补肾调经方对IVF-ET妊娠结局及颗粒细胞凋亡和OSFs的影响
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摘要
不孕不育症是影响人类生殖健康的常见病和多发病,在我国发病率约为7%-10%。体外受精-胚胎移植(in vitro fertilization-embryo transfer, IVF-ET)技术目前已成为临床治疗不孕不育症最科学、最有效的主要技术手段之一,近年来,IVF-ET取得了很大的进展,但临床妊娠率仍然较低,且多胎妊娠发生率和流产率较高。在IVF-ET过程中,卵母细胞质量与受精、胚胎发育及妊娠关系密切,目前优化控制性超促排卵方案仍然是改善卵子质量的最主要的途径。近年来,中药用于提高卵母细胞和胚胎质量以及妊娠结局的研究有很大进展。本课题组前期研究了补肾调经方对无排卵模型大鼠生殖功能的影响,证实补肾调经方可通过多环节、多靶点发挥促进卵泡发育,诱发排卵,并可促进子宫发育,调节子宫内膜微环境以利于孕卵着床的作用。本研究拟在前期研究的基础上,进一步研究补肾调经方在联合IVF-ET治疗不孕症的临床应用。
由于颗粒细胞与卵母细胞之间存在广泛的包括缝隙连接在内的复杂连接机制,在调节卵母细胞发育、排卵、卵子受精及胚胎种植等方面发挥重要的作用;卵母细胞分泌的卵母细胞分泌因子(oocyte secreted factors,OSFs)通过旁分泌作用促进颗粒细胞增生、分化并调节其功能,与颗粒细胞共同调节卵母细胞的发育过程。文献报道,卵丘细胞(cumulus cell,CC)的凋亡状态和OSFs直接影响卵母细胞的发育潜能。所以本研究以颗粒细胞凋亡和OSFs为切入点研究补肾调经方调经种子的作用机制。以期为不孕症的中医药治疗和IVF-ET的中医辅助用药提供理论基础和实验依据
第一部分:补肾调经方对体外受精-胚胎移植妊娠结局的影响
目的:观察补肾调经方对体外受精-胚胎移植超排卵周期妊娠结局的影响。
方法:收集因输卵管因素行体外受精-胚胎移植( in vitro fertilization-embryo transfer,IVF-ET)技术助孕,并符合中医肾虚不孕症辨证标准的女性患者60例,随机分为中药补肾调经方+西药控制性超排卵组(实验组)30例和单纯西药控制性超排卵组(对照组)30例。观察并记录两组患者HCG日血清E2、P、LH水平、促性腺激素(gonadotropin, Gn)使用天数和剂量、获卵数、成熟卵数、受精率、卵裂率、优质胚胎率、临床妊娠率及并发症情况。
结果:(1)HCG日两组患者血E2、LH、P值比较:两组患者在HCG日血LH、P水平无显著差异(P>0.05);实验组的E2水平明显高于对照组(P<0.05)。(2)Gn总量、Gn天数及获卵数的比较:两组患者Gn总量、Gn天数均无显著差异(P>0.05);实验组获卵数高于对照组,有显著性差异(P<0.01)。(3)实验室评定成熟卵率、受精率及优质胚胎率比较:实验组成熟卵率86.70%、2PN受精率76.84%、卵裂率96.79%、优质胚胎率64.90%。对照组成熟卵率83.80%、2PN受精率66.07%、卵裂率95.72%、优质胚胎率54.06%。统计结果显示两组在成熟卵率、卵裂率方面差异无显著性(均P >0.05)。而在受精率、优质胚胎率方面实验组高于对照组(均P <0.05)。(4)临床妊娠率比较:实验组临床妊娠22例,妊娠率73.33%;对照组临床妊娠14例,妊娠率46.67%,两组临床妊娠率差异有显著性(P<0.05)。(5)相关并发症比较:实验组发生OHSS1例,胎停育1例;对照组无OHSS病例,自然流产1例。无其它并发症发生。两组OHSS及流产发生率无统计学意义。
结论:在体外受精-胚胎移植控制性促排卵同时,加用中药补肾调经方可以增加获卵数,并提高受精率、优质胚胎率及临床妊娠率。
第二部分:人颗粒细胞凋亡及OSFs与卵母细胞发育潜能的关系
目的:通过检测人单个卵母细胞对应颗粒细胞的凋亡率和对应卵泡液中卵母细胞分泌因子GDF-9和BMP-15的含量,进一步阐明颗粒细胞凋亡及卵母细胞分泌因子GDF-9和BMP-15与卵母细胞发育潜能的关系。
方法:收集因输卵管性不孕接受IVF-ET治疗的患者。经阴道超声引导下取卵,每侧卵巢穿刺时首先选择一个直径≥18mm卵泡抽吸,将该卵泡的卵泡液单独留于一管内,置于显微镜下找卵,若找到卵-冠-丘复合物(COC),经过卵母细胞成熟度分型,并将该卵母细胞行微滴受精及培养,于受精后16-18h评估受精情况,受精24h后评估受精卵的卵裂情况,于受精后第3天(4-8细胞期),给予胚胎评分。分组方法:一组为形成优质胚胎的卵母细胞,称为优质卵母细胞组;另一组为未成熟、未受精及未形成优质胚胎的卵母细胞,称为非优质卵母细胞组。将对应的卵泡液离心,用人淋巴细胞分离液分离出颗粒细胞,采用脱氧核糖核苷转移介导的缺口末端标记法(TUNEL)检测颗粒细胞的凋亡率;采用western-blot方法检测卵泡液中GDF-9和BMP-15的含量。分别分析颗粒细胞凋亡及GDF-9和BMP-15与卵母细胞发育潜能的关系。
结果:(1)共观察了69枚卵母细胞,其中形成优质胚胎的卵母细胞36枚,未形成优质胚胎的卵母细胞33枚(其中未成熟卵5枚,未受精及异常受精卵6枚)。TUNEL法测定GC的凋亡率,形成优质胚胎的优质卵母细胞组GC凋亡率为12.746±3.73,未形成优质胚胎的非优质卵母细胞组GC凋亡率为19.88±6.36,有显著的统计学差异(P<0.01)。(2)优质卵母细胞组的GDF-9蛋白含量为2.4983±0.1328,非优质卵母细胞组为1.3052±0.406,统计学有显著差异(P=0.0084<0.01),结果显示:发育潜能较好的卵母细胞相应卵泡液中GDF-9的含量较高。(3)优质卵母细胞组的BMP-15蛋白含量为3.3785±0.4823 ,非优质卵母细胞组为1.336±0.2139,统计学有显著差异(P=0.0026<0.01),结果显示:发育潜能较好的卵母细胞相应卵泡液中BMP-15的含量较高。
结论:优质卵母细胞组颗粒细胞的凋亡率低,提示颗粒细胞凋亡可能影响卵母细胞的质量及胚胎的体外发育潜能。在优质卵母细胞组卵泡液中GDF-9和BMP-15水平及颗粒细胞中GDF-9和BMP-15mRNA的表达均较高。
提示人颗粒细胞的凋亡状态及GDF-9、BMP-15水平可以反应卵母细胞的发育潜能,颗粒细胞凋亡率及GDF-9、BMP-15水平可以做为评价卵母细胞质量的指标。
第三部分:补肾调经方对体外受精-胚胎移植超排卵周期颗粒细胞凋亡的影响
目的:研究补肾调经方对颗粒细胞凋亡的影响,探讨其提高IVF-ET超排卵周期所获卵和胚胎质量的作用机制。
方法:收集因输卵管因素行IVF-ET技术助孕,并符合中医肾虚不孕症辨证标准的女性患者60例,随机分为中药补肾调经方+西药控制性超排卵组(实验组)30例和单纯西药控制性超排卵组(对照组)30例。留取两侧卵巢所有直径≥18mm的主导卵泡的卵泡液,用人淋巴细胞分离液分离出颗粒细胞,采用脱氧核糖核苷转移介导的缺口末端标记法(TUNEL)检测颗粒细胞的凋亡率;采用免疫组化法测定颗粒细胞中凋亡相关蛋白Caspase-3的表达;采用流式细胞仪法测定颗粒细胞凋亡率。分析补肾调经方与颗粒细胞凋亡的关系。
结果:(1)TUNEL法测定两组颗粒细胞的凋亡率:实验组的凋亡率为13.61±4.53;对照组为19.41±5.95。两组比较有显著统计学差异(P<0.05),加用补肾调经方后可以降低颗粒细胞的凋亡率。(2) Caspase3在两组颗粒细胞中的表达:实验组Caspase3表达阳性率为48.08±11.24%,对照组Caspase3表达阳性率为68.58±10.31%,实验组显著低于对照组(P<0.01)。(3)流式细胞仪方法测定两组颗粒细胞凋亡细胞的DNA含量:实验组颗粒细胞凋亡细胞的DNA含量为1.26±0.24%,对照组颗粒细胞凋亡细胞的DNA含量为3.61±1.6%,实验组颗粒细胞凋亡细胞的DNA含量显著低于对照组(P<0.05)。
结论:补肾调经方可降低颗粒细胞的凋亡率及凋亡细胞DNA的含量,下调颗粒细胞中凋亡相关蛋白caspase3的表达。提示补肾调经方提高卵母细胞质量可能与降低颗粒细胞的凋亡率有关,其机制可能与抑制caspase3的表达有关。
第四部分:补肾调经方对体外受精-胚胎移植周期卵母细胞分泌因子GDF-9和BMP-15的影响
目的:研究补肾调经方对GDF-9和BMP-15的影响,探讨其提高IVF-ET超排卵周期所获卵和胚胎质量的作用机制。
方法:收集因输卵管因素行IVF-ET技术助孕,并符合中医肾虚不孕症辨证标准的女性患者60例,随机分为中药补肾调经方+西药控制性超排卵组(实验组)30例和单纯西药控制性超排卵组(对照组)30例。留取两侧卵巢所有直径≥18mm的主导卵泡的卵泡液,用人淋巴细胞分离液分离出颗粒细胞。采用western-blot方法检测卵泡液中GDF-9和BMP-15的含量,采用RT-PCR方法检测颗粒细胞中GDF-9和BMP-15的表达。分析补肾调经方与GDF-9和B MP-15的关系。
结果:(1)两组卵泡液GDF-9结果比较治疗组卵泡液GDF-9水平(2.7305±0.1260)显著高于对照组(2.0564±0.3055),差异有统计学意义(P<0.05)。(2)两组颗粒细胞GDF-9 mRNA表达的电泳结果:治疗组GDF-9mRNA表达(21807.0±1998.6)显著高于对照组(10177.0±2571.4),差异有统计学意义(P<0.01)。(3)两组卵泡液BMP-15蛋白质测定结果:治疗组卵泡液BMP-15水平(2.6454±0.4008),显著高于对照组(1.6252±0.4371),差异有统计学意义( P<0.05)。(4)颗粒细胞BMP-15 mRNA表达的电泳结果:治疗组BMP-15 mRNA表达(11894.0±865.6)显著高于对照组(6441.0±794.6),差异有统计学意义(P<0.01)。
结论:补肾调经方可增加卵泡液中GDF-9和BMP-15的含量,并上调颗粒细胞中GDF-9和BMP-15的表达。
综上所述,补肾调经方可能通过降低颗粒细胞凋亡率,增加GDF-9和BMP-15水平来改善卵母细胞质量,提高受精率、优质胚胎率及临床妊娠率,补肾调经方可以作为IVF-ET控制性促排卵的辅助用药。
In vitro fertilization-embryo transfer (IVF-ET), one of the most scientific and effective methods in treating infertility a common and frequent disease (morbidity of 7%-10%) in human reproductive health, has made great progress recent years. While the success rate still remains low with relatively high rate of multiple pregnancy and abortion. Effort to optimize the protocols of controlled ovarian hyperstimulation (COH) is still the major method to improve oocyte quality, which plays an important role in IVF-ET.Researches on Traditional Chinese Medicine (TCM) used to improve the quality of oocyte and embryo has made great progress recently.Our research team studied the effects of Bushen Tiaojing Recipe on the reproductive function of anovulatory rats and concluded that this recipe can induce follicular development and ovulation in many paths and targets, and can benefit embryo implantation by promoting uterus development and regulating the microenvironment of endometrium.
The purpose of our research is to study the effects of Bushen Tiaojing Recipe Combined with IVF-ET on treatment of infertility more deeply, on the basis of preliminary research.
It is known that bidirectional interactions between the oocytes and surrounding somatic cells through gap junctions and paracrine signaling are pivotal in maintaining the growth and development of both cell types during folliculogenesis. The oocyte plays an active role throughout folliculogenesis via secretion of oocyte-secreted factors(OSFs) that effects cumulus cell proliferation , expansion, differentiation, steriodogenesis, gene expression and maintain an appropriate microenvironment for the acquisition of its developmental competence.
It is reported that the apoptosis of cumulus cells and OSFs are associated with oocyte development directly. Therefore, in this study we observed the effects of Bushen Tiaojing Recipe on IVF-ET, granulosa cell apoptosis and OSFs and discussed the mechanisms involved in order to supply theoretical and experimental basis for traditional Chinese medicine used in infertility and assistant therapy for IVF-ET.
Part one: Clinical application of Bushen Tiaojing Recipe in IVF-ET Objective: To observe effects of Bushen Tiaojing Recipe on pregnancy outcome in patients undergoing IVF-ET.
Methods: Sixty infertile patients with tubal factors combined with kidney deficiency undergoing IVF-ET were divided into two groups at random, experimental group (Bushen Tiaojing Recipe +GnRHa/FSH/hCG) and control group (GnRHa/FSH/hCG).The content of hormones(E2、LH、P) on HCG day, days and dosage of gonadotropin, number of oocytes and mature oocytes, fertilization rate, high quality embryos rate ,pregnancy rate and complication were observed.
Results: (1) The content of E2 was significantly higher in experimental group than in control group(P<0.05).The level of P、LH was no statistical significance between two groups(P>0.05).(2) The time and dosage of gonadotropin were no statistical significance between two groups(P >0.05),while the oocyte number was significantly larger in experimental group than in control group(P<0.05).(3) The rate of mature oocyte and oocyte cleavage were no statistical significance between two groups(P>0.05),while the rate of fertilization , high quality embryo were significantly higher in experimental group than in control group(P<0.05).(4) The clinical pregnancy rate of two groups respectively were 73.33%(22/30) and 46.67%(14/30). In experimental group, the pregnancy rate was significantly higher than in control group(P<0.05).(5) No differences were observed between the two groups in complications including OHSS and abortion.
Conclusions: It comes into the conclusion that combined use of Bushen Tiaojing Recipe during COH of IVF-ET can increase oocyte number and improve fertilization rate, high quality embryo rate and clinical pregnancy rate.
Part two: The relationship human granulosa cells apoptosis and OSFs to oocyte development potential
Objective: To investigate the incidence of apoptosis in human single oocyte’s granulosa cells and the level of GDF-9 and BMP-15 in corresponding follicular fluid(FF)and evaluate the relationship granulosa cells apoptosis and OSFs to oocyte development potential.
Methods: A total of 69 occytes and their corresponding FF from women undergoing IVF-ET because of tubal infertility were examined. FF was sampled by trans-vaginal ultrasound-guided puncture and aspiration of >18 mm diameter follicles. Fluid from the first aspirated follicle of each ovary was carefully collected in a tube, and the oocyte-cumulus- complex (COC) that was used for IVF was identified in the fluid sample coming from the same follicle and maintained in isolation from that coming from other follicles. Individual oocytes were inseminated and subsequently assessed on the basis of their maturation , fertilization, cleavage and preimplantation development. The 69 occytes were divided into good quality oocytes group which divided into high-quality embryos and low-quality oocytes group including immature, nonfertilized, developed bad embryos. FF samples were centrifuged and collected granulosa cells used for the measurement of apoptosis rate by TUNEL.The GDF-9 and BMP-15 levels in FF were measured by western-blot.
Results: (1)69 oocytes were observed including 36 high-quality oocytes and 33 low-quality oocytes. The average of apoptosis rate in granulosa cells was 12.746±3.73 in high-quality oocytes group and 19.88±6.36 in low-quality oocytes group. The apoptosis rate in high-quality oocytes group was significantly lower than in low-quality oocytes group (P<0.01)(.2)The level of GDF-9 in FF was 2.4983±0.1328 in high-quality oocytes group and 1.3052±0.406 in low-quality oocytes group. The level of GDF-9 in high-quality oocytes group was significantly higher than in low-quality oocytes group (P<0.01).(3) The level of BMP-15in FF was 3.3785±0.4823 in high-quality oocytes group and 1.336±0.2139 in low-quality oocytes group. The level of BMP-15 in high-quality oocytes group was significantly higher than in low-quality oocytes group (P<0.01).
Conclusions: It comes into the conclusion that the incidence of apoptosis in human granulosa cells shows negative correlation with oocyte development potential ; and GDF-9 and BMP-15 level with good oocyte quality show significant positive correlation. The present study demonstrates that the incidence of apoptosis in human granulosa cells and the GDF-9 and BMP-15 level in FF appears to be potential factors in predicting oocyte quality and subsequent embryo development.
Part three: The effect of Bushentiaojing Recipe on apoptosis in human granulosa cells in Patients Undergoing in Vitro Fertilization
Objective: To investigate the effect of Bushentiaojing Recipe on apoptosis in human granulosa cells and to explore the mechanisms of improvement on oocyte and embryo quality in Patients Undergoing IVF
Methods: Sixty infertile patients with tubal factors undergoing IVF-ET were divided into two groups at random, 30 cases in the study group treated with Bushentiaojing Recipe +GnRHa/FSH/hCG, and 30 cases in control group treated with GnRHa/FSH/hCG. FF was sampled by trans-vaginal ultrasound- guided puncture and aspiration of all≥18–20 mm diameter follicles. FF samples were centrifuged and collected granulosa cells. The apoptosis rate of granulosa cells measured by TUNEL. The immunocytochemistry was performed to determine the expression of apoptosis-related protein Caspase 3.The DNA of apoptosis cell was detected by flow cytometry .
Results: (1) The average of apoptosis rate in granulosa cells was 13.61±4.53 in experimental group and 19.41±5.95 in control group. The apoptosis rate in experimental group was significantly lower than in control group (P<0.01 ).(2) The expression positive rate of Caspase 3 was 48.08±11.24% in experimental group and 68.58±10.31% in control group. The expression positive rate of Caspase 3 in experimental group was significantly lower than in control group (P<0.01).(3) The average value of the DNA of apoptosis cell in experimental group and control group was 1.26±0.24%、3.61±1.6% respectively, and the former were significantly lower than the latter.
Conclusions: Bushen Tiaojing Recipe can decrease the apoptosis rate of granulosa cells and the content of DNA in apoptosis cells and can inhibit the expression of apoptosis-related protein Caspase 3. All of the above indicates that the mechanisms of Bushen Tiaojing Recipe can decrease apoptosis rate in granulosa cells is related to inhibit the expression of apoptosis-related protein Caspase 3.
Part four:Effects of Bushen Tiaojing Recipe on GDF-9 and BMP-15 in Patients Undergoing in Vitro Fertilization
Objective: To explore the mechanisms involved in the improvement of oocyte and embryo quality in patients undergoing IVF by investigating the effects of Bushen Tiaojing Recipe on OSFs GDF-9 and BMP-15.
Methods: Sixty infertile patients with tubal factors undergoing IVF-ET were divided into two groups at random, 30 cases in the study group treated with Bushentiaojing Recipe +GnRHa/FSH/hCG, and 30 cases in control group treated with GnRHa/FSH/hCG. FF was sampled by trans-vaginal ultrasound-guided puncture and aspiration of all≥18-20 mm diameter follicles. FF samples were centrifuged and collected granulosa cells. The level of GDF-9 and BMP-15 in FF was analyzed by western-blot. The expression of GDF-9 and BMP-15 in granulosa cells was measured by RT-PCR.
Results: (1) Compared with control group, The average value of GDF-9 in FF of experimental group(2.7305±0.1260) was higher than in control group(2.0564±0.3055) (P<0.05). (2) The expression of GDF-9 mRNA in human granulosa cells of experimental group and control group were 21807.0±1998.6 and 10177.0±2571.4 respectively. The expression of GDF-9 mRNA in experimental group was significantly higher than in control group(P<0.01). (3) Compared with control group, BMP-15 in FF of experimental group (2.6454±0.4008) was higher than in control group (1.6252±0.4371). (P<0.05).(2) The expression of BMP-15 mRNA in human granulosa cells of experimental group and control group were 11894.0±865.6 and 6441.0±794.6 respectively. The expression of BMP-15mRNA in experimental group was significantly higher than in control group(P<0.05).
Conclusions: Bushen Tiaojing Recipe can increase the level of GDF-9 and BMP-15 in FF and the expression of GDF-9 and BMP-15 in granulosa cells.
All of the above demonstrates that Bushen Tiaojing Recipe can improve oocyte quality, increase fertilization rate, high quality embryo rate and clinical pregnancy by decreasing granulosa cells apoptosis rate and increasing OSFs GDF-9 and BMP-15. Bushen Tiaojing Recipe may be the adjuvant treatment during IVF.
由于颗粒细胞与卵母细胞之间存在广泛的包括缝隙连接在内的复杂连接机制,在调节卵母细胞发育、排卵、卵子受精及胚胎种植等方面发挥重要的作用;卵母细胞分泌的卵母细胞分泌因子(oocyte secreted factors,OSFs)通过旁分泌作用促进颗粒细胞增生、分化并调节其功能,与颗粒细胞共同调节卵母细胞的发育过程。文献报道,卵丘细胞(cumulus cell,CC)的凋亡状态和OSFs直接影响卵母细胞的发育潜能。所以本研究以颗粒细胞凋亡和OSFs为切入点研究补肾调经方调经种子的作用机制。以期为不孕症的中医药治疗和IVF-ET的中医辅助用药提供理论基础和实验依据
第一部分:补肾调经方对体外受精-胚胎移植妊娠结局的影响
目的:观察补肾调经方对体外受精-胚胎移植超排卵周期妊娠结局的影响。
方法:收集因输卵管因素行体外受精-胚胎移植( in vitro fertilization-embryo transfer,IVF-ET)技术助孕,并符合中医肾虚不孕症辨证标准的女性患者60例,随机分为中药补肾调经方+西药控制性超排卵组(实验组)30例和单纯西药控制性超排卵组(对照组)30例。观察并记录两组患者HCG日血清E2、P、LH水平、促性腺激素(gonadotropin, Gn)使用天数和剂量、获卵数、成熟卵数、受精率、卵裂率、优质胚胎率、临床妊娠率及并发症情况。
结果:(1)HCG日两组患者血E2、LH、P值比较:两组患者在HCG日血LH、P水平无显著差异(P>0.05);实验组的E2水平明显高于对照组(P<0.05)。(2)Gn总量、Gn天数及获卵数的比较:两组患者Gn总量、Gn天数均无显著差异(P>0.05);实验组获卵数高于对照组,有显著性差异(P<0.01)。(3)实验室评定成熟卵率、受精率及优质胚胎率比较:实验组成熟卵率86.70%、2PN受精率76.84%、卵裂率96.79%、优质胚胎率64.90%。对照组成熟卵率83.80%、2PN受精率66.07%、卵裂率95.72%、优质胚胎率54.06%。统计结果显示两组在成熟卵率、卵裂率方面差异无显著性(均P >0.05)。而在受精率、优质胚胎率方面实验组高于对照组(均P <0.05)。(4)临床妊娠率比较:实验组临床妊娠22例,妊娠率73.33%;对照组临床妊娠14例,妊娠率46.67%,两组临床妊娠率差异有显著性(P<0.05)。(5)相关并发症比较:实验组发生OHSS1例,胎停育1例;对照组无OHSS病例,自然流产1例。无其它并发症发生。两组OHSS及流产发生率无统计学意义。
结论:在体外受精-胚胎移植控制性促排卵同时,加用中药补肾调经方可以增加获卵数,并提高受精率、优质胚胎率及临床妊娠率。
第二部分:人颗粒细胞凋亡及OSFs与卵母细胞发育潜能的关系
目的:通过检测人单个卵母细胞对应颗粒细胞的凋亡率和对应卵泡液中卵母细胞分泌因子GDF-9和BMP-15的含量,进一步阐明颗粒细胞凋亡及卵母细胞分泌因子GDF-9和BMP-15与卵母细胞发育潜能的关系。
方法:收集因输卵管性不孕接受IVF-ET治疗的患者。经阴道超声引导下取卵,每侧卵巢穿刺时首先选择一个直径≥18mm卵泡抽吸,将该卵泡的卵泡液单独留于一管内,置于显微镜下找卵,若找到卵-冠-丘复合物(COC),经过卵母细胞成熟度分型,并将该卵母细胞行微滴受精及培养,于受精后16-18h评估受精情况,受精24h后评估受精卵的卵裂情况,于受精后第3天(4-8细胞期),给予胚胎评分。分组方法:一组为形成优质胚胎的卵母细胞,称为优质卵母细胞组;另一组为未成熟、未受精及未形成优质胚胎的卵母细胞,称为非优质卵母细胞组。将对应的卵泡液离心,用人淋巴细胞分离液分离出颗粒细胞,采用脱氧核糖核苷转移介导的缺口末端标记法(TUNEL)检测颗粒细胞的凋亡率;采用western-blot方法检测卵泡液中GDF-9和BMP-15的含量。分别分析颗粒细胞凋亡及GDF-9和BMP-15与卵母细胞发育潜能的关系。
结果:(1)共观察了69枚卵母细胞,其中形成优质胚胎的卵母细胞36枚,未形成优质胚胎的卵母细胞33枚(其中未成熟卵5枚,未受精及异常受精卵6枚)。TUNEL法测定GC的凋亡率,形成优质胚胎的优质卵母细胞组GC凋亡率为12.746±3.73,未形成优质胚胎的非优质卵母细胞组GC凋亡率为19.88±6.36,有显著的统计学差异(P<0.01)。(2)优质卵母细胞组的GDF-9蛋白含量为2.4983±0.1328,非优质卵母细胞组为1.3052±0.406,统计学有显著差异(P=0.0084<0.01),结果显示:发育潜能较好的卵母细胞相应卵泡液中GDF-9的含量较高。(3)优质卵母细胞组的BMP-15蛋白含量为3.3785±0.4823 ,非优质卵母细胞组为1.336±0.2139,统计学有显著差异(P=0.0026<0.01),结果显示:发育潜能较好的卵母细胞相应卵泡液中BMP-15的含量较高。
结论:优质卵母细胞组颗粒细胞的凋亡率低,提示颗粒细胞凋亡可能影响卵母细胞的质量及胚胎的体外发育潜能。在优质卵母细胞组卵泡液中GDF-9和BMP-15水平及颗粒细胞中GDF-9和BMP-15mRNA的表达均较高。
提示人颗粒细胞的凋亡状态及GDF-9、BMP-15水平可以反应卵母细胞的发育潜能,颗粒细胞凋亡率及GDF-9、BMP-15水平可以做为评价卵母细胞质量的指标。
第三部分:补肾调经方对体外受精-胚胎移植超排卵周期颗粒细胞凋亡的影响
目的:研究补肾调经方对颗粒细胞凋亡的影响,探讨其提高IVF-ET超排卵周期所获卵和胚胎质量的作用机制。
方法:收集因输卵管因素行IVF-ET技术助孕,并符合中医肾虚不孕症辨证标准的女性患者60例,随机分为中药补肾调经方+西药控制性超排卵组(实验组)30例和单纯西药控制性超排卵组(对照组)30例。留取两侧卵巢所有直径≥18mm的主导卵泡的卵泡液,用人淋巴细胞分离液分离出颗粒细胞,采用脱氧核糖核苷转移介导的缺口末端标记法(TUNEL)检测颗粒细胞的凋亡率;采用免疫组化法测定颗粒细胞中凋亡相关蛋白Caspase-3的表达;采用流式细胞仪法测定颗粒细胞凋亡率。分析补肾调经方与颗粒细胞凋亡的关系。
结果:(1)TUNEL法测定两组颗粒细胞的凋亡率:实验组的凋亡率为13.61±4.53;对照组为19.41±5.95。两组比较有显著统计学差异(P<0.05),加用补肾调经方后可以降低颗粒细胞的凋亡率。(2) Caspase3在两组颗粒细胞中的表达:实验组Caspase3表达阳性率为48.08±11.24%,对照组Caspase3表达阳性率为68.58±10.31%,实验组显著低于对照组(P<0.01)。(3)流式细胞仪方法测定两组颗粒细胞凋亡细胞的DNA含量:实验组颗粒细胞凋亡细胞的DNA含量为1.26±0.24%,对照组颗粒细胞凋亡细胞的DNA含量为3.61±1.6%,实验组颗粒细胞凋亡细胞的DNA含量显著低于对照组(P<0.05)。
结论:补肾调经方可降低颗粒细胞的凋亡率及凋亡细胞DNA的含量,下调颗粒细胞中凋亡相关蛋白caspase3的表达。提示补肾调经方提高卵母细胞质量可能与降低颗粒细胞的凋亡率有关,其机制可能与抑制caspase3的表达有关。
第四部分:补肾调经方对体外受精-胚胎移植周期卵母细胞分泌因子GDF-9和BMP-15的影响
目的:研究补肾调经方对GDF-9和BMP-15的影响,探讨其提高IVF-ET超排卵周期所获卵和胚胎质量的作用机制。
方法:收集因输卵管因素行IVF-ET技术助孕,并符合中医肾虚不孕症辨证标准的女性患者60例,随机分为中药补肾调经方+西药控制性超排卵组(实验组)30例和单纯西药控制性超排卵组(对照组)30例。留取两侧卵巢所有直径≥18mm的主导卵泡的卵泡液,用人淋巴细胞分离液分离出颗粒细胞。采用western-blot方法检测卵泡液中GDF-9和BMP-15的含量,采用RT-PCR方法检测颗粒细胞中GDF-9和BMP-15的表达。分析补肾调经方与GDF-9和B MP-15的关系。
结果:(1)两组卵泡液GDF-9结果比较治疗组卵泡液GDF-9水平(2.7305±0.1260)显著高于对照组(2.0564±0.3055),差异有统计学意义(P<0.05)。(2)两组颗粒细胞GDF-9 mRNA表达的电泳结果:治疗组GDF-9mRNA表达(21807.0±1998.6)显著高于对照组(10177.0±2571.4),差异有统计学意义(P<0.01)。(3)两组卵泡液BMP-15蛋白质测定结果:治疗组卵泡液BMP-15水平(2.6454±0.4008),显著高于对照组(1.6252±0.4371),差异有统计学意义( P<0.05)。(4)颗粒细胞BMP-15 mRNA表达的电泳结果:治疗组BMP-15 mRNA表达(11894.0±865.6)显著高于对照组(6441.0±794.6),差异有统计学意义(P<0.01)。
结论:补肾调经方可增加卵泡液中GDF-9和BMP-15的含量,并上调颗粒细胞中GDF-9和BMP-15的表达。
综上所述,补肾调经方可能通过降低颗粒细胞凋亡率,增加GDF-9和BMP-15水平来改善卵母细胞质量,提高受精率、优质胚胎率及临床妊娠率,补肾调经方可以作为IVF-ET控制性促排卵的辅助用药。
In vitro fertilization-embryo transfer (IVF-ET), one of the most scientific and effective methods in treating infertility a common and frequent disease (morbidity of 7%-10%) in human reproductive health, has made great progress recent years. While the success rate still remains low with relatively high rate of multiple pregnancy and abortion. Effort to optimize the protocols of controlled ovarian hyperstimulation (COH) is still the major method to improve oocyte quality, which plays an important role in IVF-ET.Researches on Traditional Chinese Medicine (TCM) used to improve the quality of oocyte and embryo has made great progress recently.Our research team studied the effects of Bushen Tiaojing Recipe on the reproductive function of anovulatory rats and concluded that this recipe can induce follicular development and ovulation in many paths and targets, and can benefit embryo implantation by promoting uterus development and regulating the microenvironment of endometrium.
The purpose of our research is to study the effects of Bushen Tiaojing Recipe Combined with IVF-ET on treatment of infertility more deeply, on the basis of preliminary research.
It is known that bidirectional interactions between the oocytes and surrounding somatic cells through gap junctions and paracrine signaling are pivotal in maintaining the growth and development of both cell types during folliculogenesis. The oocyte plays an active role throughout folliculogenesis via secretion of oocyte-secreted factors(OSFs) that effects cumulus cell proliferation , expansion, differentiation, steriodogenesis, gene expression and maintain an appropriate microenvironment for the acquisition of its developmental competence.
It is reported that the apoptosis of cumulus cells and OSFs are associated with oocyte development directly. Therefore, in this study we observed the effects of Bushen Tiaojing Recipe on IVF-ET, granulosa cell apoptosis and OSFs and discussed the mechanisms involved in order to supply theoretical and experimental basis for traditional Chinese medicine used in infertility and assistant therapy for IVF-ET.
Part one: Clinical application of Bushen Tiaojing Recipe in IVF-ET Objective: To observe effects of Bushen Tiaojing Recipe on pregnancy outcome in patients undergoing IVF-ET.
Methods: Sixty infertile patients with tubal factors combined with kidney deficiency undergoing IVF-ET were divided into two groups at random, experimental group (Bushen Tiaojing Recipe +GnRHa/FSH/hCG) and control group (GnRHa/FSH/hCG).The content of hormones(E2、LH、P) on HCG day, days and dosage of gonadotropin, number of oocytes and mature oocytes, fertilization rate, high quality embryos rate ,pregnancy rate and complication were observed.
Results: (1) The content of E2 was significantly higher in experimental group than in control group(P<0.05).The level of P、LH was no statistical significance between two groups(P>0.05).(2) The time and dosage of gonadotropin were no statistical significance between two groups(P >0.05),while the oocyte number was significantly larger in experimental group than in control group(P<0.05).(3) The rate of mature oocyte and oocyte cleavage were no statistical significance between two groups(P>0.05),while the rate of fertilization , high quality embryo were significantly higher in experimental group than in control group(P<0.05).(4) The clinical pregnancy rate of two groups respectively were 73.33%(22/30) and 46.67%(14/30). In experimental group, the pregnancy rate was significantly higher than in control group(P<0.05).(5) No differences were observed between the two groups in complications including OHSS and abortion.
Conclusions: It comes into the conclusion that combined use of Bushen Tiaojing Recipe during COH of IVF-ET can increase oocyte number and improve fertilization rate, high quality embryo rate and clinical pregnancy rate.
Part two: The relationship human granulosa cells apoptosis and OSFs to oocyte development potential
Objective: To investigate the incidence of apoptosis in human single oocyte’s granulosa cells and the level of GDF-9 and BMP-15 in corresponding follicular fluid(FF)and evaluate the relationship granulosa cells apoptosis and OSFs to oocyte development potential.
Methods: A total of 69 occytes and their corresponding FF from women undergoing IVF-ET because of tubal infertility were examined. FF was sampled by trans-vaginal ultrasound-guided puncture and aspiration of >18 mm diameter follicles. Fluid from the first aspirated follicle of each ovary was carefully collected in a tube, and the oocyte-cumulus- complex (COC) that was used for IVF was identified in the fluid sample coming from the same follicle and maintained in isolation from that coming from other follicles. Individual oocytes were inseminated and subsequently assessed on the basis of their maturation , fertilization, cleavage and preimplantation development. The 69 occytes were divided into good quality oocytes group which divided into high-quality embryos and low-quality oocytes group including immature, nonfertilized, developed bad embryos. FF samples were centrifuged and collected granulosa cells used for the measurement of apoptosis rate by TUNEL.The GDF-9 and BMP-15 levels in FF were measured by western-blot.
Results: (1)69 oocytes were observed including 36 high-quality oocytes and 33 low-quality oocytes. The average of apoptosis rate in granulosa cells was 12.746±3.73 in high-quality oocytes group and 19.88±6.36 in low-quality oocytes group. The apoptosis rate in high-quality oocytes group was significantly lower than in low-quality oocytes group (P<0.01)(.2)The level of GDF-9 in FF was 2.4983±0.1328 in high-quality oocytes group and 1.3052±0.406 in low-quality oocytes group. The level of GDF-9 in high-quality oocytes group was significantly higher than in low-quality oocytes group (P<0.01).(3) The level of BMP-15in FF was 3.3785±0.4823 in high-quality oocytes group and 1.336±0.2139 in low-quality oocytes group. The level of BMP-15 in high-quality oocytes group was significantly higher than in low-quality oocytes group (P<0.01).
Conclusions: It comes into the conclusion that the incidence of apoptosis in human granulosa cells shows negative correlation with oocyte development potential ; and GDF-9 and BMP-15 level with good oocyte quality show significant positive correlation. The present study demonstrates that the incidence of apoptosis in human granulosa cells and the GDF-9 and BMP-15 level in FF appears to be potential factors in predicting oocyte quality and subsequent embryo development.
Part three: The effect of Bushentiaojing Recipe on apoptosis in human granulosa cells in Patients Undergoing in Vitro Fertilization
Objective: To investigate the effect of Bushentiaojing Recipe on apoptosis in human granulosa cells and to explore the mechanisms of improvement on oocyte and embryo quality in Patients Undergoing IVF
Methods: Sixty infertile patients with tubal factors undergoing IVF-ET were divided into two groups at random, 30 cases in the study group treated with Bushentiaojing Recipe +GnRHa/FSH/hCG, and 30 cases in control group treated with GnRHa/FSH/hCG. FF was sampled by trans-vaginal ultrasound- guided puncture and aspiration of all≥18–20 mm diameter follicles. FF samples were centrifuged and collected granulosa cells. The apoptosis rate of granulosa cells measured by TUNEL. The immunocytochemistry was performed to determine the expression of apoptosis-related protein Caspase 3.The DNA of apoptosis cell was detected by flow cytometry .
Results: (1) The average of apoptosis rate in granulosa cells was 13.61±4.53 in experimental group and 19.41±5.95 in control group. The apoptosis rate in experimental group was significantly lower than in control group (P<0.01 ).(2) The expression positive rate of Caspase 3 was 48.08±11.24% in experimental group and 68.58±10.31% in control group. The expression positive rate of Caspase 3 in experimental group was significantly lower than in control group (P<0.01).(3) The average value of the DNA of apoptosis cell in experimental group and control group was 1.26±0.24%、3.61±1.6% respectively, and the former were significantly lower than the latter.
Conclusions: Bushen Tiaojing Recipe can decrease the apoptosis rate of granulosa cells and the content of DNA in apoptosis cells and can inhibit the expression of apoptosis-related protein Caspase 3. All of the above indicates that the mechanisms of Bushen Tiaojing Recipe can decrease apoptosis rate in granulosa cells is related to inhibit the expression of apoptosis-related protein Caspase 3.
Part four:Effects of Bushen Tiaojing Recipe on GDF-9 and BMP-15 in Patients Undergoing in Vitro Fertilization
Objective: To explore the mechanisms involved in the improvement of oocyte and embryo quality in patients undergoing IVF by investigating the effects of Bushen Tiaojing Recipe on OSFs GDF-9 and BMP-15.
Methods: Sixty infertile patients with tubal factors undergoing IVF-ET were divided into two groups at random, 30 cases in the study group treated with Bushentiaojing Recipe +GnRHa/FSH/hCG, and 30 cases in control group treated with GnRHa/FSH/hCG. FF was sampled by trans-vaginal ultrasound-guided puncture and aspiration of all≥18-20 mm diameter follicles. FF samples were centrifuged and collected granulosa cells. The level of GDF-9 and BMP-15 in FF was analyzed by western-blot. The expression of GDF-9 and BMP-15 in granulosa cells was measured by RT-PCR.
Results: (1) Compared with control group, The average value of GDF-9 in FF of experimental group(2.7305±0.1260) was higher than in control group(2.0564±0.3055) (P<0.05). (2) The expression of GDF-9 mRNA in human granulosa cells of experimental group and control group were 21807.0±1998.6 and 10177.0±2571.4 respectively. The expression of GDF-9 mRNA in experimental group was significantly higher than in control group(P<0.01). (3) Compared with control group, BMP-15 in FF of experimental group (2.6454±0.4008) was higher than in control group (1.6252±0.4371). (P<0.05).(2) The expression of BMP-15 mRNA in human granulosa cells of experimental group and control group were 11894.0±865.6 and 6441.0±794.6 respectively. The expression of BMP-15mRNA in experimental group was significantly higher than in control group(P<0.05).
Conclusions: Bushen Tiaojing Recipe can increase the level of GDF-9 and BMP-15 in FF and the expression of GDF-9 and BMP-15 in granulosa cells.
All of the above demonstrates that Bushen Tiaojing Recipe can improve oocyte quality, increase fertilization rate, high quality embryo rate and clinical pregnancy by decreasing granulosa cells apoptosis rate and increasing OSFs GDF-9 and BMP-15. Bushen Tiaojing Recipe may be the adjuvant treatment during IVF.
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