摘要
目的：观察补肾中药对超排卵周期颗粒细胞胶质细胞源性神经营养因子（glial cellline-derived neurotrophic factor，GDNF）、GDNF家族特异性受体α-1（GDNF familyreceptor α-1，GFRα-1）的影响，从卵母细胞生殖泡裂解、第一极体的排出及其成熟旁分泌调节的角度，探讨补肾改善卵母细胞质量，提高控制性超排卵妊娠结局的机制。
方法：临床研究：将80例因输卵管因素行体外受精－胚胎移植（in vitrofertilization-embryo transfer，IVF-ET）的患者按照随机数字表法分为治疗组40例（二至天癸颗粒联合促性腺激素（Gn），40个周期）和对照组40例（安慰剂颗粒联合Gn，40个周期）。采用IVF-ET治疗中目前常规应用的控制性超排卵方案，治疗组在IVF治疗周期加用二至天癸颗粒；对照组则加用安慰剂颗粒。观察临床肾虚证候积分变化情况，Gn用量及用药天数，取卵数、优质卵率、受精率、优质胚胎率、临床妊娠率及颗粒细胞GDNF、GFR α-1mRNA的表达情况。同时行颗粒细胞体外培养24h、48h后观察E2、P的分泌情况以了解颗粒细胞的分泌功能。
实验研究：将165只8-9周龄、健康昆明种雌性小鼠按照随机法分为空白组、治疗组及对照组，每组各55只。治疗组及对照组进行控制性超排卵，连续灌胃给药（治疗组用二至天癸颗粒，对照组用等量生理盐水），三组又各分为1、2两组。1组50只脱颈处死小鼠，取生发泡(GV)期裸卵(DOs),观察生殖泡破裂（GVBD）和第一极体（PB1）释放；之后成熟卵母细胞行GDNF、GFR α-1mRNA的表达检测；2组各5只雌雄鼠合笼饲养，取受精卵，观察卵裂率、成胚率。
结果：临床研究：治疗组患者肾虚症状得到明显改善，治疗组Gn用量及用药天数均低于对照组，单个卵雌激素（E_2/卵泡）水平高于对照组，优质卵率、优质胚胎率及临床妊娠率均高于对照组，差异均有统计学意义（P＜0.05）；治疗组颗粒细胞培养24h、48h生长情况，培养24h、48h后收集的培养液E_2、P检测及颗粒细胞GDNF、GFR α-1mRNA表达均优于对照组，差异均有统计学意义(P＜0.05)。实验研究：治疗组GV期卵母细胞GVBD率、PB1排出率均高于对照组，差异有统计学意义（P＜0.05）；各组小鼠受精卵卵裂率及成胚率比较，对照组低于空白组，治疗组高于对照组，差异有统计学意义（P＜0.05）。治疗组卵母细胞GFR α-1mRNA的表达高于对照组，差异有统计学意义(P＜0.05)。
结论：1.获得与既往国家级和省级课题相同的临床妊娠率（54%左右）。在IVF-ET治疗周期，应用补肾中药可明显改善患者肾虚症状，减少Gn用量，提高优质卵率、优质胚胎率，进而改善临床妊娠率，是临床常用的有效药物。2.补肾中药提高颗粒细胞GDNF、GFRα-1mRNA的表达，改善颗粒细胞分泌功能，这可能是补肾中药改善IVF结局的机制之一。3.补肾中药促进小鼠GV期卵母细胞生殖泡的破裂（GVBD）和第一极体（PB1）的排出；提高小鼠卵母细胞GFRα-1mRNA的表达，并改善了小鼠受精卵的卵裂、形成囊胚的能力。推断：补肾中药提高颗粒细胞GDNF、GFRα-1mRNA的表达，提高小鼠卵母细胞GFRα-1mRNA的表达，促进卵母细胞GVBD和PB1的排出，从而改善了卵母细胞成熟度，提高卵细胞质量，从而提高胚胎质量及临床妊娠率。这可能又是补肾中药改善IVF结局的机制之一。
Objective：To investigate the effects of reinforcing kidney on the expression ofGDNF、GFRα-l in granulosa cell of superovulation cycle, In view of GVBD、extrusion ofPB1and oocyte maturation paracrine, to explore the mechanism of reinforcing KidneyChinese herbs Erzhitiangui(ETG) Granule to improve the treatment outcome of insuperovulation cycle.
Method： Clinical Study:80infertile women with tubal factor undergoing vitrofertilization-embryo transfer (IVF-ET) were randomized into the study group, treated withETG and Gn(40cases), and the control group, treated with placebos granules and Gn (40cases). The long term protocol was adopted. The E2and P secretion in granule cell aftercultured for24h、48h in vitro was observed to learn about the secreting function of granulecell. The expression of GDNF、GFR-1mRNA in luteinizing granulosa cells were alsodetected. The syndrome scores of TCM, administration time and number of Gn, number ofretrieved oocytes, fertilization rate, high quality embryo rate and pregnancy rate wereevaluated.
Experimental Study:165Kunming female mice of8-9weeks old were allocated intothree groups randomly: control group(group HMG＋HCG＋NS), HMG and HCG weregiven for ovarian stimulation, performed abdomen injection with0.9%salt solution ofsame as study group on the same time schedule; study group(group HMG＋HCG＋ETG);ETG combined with HMG and HCG were given; blank group(group nothing). Three groupwere divided into1、2groups each.1group were taked denuded on germinal vesiclestage，observed the germinal veicles breakdowm and extrusion of the first polar body, The expression of GDNF、GFR-1mRNA in the oocytes were also detected;;2group were retrieved to evaluate the ability of merogenesis and forming the embryo.
Result：Symptoms of kidney asthenia of the study group was significantly improved.The level of E2per occtye, the ratio of high quality occtye and embryo, the clinicalpregnancy rate were higher than those of the control group. The difference werestatistically significant (P＜0.05). The E2and P secretion in granule cell after cultured for24h、48h in vitro were higher than the control group. The expression of GDNF、GFRα-lmRNA in granule cell of the study group was higher than that of the control group, Thedifference were statistically significant (P＜0.05). The study group was significant higherthan control group in the frequency of the germinal veicles breakdowm and extrusion ofthe first polar body. The expression of GFRa-l mRNA in in the oocytes of the study groupwas higher than that of the control group. The number ability of merogenesis and formingthe embryo of fertilized ovum in control group were lower than those of the study andblank group. The difference was statistically significant (P＜0.05).
Conclusion：1. Reinforcing Kidney Chinese herbs combined with western medicinecan decrease the dosage of Gn obviously and improve symptoms of kidney asthenia, thequality of oocyte, the ratio of high quality occtye and embryo, the ratio of pregnancy.2.Reinforcing Kidney Chinese herbs can also improve the secreting function of granule cell,the expression of GDNF、GFRα-1mRNA in luteinizing granulosa cells. That maybe oneof the mechanism of reinforcing Kidney Chinese herbs to improve the ratio of pregnancy.and raise the quality of oocytes.3. Reinforcing Kidney Chinese herbs can improve thegerminal veicles breakdowm and extrusion of the first polar body, the ability ofmerogenesis and forming the embryo of fertilized ovum, and the expression of GFRα-1mRNA in oocytes., which may suggest that Reinforcing Kidney Chinese herbs raise thequality of oocytes、embryo and the ratio of pregnancy by improve the germinal veiclesbreakdowm, extrusion of the first polar body, the expression of GDNF、GFRα-1mRNAin luteinizing granulosa cells and the expression of GFRα-1mRNA in oocytes. Thatmaybe another one of the mechanism of reinforcing Kidney Chinese herbs to improve theratio of pregnancy.
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