医院由专人注射。于月经周期第2-3天,抽血测定基础血清FSH、LH、E2水平,阴道B超监测基础窦卵泡数。根据患者的年龄、窦卵泡数目,决定起始FSH剂量。从Gn第5天开始定期进行阴道B超监测及血清LH,E2、P、HCG激素水平测定。在卵巢刺激6天后开始应用促性腺激素释放激素拮抗剂(思则凯)0.25mg/d。根据卵泡大小及激素水平调整FSH剂量及小剂量HCG的用药天数。当1个卵泡平均直径≥18mm,3个卵泡平均直径>16mm,于当日晚9点注射重组人绒毛膜促性腺激素(艾泽)250μg诱发卵泡成熟,扳机后36小时取卵。取卵后采用常规体外受精和胚胎培养。培养72小时行胚胎移植(ET),剩余优质胚胎行玻璃化冷冻。给予每日经阴道黄体酮600mg作为黄体支持,ET后12天测定血β-hCG值。对于妊娠测定阳性的病人进行随访,6-7周超声见孕囊确定为临床妊娠、10-12周为继续妊娠。资料收集：采集两组病人1.临床资料：重组FSH药物的用药天数,总剂量。2.实验室资料：取卵数,成熟卵数,受精数,正常受精数,可用胚胎数,优质胚胎数,优质胚胎率,移植胚胎数,冷冻胚胎数。3.血清激素情况：采用化学发光免疫法测定基础FSH、LH、E2；刺激后5天开始,隔天至HCG扳机日E2、P、LH、HCG水平。4.妊娠情况：妊娠HCG阳性率,种植率,临床妊娠率,流产率以及继续妊娠率。5.并发症的发生。研究终点：比较两组间各临床,实验室,妊娠指标,激素变化。1.基本观察指标：D3优质胚胎数2.二级观察指标：总Gn量、用药天数,取卵数、受精数、可利用胚胎数、总种植率、HCG阳性妊娠率、临床妊娠率,流产率,继续妊娠率,HCG日两组间血清E2,P,LH,HCG值,内分泌激素情况。结果：主要观察指标添加HCG组优质胚胎数为4.4±3.79,显著高于FSH组3.14±2.46,(t=-2.028,P=0.045),次级观察指标Gn用量上,添加小剂量HCG组为2348±726IU,明显低于对照组2688±777IU,(t=2.26,P=0.026)。小剂量HCG组取卵数、成熟卵数及正常受精数分别为10.26±6.72,9.26±6.05,及6.78±5.17。均比对照组高,9.08±6.08,8.04±5.46及5.56±3.94。但是两组无统计学差异。试验组可用胚胎数,移植胚胎数及冷冻胚胎数分别为6.18±4.76,2.14±0.65及4.64±5.18,均较对照组高,4.64±3.51,2.00±0.46及3.06±3.55。但是两组无统计学差异。妊娠结局试验组35例移植,共移植胚胎数目75个,成功种植22个胚胎,种植率为29%；20例HCG阳性,5例生化妊娠,临床妊娠率为42.9%,流产率13.3%,继续妊娠率为37.1%。对照组39例移植,共移植胚胎数目78个,成功种植17个胚胎,种植率为22%。19例HCG阳性,4例生化妊娠,临床妊娠率38.5%。流产率20%,继续妊娠率为30.7%。两组在临床妊娠率,种植率,流产率,继续妊娠率上均无统计学差异,P>0.05。血清内分泌情况血清HCG值：试验组HCG平均用药天数为8.86±1.85,血清HCG值逐步上升,到第6天后达到稳态,上升至8.77IU。HCG日血清E2值试验组为4112±1186pg/ml,对照组为3097±±1566pg/mLp<0.01,t=-3.65。HCG日直径>14mm卵泡平均E2值分别为试验组398.72±163.03pg/ml,对照组308.54±94.00pg/mL,p=0.01,有显著性差异。HCG日P值,试验组及对照组分别为1.65±±0.96mg/ml,1.40±0.62mg/ml,p=0.13,试验组在HCG日P值水平稍高于对照组,但是两组间无统计学差异。HCG日LH值,试验组1.70±±1.02mIU/mL,明显低于对照组2.9±3.03mIU/mL,p=0.006。不良反应OHSS在小剂量HCG组中未发生。对照组中发生1例中度OHSS。小结1.拮抗剂超促排卵方案中早期添加小剂量HCG,提高卵泡雌激素水平,获得更多的优质胚胎数。2.拮抗剂超促排卵方案中早期添加小剂量HCG,可减少促排卵药物的使用,未引起显著性孕酮升高及其他不良反应,是一种可供选择的促排卵方案。第二部分人胚胎源性HCG在胚胎培养液中的表达及其与胚胎发育种植关系的研究材料与方法研究对象及分组：病人分组及治疗情况同第一部分,采集行第三天新鲜周期移植的胚胎培养液,检测其中HCG的表达水平。胚胎培养及胚胎培养液的收集所有胚胎选择单一培养体系Quinn系列(SAGE美国)。取卵后第三天选择优质胚胎进行胚胎移植,移植后15分钟内将移植胚胎培养上清分别收集于微量eppendorf管中,置于-80℃冰箱待测。同一培养条件的无胚胎的培养滴也被收集以做对照。胚胎的评分记录各胚胎的评分情况。胚胎质量评估包括细胞数、3级形态、碎片分级。妊娠及着床的确定移植后第12天测定血β-hCG值。对于阳性妊娠病人随访,记录临床妊娠,种植胚胎数。胚胎培养液中HCG水平的检测采用体外酶联免疫吸附试验ELISA方法检测胚胎培养液中HCG水平。RayBio试剂(美国)。变异系数控制在15%之内。研究终点1.主要观察指标为第三天胚胎培养液中HCG水平与胚胎形态评估、种植率、妊娠结局间的关系。2.二级观察指标为COH中添加及未添加小剂量HCG,胚胎培养液中HCG水平的差异。结果一般资料163份第三天的胚胎培养液标本,10份标本因样本量太少未能测出,153份胚胎培养液滴能测出微量的阳性结果,检出率为93.8%。其中75份来自试验组为添加小剂量HCG组,78份来自对照组单纯FSH组。15份标本为空白对照,标本包括生理盐水,卵裂液(Quinn’s),及同一病人共同培养皿中空白对照液滴,其HCG的测量值均<0.100mIU/ml。同一病人的D3胚胎培养液HCG值用该病人各移植胚胎HCG值的均数表达。两组病人D3胚胎培养液HCG水平试验组COH中添加小剂量HCG组,第三天胚胎培养液HCG均值为0.93±0.25mIU/ml,未添加组为0.81±0.21mIU/ml。添加组较未添加组高,有显著性差异t=-2.39,p=0.02。D3胚胎培养液HCG水平与胚胎形态评估间的关系胚胎卵裂球数目不同,D3培养液中HCG表达值也相应有差异,F=2.273,p=0.03,提示D3培养液HCG值与胚胎卵裂球细胞数有正相关,随着细胞数增加,D3培养液中HCG表达也提高。随着胚胎形态评分级别的下降,培养液中表达值也相应有所下降,F=3.90,P=0.02,提示培养液HCG表达与分级成负相关,随着分级增加,培养液中HCG表达下降。两组病人D3胚胎培养液HCG水平与种植率的关系按胚胎着床与胚胎移植的百分比计算出每个病人的种植率,分为0,33.3%,50%,100%四个等级,添加组各种植率相应的D3胚胎培养液HCG值分别为0.89±0.18；0.63±0.31；0.91±0.11；1.15±0.7(F=4.07,p=0.015)。随着种植率的提高,培养液中HCG表达也提高。未添加组中各种植率相应的D3培养液HCG值分别为0.73±0.20；0.86±0.15；0.91±0.11；1.15±0.70,F=5.75,p=0.003。随着种植率的提高,培养液中HCG表达也提高。两组病人D3培养液HCG水平及妊娠的关系两组中妊娠病人的胚胎培养液HCG值均较非妊娠病人高,虽然在添加组中未见统计学差异,但在未添加组中,妊娠患者D3培养液HCG值为0.88±0.18,非妊娠患者为0.74±0.21。有显著性的差异。t=-2.15,p=0.038。小结1.通过ELISA方法能检测出D3胚胎培养液中胎源性HCG的微量表达。2.D3胚胎培养液中胎源性HCG的水平与胚胎形态评估,种植率及妊娠结局存在相关性,提示它可作为新的评估胚胎发育及种植的无创性方法。3.COH中添加小剂量HCG促进D3胚胎培养液中胎源性HCG的分泌增高。第三部分人胚胎卵裂球中LH/HCG受体蛋白水平表达的研究材料与方法：研究对象及分组：病人分组治疗同第一部分,拮抗剂COH中加入小剂量HCG组为试验组,对照组为未添加HCG组。胚胎培养至第三天,采集其中不适合新鲜周期移植及冷冻标准的废弃胚胎。通过卵裂球活检技术提取单个卵裂球细胞,经固定后免疫组化荧光染色,分析卵裂球LH/HCG受体的蛋白表达。临床资料的收集详细记录病人临床、实验室资料,妊娠情况等。废胚收集：选择正常受精,已卵裂的第三天不适合新鲜周期移植及冷冻标准的废弃胚胎,进行冷冻保存,集中复苏后进行后续的实验。当第三天胚胎评分细卵裂球数目少于4个时,或者卵裂球为4但形态分级或碎片为2级以上时,或者卵裂球少于6个(或多于10个)而分级为3级或者碎片为3级及3级以上时,或者卵裂球为7、8、9个但分级4级,碎片3级以上均可视为不可胚胎。胚胎活检技术提取卵裂球胚胎冷冻复苏后,利用胚胎活检技术提取单个卵裂球,并固定。细胞免疫组化荧光染色方法检测单卵裂球LH/HCG受体表达一抗：兔抗人LH/HCGR (H-50),稀释比例为1：50(美国santa cruz sc25828);二抗：DyLight(?)594(红色荧光)标记的羊抗兔IgG。蓝色荧光DAPI染卵裂球细胞核。结果判定：染色后标本分别于低倍及高倍镜下观察,根据拍摄图片判定单卵裂球上LH/HCGR的表达。蓝色荧光代表细胞核,红色荧光代表LH/HCGR的表达,观察LH/HCGR在卵裂球细胞不同区域的表达分布及强度。卵裂球上LH/HCG受体表达半定量转换：用Image J软件分别测量细胞核区域和细胞膜区域的红色荧光信号强度。具体操作方法如下：首先把图片转换为灰度图像,用选择工具选定需要测量的特定区域,通过测量工具,测算荧光相对强度累积值。分析指标：1.第三天胚胎卵裂球中LH/HCG受体的具体定位及半定量表达。2.第三天胚胎卵裂球中LH/HCG受体的表达与废胚细胞数的关系。3.比较超促排卵中添加小剂量HCG组及无添加组中,卵裂球中LH/HCG受体的表达差异。结果1.一般资料共收集来自32位病人的40个废胚,有13张玻片在检测过程中细胞脱落,其余27张成功检测到卵裂球上LH/HCG受体的表达。检出率为67.5%,7个废胚取自实验组,20个取自对照组2.卵裂球上LH/HCG受体的表达定位：LH/HCG受体标志为红色荧光,细胞核定位为蓝色荧光,免疫荧光显示LH/HCG受体主要在细胞膜上表达,细胞浆上也见较弱的表达。3.卵裂球上LH/HCG受体的半定量测定：卵裂球上LH/HCG受体的半定量测定值均数为1.49±0.621,最大值为2.04,最小值为0.83。4.两组中LH/HCG受体的表达差异：添加小剂量HCG组卵裂球上LH/HCG受体的表达1.793±0.210,明显高于无添加组1.394±0.318。(t=-3.073,P=0.005)。小结1.运用卵裂球活检加单细胞免疫组织化学荧光染色技术可以成功检测到第三天胚胎卵裂球细胞上LH/HCG受体的蛋白水平的表达。2.第三天胚胎卵裂球上LH/HCG受体表达主要定位在细胞膜上。细胞浆上也见微弱表达。3.超促排卵周期中早期添加HCG组卵裂球上LH/HCG受体表达增强。全文小结本研究在拮抗剂超促排卵周期中早期添加小剂量HCG,与配对设计的对照组相比较,分析各项临床及实验室指标,Gn用量,优质胚胎数,妊娠率,着床率,激素内分泌改变及并发症等；采集D3胚胎培养液,ELISA方法检测其中胚胎源性HCG水平,分析两组间D3培养液HCG值表达差异及其与传统胚胎形态评估,种植率、妊娠结局间的关系；应用卵裂球活检技术,提取第三天废弃胚胎卵裂球细胞,运用单细胞免疫组化荧光染色技术检测卵裂球上LH/HCG受体蛋白水平的表达,研究早期胚胎LH/HCG受体定位及半定量表达,并分析两组间的表达差异。本研究是国内首次小剂量HCG应用在促排卵方案上的前瞻性研究。首次利用D3胚胎培养液HCG水平作为无创性指标评估比较不同促排卵方案的胚胎质量。采用的卵裂球活检及单细胞免疫组化荧光染色技术检测单卵裂球细胞LH/HCG受体的蛋白水平表达,在国内外为首次相关报道。我们的研究表明：1.拮抗剂超促排卵方案中早期添加小剂量HCG,改善胚胎质量,获得更多的优质胚胎数。2.运用ELISA方法可检测出第三天胚胎培养液中胎源性HCG的微量表达,其与胚胎形态评估、种植率,妊娠率间存在相关性,可作为新的评估胚胎发育潜能及种植的无创性方法。3.运用卵裂球活检加单细胞免疫组织化学荧光染色技术可以检测到第三天胚胎卵裂球上LH/HCG受体的蛋白水平表达。LH/HCG受体表达主要定位在细胞膜上,细胞浆上也见微弱表达。4.拮抗剂超促排卵方案中早期添加小剂量HCG,第三天胚胎培养液中HCG的分泌增高,胚胎卵裂球上LH/HCG受体表达增强,可能通过自分泌调节及受体调节,提高胚胎质量及种植。5.拮抗剂超促排卵方案中早期添加小剂量HCG,可减少促排卵药物的使用,未引起显著性孕酮升高及其他不良反应,是一种可供选择的促排卵方案。更多还原

【Abstract】 Controlled ovarian hyperstimulation (COH) was the most important part in assisted reproduction techniques (ART). Gonadotrophin-releasing hormone agonists (GnRHa) have been the gold standard in stimulation protocols for many years. Gonadotrophin-releasing hormone antagonists (GnRH-A) can be used to prevent a luteinizing hormone (LH) surge during COH without the hypo-estrogenic side-effects, flare-up, or long down-regulation period associated with GnRH agonists. The updated Cochrane database in2011suggests that GnRH-A protocol is associated with a significant reduction in OHSS and comparable clinical outcomes. The future paramount aim should be to further improve the reproductive outcome by optimizing the details of the GnRH-A protocol. LH supplementation in GnRH-A protocol was studied widely in improving the impaired follicle development due to low LH level induced by GnRH-A.Human chorionic gonadotropin (hCG) was called the wonder of today’s science. Firstly, because hCG is such an extreme molecule. hCG is the most acidic glycoprotein containing the highest proportion of sugars. Secondly, hCG exists in5common forms. Finally, it has so many functions ranging from control of human pregnancy to human cancer.HCG has been used as a substitute for the luteinizing hormone (LH) surge because of the degree of homology between the two hormones. Recently, the LH/hCG receptor has been found an almost ubiquitous distribution in reproductive organs, thus suggesting that the actions of hCG might be more extensive than previously thought. In addition, hCG has a slower plasma metabolic clearance, and may be more effective than LH. The longer half-life and greater affinity for the LH/hCG receptor of hCG account for a potency ratio estimate of hCG-to-LH of around1：6. The application of hCG in GnRH-A protocol need further investigation.It has been reported that low-dose hCG can support development and maturation of larger ovarian follicles independently of follicular stimulation hormone (FSH), and the pre-treatment of low-dose hCG before or in the early stage of controlled ovarian hyperstimulation (COH) might provide an effective way in reducing rFSH use and enhancing oocyte developmental competence to obtain top-quality embryos, and improves implantation and on-going pregnancy rates.Several studies have shown that hCG can replace rFSH during the final days of controlled ovarian stimulation (COS)(Filicori et al.,2005; Blockeel et al.,2009; Kosmas et al.,2009), but seldom study has investigated the effects of addition of hCG to rFSH from the first day of stimulation.Prospective multicentre studies and meta-analyses have shown that pregnancy and live birth rates may be improved after IVF by using highly purified-human menopausal gonadotrophins (HP-hMG) rather than r-FSH (Andersen et al.,2006; Platteau et al.,2008; van Wely et al.,2011). HP-hMG contains hCG and compared with rFSH, COS with HP-hMG has been shown to induce a different endocrine profile, different follicular dynamics, a larger proportion of top-quality embryos and more favourable endometrial receptivity (Andersen et al.,2006; Smitz et al.,2007; Ziebe et al.,2007).The LH activity of urinary-derived commercially available menotrophins varies, but in the HP menotrophins (hMG; Menopur), the main LH activity is related to hCG and for75IU of FSH, the drug contains around10IU of hCG (Wolfenson et al.,2005). When using HP-hMG for COS, hCG will be present throughout the stimulation. The differences in endocrine profiles observed using HP-hMG versus rFSH have in part been attributed to the hCG content. The circulating levels of hCG on Day6in the long agonist protocol have been shown to be positively correlated with live birth rates and number of top-quality embryos (Smitz et al.,2007).HCG was the first reported bio-marker embryonic secretion, which is primarily produced by the embryo and later by the syncytiotrophoblast. The hormone was detected at various levels in embryonic culture media from day2to blastocyst stage by different assays. The ability to dectect hCG from day2spent culture media may be used as a marker for embryo competence. However, they were only used in research now, and no use to compare the different COH protocols. Recently, the LH/hCG receptor has been found an almost ubiquitous distribution in reproductive organs. The study demonstrates the mRNA expression of the LHR in human oocytes and preimplantation embryo. But gene expression does not necessarily imply that the transcripts are translated in protein or the receptors are functionally invoved in signal transduction.The purpose of the present study was to determine whether low dose hCG early added to rFSH in regimens of ovarian stimulation could produce better results compare to rFSH alone in IVF-ET antagonist protocol.This was investigated through a regular study analysing the clinical, embryological and endocrine aspects. An additional aim was to define possible ceiling levels, i.e. levels above which there were no additional beneficial effects or potentially harmful effects of supplementation with hCG. The primary end-point was the number of top-quality embryos per patient on Day3after fertilization. The second objective of our study was to develop an in-house enzyme-linked immunoscorbent assay (ELASA) procedure to detect and qualify hCG from day3spent embryo culture media and to determine the association of hCG with the development potencial of the embryo and compare the outcome of different COH protocols. The third objective of the study was to develop a blastocyst biopsy combined immunohistochemical technique procedure to detect the protein expression of LH/HCG receptor in human preimplation embryo. To evaluate the effluence on embryo development by the number of top-quality embryos, the value of hCG from day3spent embryo culture media and the protein expression of LH/HCG receptor in day3discarded embryos in early addition of low dose human chorionic gonadotropin during controlled ovarian hyperstimulation antagonist protocol.Materials and methodsStudy design:This prospective, single center, paired design controlled trial was performed on100patients undergoing IVF treatment in Reproductive Center,Guangdong General Hospital,from March2012to November2012. The study was approved by the institutional review board and Ethics Committee. All the participants were informed and signed the consent.Patients and treatment protocol100patients undergoing COH in a GnRH-antagonist protocol was paired designed into two group according to age, primary infertility, infertility duration, the number of antral follical count and infertility factor.The Control group received a standard treatment with rFSH (Purgen) plus a GnRH-antagonist, daily from Day6of stimulation. In the study group, low dose HCG100IU was added to rFSH in the first day of stimulation.Both groups were started at a dose of150-600IU per day according the AFC on the2day of period. Transvaginal ultrasound was performed after4days of stimulation, and the dose of recombinant FSH and hCG was adjusted based on the number and size of follicles and the estradiol level. A daily morning dose of250mg of ganirelix acetate was started after Day6of stimulation. When there were at least two follicles with a mean diameter of18mm, with at least two additional follicles sized16mm, hCG was administered (Ovidel250ug). Oocyte retrieval was performed36hours later, and the embryos were transferred either3days after retrieval, depending on embryo number and quality. Luteal phase support was maintained with600mg of progesterone intravaginal daily beginning the evening after the oocyte retrieval and continuing until7to8weeks’ estimated gestational age.Serum LH, progestone and estradiol levels were measured at several intervals after the start of GnRH antagonist, and on the day of hCG administration for final oocyte maturation. All serum tests were drawn in the morning before the morning doses of gonadotropins or ganirelix acetate were administered. Luteinizing hormone and estradiol were measured by using an electrochemiluminescence immunoassay (Modular Analytics E170module; Roche).Data collection:1. Clinical outcome:duration of stimulation, total dose of rFSH,2. Lab outcome:follicular development, number of oocytes retrieved, number of M2oocytes, fertilization, fertilization rate, number of embryos transferred, implantation rate, number of top-quality embryos on Day3.3. Serum endocrinology:basal FSH and LH, stimulate day LH, P, E2, HCG value; HCG day LH, E2, P and HCG value.4. Pregnancy:pregnancy rates and miscarriage rates5. Side effect and OHSS case.Study end-pointsThe primary end-point was the total number of top-quality embryos on Day3. A top-quality embryo was defined as four to five blastomeres on Day2, seven or more blastomeres on Day3, equally sized blastomeres and≤20%fragmentation on Day3and no multinucleation. These endpoints were the same as those used in the MERIT trial (Ziebe et al.,2007).Secondary end-points included follicular development, number of oocytes retrieved, number of oocytes, fertilization, fertilization rate, number of embryos transferred, implantation rate, duration of stimulation, total dose of rFSH, serum levels of endocrine parameters, pregnancy rates and miscarriage rates.Result: Primary endpoint:The number of top-quality embryos per patient was analysed as a Poisson-distributed count. The mean numbers of top-quality embryos was4.44±3.79in study group, with a statistically significant higher than in control group3.14±2.46,(t=-2.028,P=0.045).Secondary end-pointsClinical outcomes:1. The total dose of rFSH consumed was significantly lower in the hCG group:2348±726IU versus2688±777IU,(t=2.26,P=0.026)。2. The number of oocytes retrieved, number of M2oocytes and fertilization were10.26±6.72,9.26±6.05, and6.78±5.17in hCG group,9.08±6.08,8.04±5.46and5.56±3.94。There were no significant differences between two group.3. The number of transferable embryos, transferal embryo and frozen embryos in study group were6.18±4.76,2.14±0.65and4.64±5.18. The corresponding number in control group were4.64±3.51,2.00±0.46and3.06±3.55in control group。Although the level was higher in study group, but no significant differences.4. The implantation rate, pregnancy rate and miscarriage rate, ongoing pregnancy rate were comparable in both groups. P>0.05.Serum endocrinology1. Estradiol:The FSH with low dose hCG group had higher peak estradiol level (4112±1186Vs3097±1566pg/mL, p<0.01,t=-3.65) than the FSH only group.2. Progesterone:There were no significant differences between two group in P level on HCG day. Although which was1.65±0.96mg/ml in study group, higher than in control group1.40±0.62mg/ml，p=0.13.3. LH level:The LH level on HCG triggering day was1.70±1.02mIU/mL in study group, which is lower than in control group2.9±3.03mIU/mL, p=0.006。4. HCG level:Steady state level of s-hCG was reached8.77IU on Day6of stimulation in study group.Adverse reactions events OHSS was not seen in study group, however, one patient in control group was diagnosed with morderate OHSS (outpatient).Summary:1. Supplementation with hCG from the first day of stimulation in GnRH antagonist protocol may increase the number of top-quality embryos.2. Supplementation with hCG from the first day of stimulation in GnRH antagonist protocol has the advantage of decreasing the dose of FSH and no obvious side effect, which is an new alternative COH protocol.Part two Human chorionic gonadotropin from embryo culture media and its relationship to embryo developmentMaterials and methodsPatients and treatment protocol:same in part oneSpent Embryo culture Media collection:A total of178day3spent embryo culture media from the patient IVF treatment, which inclusion in part one. The Day3embryos were transferred into uterus. The spent culture media were collected into eppendorf tube within15minutes, which was kept frozen individually in labeled microcentrifuge vials at--80℃until analysis. A sample of pure clture media incubated under the same condiction but without an embryo was also kept frozen and used as a blank control.HCG determination by ELISAA quantitative sandwich ELISA was developed in house and was performed following the optimization on spent embryo culture media. RayBio Amercian.Embryo Development monitoringThe embryo quality evaluation consisted of assessment of cell number and three parameters of embryo morphology, degree of fragmentation.Following the pregnancy rate and implantation rate Study end-pointsThe primary end-point evaluated the relationship of HCG value in day3embryo culture with the embryo development, pregnancy outcome, and implantation rate.Secondary end-point compared the HCG value in day3embryo culture in low-dose HCG group and rFSH group.Result:1. Of165spent embryo culture,10had no detectable amounts of hCG15were control samples, which include saline, Cleavage liquid and no embryo control sample. The value of hCG in control samples were lower0.1mIU/ml.75samples were from study group low-dose HCG group and78samples were from FSHgroup.2. The value of hCG in embryo culture between two group:In study group0.93±0.25mIU/ml, significant higher than FSH group0.81±0.21mIU/ml。 t=-2.39, p=0.02。3. The concentration of hCG in culture media increased gradually along with the increase number of blastomeres and decreased with the morphology grade. F=2.273, p=0.03and F=3.900, P=0.024. The amount of hCG correlated positively with implantation rate in both group. F=4.07, p=0.0155. The relationship between pregnancy outcome and hCG value in embryo culture: A total of74patients underwent embryo-transfer procedure. In FSH group, hCG value was higher in pregnancy group0.88±0.18IU than in non-pregnancy group0.74±0.21IU。t=-2.15, p=0.038.Summary:1. ELISA may be an optimal choice for detecting hCG in D3spent culture media.2. The concentration of hCG in spent culture media was correlated positively with embryo development monitoring, implantation rate, which may be as a useful marker for embryo selection in IVF-ET procedure. 3. The concentration of hCG in D3spent culture media was increase in the group which addition hCG in COH procedure.Part Three LH/HCG receptor protein expression in blastomere from day3human embryoMaterials and methodsPatients and treatment protocol:same in part oneInstitutional approval and informed consent For both ethical and practical reasons, this investigation was performed on normal fertilized but discarded embryos due to poor embryo score in day3embryo.Cleavage stage embryo biopsy and hCG-receptor FluorescenceImmunocytochmistry on blastomeres Preparation:D3discarded embryos for both experimental and control groups were collected and cryopreserved for test. Before Fluorescencelmmunocytochmistry to be performed, all of the embryos were thawed and cultured in blastycyst medium at370C and6%CO2for at lest1hr. Operation dishes containing Ca/Mg free HEPES buffered HTF droplet covered with oil were prepared a half hour before biopsy operation and warmed in desktop incubator, without CO2gas. Holding and biopsy needles were fixed on micro-operating system, adjusted to horizontal position and opposite direction, and elevated to the level under which operation dishes can easily put on the objective stage. Blastomere washing dishes with PBS droplets were prepaired just by blastomere fixation.Biopsy:Transfer each embryo to be biopsied into the individual droplet labeled with corresponding serial number, and put the dish on the objective stage. Lower the needles and flush them in the droplet without embryo. Locate and aspriate to fix the embryo on the top of the holding needle in the position with maxmal perivitelline space is opposite, considering minimizing the heat damage of laser to blastomere. Punch a hole with laser in a minimum diameter about30-40uM for the biopsy needle to enter and aspirate the each blastomeres gently out succesively.Blastomere fixation:First transfer blastomeres to droplets in the washing dishes and flush intensively, and then transfer to slides within the circle made by marker pen, naturally dried in room temperature.Fluorescence Immunocytochmistry on blastomeres Immunochemistry Blue fluorescent DAPI tags blatomere nuclei. Immunochemistry red fluorescent tags HCG receptor in blstomere.(DyLight(?)594).Collect the clinical data and following the pregnancy outcomeResult:Total40discarded D3embryos were collected from32patients. There were13embryos failed to perform immunohistochemical staining due to cell dropping out during the procedure. There were27embryos complete the procedure.7embryos were from the low-dose HCG group and20embryos were from rFSH group.Immunohistochemical StudyImmunohistochemical localiztiong of LH/HCG receptor showed intense staining of the different blastomere at Day3embryo. Higher expression on membrane than on cytoplasm..The HSCORE for LH/HCG receptor staining was higher in low-dose HCG group (1.793±0.210)than in rFSH group(1.394±0.318), t=-3.073, P=0.005Summary:1. Blatomere biopsy plus immunohistochemical localization of Lh/HCG receptors showed intense staining in the plasma membrane in all the blstomere at day3embryos, cytoplasm also low express.2. The higher expression of Lh/HCG receptor in D3embryo in the group which addition hCG in COH procedure. But the sample size was limited. Conclusion:1. Supplementation with hCG from the first day of stimulation in GnRH antagonist protocol may increase the number of top-quality embryos.2. ELISA may be an optimal choice for detecting hCG in D3spent culture media. The concentration of hCG in spent culture media was correlated positively with embryo development monitoring, implantation rate, which may be as a useful marker for embryo selection in IVF-ET procedure.3. Blatomere biopsy plus immunohistochemical localization of LH/HCG receptors showed intense staining in the plasma membrane in all the blstomere at day3embryos.4. The concentration of hCG in D3spent culture media and the expression of Lh/HCG receptor in D3embryo were increase in the group which addition hCG in COH procedure.5. Supplementation with hCG from the first day of stimulation in GnRH antagonist protocol has the advantage of decreasing the dose of FSH and no obvious side effect, which may be a new alternative COH protocol.更多还原