控制性促排卵黄体期添加雌激素对子宫内膜容受性影响的实验研究
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摘要
1.研究背景
不孕症(Infertility)发病率约占育龄妇女的10～15%,是影响家庭乃至社会和谐与稳定的重要因素之一。受孕成功的四大因素是:高质量的卵子和精子、通畅的输卵管和良好的子宫内膜,四者缺一不可。其中子宫内膜是胚胎种植的“土壤”,良好的子宫内膜环境是受孕成功的关键。所以,子宫内膜容受性(Endometrialreceptivity)是目前生殖领域研究的热点。
正常子宫内膜仅在一特定时期允许着床,这一时期子宫内膜容受性最高,称为着床窗口期(Window of implantation),一般为排卵后6～10d,即正常月经周期的19～24d。子宫内膜容受性的评价标准包括子宫内膜厚度、形态、母体内分泌状况和子宫内膜的生殖调控因子,包括细胞形态学、分子生物学和基因水平的研究,如:胞饮突(pinopode,pp)、白血病抑制因子(Leukaemia inhibitory factor,LIF)、整合素家族(Integrins)、白细胞介素1(IL-1)、表皮生长因子(EGF)、集落刺激因子(CSF-1)、雌孕激素受体等。
控制性促排卵(Contral Ovarian Stimulation,COS)体外受精-胚胎移植(InVitro Fertilization and Embryo Transfer,IVF-ET)技术已较广泛地用于治疗难治性不孕症,其临床妊娠成功率受诸多因素的影响。如何提高其胚胎种植率和临床妊娠率是一个亟待解决的难题。子宫内膜容受性与胚胎质量是影响IVF-ET成功率的两大主要因素。而胚胎着床前子宫内膜的容受状态受雌、孕激素及生殖调控因子的协同调节。在IVF-ET控制性促排卵中大多数应用促性腺激素释放激素协同剂(Gonadotropicreleasingllormoneagonist,GnRHa)进行降调节,通过抑制内源性的促黄体生成素(Lutenizing Hormone,LH)峰的出现,使多个卵泡募集,获得多个高质量的卵子,与精子结合形成多个胚胎,选择优秀的胚胎进行移植,从而提高妊娠成功率,改善妊娠结局。但是,这种抑制作用可使排卵后黄体期的LH分泌也受到抑制,导致黄体功能不足,胚胎种植率和妊娠率的降低。
对接受控制性促排卵WF-ET患者进行黄体支持已得到大家的公认。传统的黄体支持方法是黄体期应用黄体酮(Progesterone,p),如何改进黄体期支持方案增加子宫内膜容受性以提高胚胎种植率和WF-ET妊娠成功率是近年来生殖领域研究的热点。近来有学者试着在传统方案的基础上添加小剂量雌激素,但只是在初步探索阶段,尚无确切的定论。到目前为止,有关黄体期支持添加雌激素与子宫内膜容受性关系的研究,报道甚少,尚未见运用细胞形态学、免疫组化、分子生物学手段及临床资料统计分析联合证实黄体期添加雌激素对子宫内膜容受性影响的研究。
2.研究目的
为探讨COS黄体期支持添加小剂量雌激素对子宫内膜容受性的影响,本课题首先通过建立动物模型,采用细胞形态学、免疫组化和分子生物学技术,观察不同COS黄体期支持方案小鼠子宫内膜胞饮突、LIF和LIFmRNA的表达,从而总结添加雌激素对小鼠子宫内膜容受性的影响。在此基础上,对临床进行控制性促排卵IVF-ET的患者进行回顾性分析,观察其胚胎移植(Embryo transfer,ET)日血清雌二醇(Estradiol,E_2)水平较注射绒毛膜促性腺激素(hCG)日下降幅度对人胚胎种植率和临床妊娠率的影响,进而对临床接受控制性促排卵IVF-ET患者采用不同方案进行黄体期支持,对各组患者IVF-ET实验室和临床资料进行研究分析,探讨ET日E_2下降幅度对人胚胎种植率和临床妊娠率的影响及黄体期支持添加雌激素的必要性。通过系列研究,深入探讨雌激素与子宫内膜容受性关系的内在分子机制,从而揭示控制性促排卵黄体期支持添加雌激素对子宫内膜容受性的影响,为改进临床治疗方案、提高IVF-ET技术妊娠成功率提供一定的理论依据。
3.材料与方法
3.1动物实验材料与方法
3.1.1分组方法:将正常雌性小鼠随机分为四组:A组、B组和C组均采用控制性促排卵,用hCG后分别与正常雄性小鼠2:1合笼,次日清晨检查阴栓,有阴栓者为受孕第1d;D组:自然周期组,与正常雄性小鼠2:1合笼,次日清晨检查阴栓,有阴栓者为受孕第1d。
3.1.2用药方法:控制性促排卵的雌性小鼠从受孕第1d开始接受不同的黄体期支持方法:A组:不采用黄体期药物支持;B组:单用黄体酮进行黄体期支持;C组:在应用黄体酮的基础上添加雌激素;D组:同时应用同等量的生理盐水。
3.1.3取材:A、B、C、D组小鼠分别于孕第3、4、5d剖腹取子宫。
3.1.4扫描电镜(scanning electronic microscopy,SEM)观察各组受孕小鼠围植入期胞饮突的表达。
3.1.5采用免疫组化(Immunohistochemistry,SP)法检测LIF蛋白在各组受孕小鼠围植入期子宫内膜的表达情况。
3.1.6采用逆转录聚合酶链反应(Reverse transcriptional-polymerise chain reaction,RT-PCR)法检测LIFmRNA在各组受孕小鼠围植入期子宫内膜的表达情况。
3.2临床材料与方法
3.2.1回顾性分析97个接受长方案控制性超排卵(COS)及IVF-ET治疗周期。应用电化学发光技术分别测定hCG日和ET日血清雌二醇(E_2)水平。根据ET日血清雌二醇(E_2)下降幅度分为6组:组1:12个治疗周期,E_2下降幅度＜20%;组2:13个,E_2下降幅度20%～29%;组3:11个周期,E_2下降幅度30%～39%;组4:16个周期,E_2下降幅度40%～49%;组5:24个周期,E_2下降幅度50%～59%;组6:21个周期,E_2下降幅度≥60%。观察ET日血清雌二醇(E_2)下降幅度对胚胎种植率和临床妊娠率的影响。
3.2.2为探讨雌激素添加是否可以改善IVF-ET的结局,回顾性分析216个长方案控制性超排卵IVF-ET周期的治疗结果,根据其黄体期支持用药情况分为两组:A_1组:97个周期,单用黄体酮进行黄体期支持;B_1组:119个周期,对移植日血清雌二醇(E_2)水平下降幅度≥30%的89个周期采用黄体酮加小剂量雌激素进行黄体期支持。
3.2.3为了探讨雌激素添加的剂量及方案,回顾性分析104个长方案控制性促排卵IVF-ET周期的治疗结果,根据其移植日血清E_2下降幅度黄体期支持添加雌激素的剂量分为四组:A_2组:12个周期,单用黄体酮进行黄体期支持;B_2组:18个周期,移植日血清E_2水平较hCG日下降幅度在30%～390,其黄体期支持采用黄体酮加3mg雌激素;C组:16个周期,移植日血清E_2水平下降幅度40%～49%;D组:58个周期,移植日血清E_2水平下降幅度≥50%;对于C组、D组患者采用黄体酮加4mg雌激素进行黄体期支持。
3.2.4对比雌激素添加与传统黄体期支持方案胚胎种植率和临床妊娠率的差异。
3.2.5对比添加不同剂量的雌激素其胚胎种植率和临床妊娠率的差异。
3.3数据处理与统计学分析
采用SPSS11.0统计软件进行分析,数值变量采用t检验或方差分析,分类变量采用卡方检验或精确概率法检验,P＜0.05为差异有统计学意义。
4.结果
4.1动物实验结果
4.1.1子宫内膜胞饮突表达结果:B、C、D组小鼠子宫内膜孕第3d为发育中的胞饮突,孕第4d子宫内膜表面胞饮突完全发育,边界清楚,大小一致,呈蘑菇样外观,5d子宫内膜可见退化的胞饮突;C和D组较好,B组稍次之。A组于孕3d即可见少量完全发育的胞饮突,但凸起较小,大小不均,第4d胞饮突开始退化。
4.1.2子宫内膜LIF蛋白定性表达结果:在C、D组受孕小鼠,孕3d腺上皮和腔上皮LIF蛋白表达水平呈弱阳性着色,孕4d时LIF蛋白表达水平显著上升达峰值,呈强阳性着色(++),孕5d时呈阳性着色(+)。B组孕3d腺上皮和腔上皮呈弱阳性着色,孕4d时,LIF蛋白表达水平呈阳性着色(+),孕5d时呈弱阳性着色。A组孕3d腺上皮和腔上皮LIF蛋白表达水平呈阳性着色(+),孕4d和5d时呈弱阳性着色。
4.1.3子宫内膜LIF蛋白半定量表达结果:图像分析和统计学处理结果显示小鼠C、D组受孕第3、4、5d子宫内膜LIF蛋白的表达水平均明显高于A组(P＜0.05);B组第3、4、5d子宫内膜LIF蛋白的表达水平高于A组(P＜0.05),但明显低于C、D组,差异有统计学意义(P＜0.05)。D组与C组相比无统计学差异。
4.1.4子宫内膜LIF mRNA表达组内比较结果:B、C、D组孕鼠子宫内膜LIFmRNA表达均为阳性,A组孕鼠LIF mRNA的表达极弱。半定量分析表明:组内比:B、C、D组受孕小鼠均于孕4d时LIF蛋白表达水平达峰值,而A组于孕3d腺上皮和腔上皮LIF蛋白表达水平最高,孕4d和5d时较低。
4.1.5 LIF mRNA表达各组间比较结果:各组小鼠子宫内膜LIF mRNA表达半定量分析组间比结果:小鼠C、D组明显高于A组;B组高于A组(P＜0.05),但明显低于C、D组,差异有统计学意义(P＜0.05)。C组与D组相比无统计学差异。
4.1.6 LIF mRNA表达高峰期比较结果:B、C、D组小鼠孕4d与A组孕3天相比,其子宫内膜胞饮突表达、腺上皮和腔上皮LIF蛋白表达、LIF mRNA表达结果:C和D组最好,B组稍次之,A组最差。LIF蛋白图像分析、LIF mRNA半定量分析和统计学处理结果显示:小鼠C、D组明显高于A组,差异有统计学意义(P＜0.05);B组高于A组(P＜0.05),但明显低于C、D组,差异有统计学意义P＜0.05);C组与D组相比无统计学差异。
4.2临床实验结果
4.2.1 ET日血清E_2水平均呈不同程度的下降。
4.2.2当ET日血清E_2水平下降幅度≥30%时胚胎种植率和妊娠率明显降低,差异出现有统计学意义(P＜0.05)。
4.2.3其中下降幅度≥30%者占总周期数的74.23%。
4.2.4年龄大的患者其下降幅度偏大,但无统计学差异。
4.2.5各组参数比较结果:A_1组与B_1组的取卵数目、受精率、卵裂率、人绒毛膜促性腺激素(hCG)日E_2水平、优质胚胎数、内膜厚度和移植胚胎数目相比差异无统计学意义(P＞0.05);A_2组、B_2组、C组、D组的取卵数目、受精率、卵裂率、人绒毛膜促性腺激素(HCG)日E_2水平、优质胚胎数、内膜厚度和移植胚胎数目相比差异无统计学意义(P＞0.05)
4.2.6 A_1组与B_1组结果比较:A_1组当胚胎移植日血清E_2下降幅度≥30%时,胚胎种植率和临床妊娠率下降,差异有统计学意义(P＜0.05);B_1组当E_2下降幅度≥40%时,胚胎种植率和临床妊娠率差异有统计学意义(P＜0.05);说明给予3mg/d戊酸雌二醇补充改善了E_2下降幅度在30～40%的患者,而下降幅度≥40%的患者需较大量的雌激素补充。两组整体相比,B_1组胚胎种植率明显高于A_1组(P＜0.05),妊娠率亦高于A_1组,但无统计学差异(P=0.15)。
4.2.8添加不同剂量雌激素结果比较:根据E_2下降幅度添加不同剂量的雌激素,C组当E_2下降幅度≥40%时,IVF的妊娠率和种植率的差异无统计学意义(P＞0.05):D组当E_2下降幅度≥50%时,IVF的妊娠率和种植率的差异有统计学意义(P＜0.05)。添加戊酸雌二醇3mg/d改善了E_2下降幅度在30～39%的患者胚胎种植率和临床妊娠率;添加戊酸雌二醇4mg/d改善了E_2下降幅度在40～49%的患者的胚胎种植率和临床妊娠率;而添加戊酸雌二醇4mg/d对于E_2下降幅度在≥50%的患者其胚胎种植率和临床妊娠率仍无明显的改善。
5.结论
5.1控制性促排卵使胞饮突、LIF蛋白和LIFmRNA在小鼠子宫内膜的表达减弱,且使子宫内膜着床窗口期提前,从而对子宫内膜容受性有负面影响。
5.2控制性促排卵黄体期给与黄体酮能明显改善小鼠子宫内膜容受性。
5.3控制性促排黄体期支持添加雌激素和单用黄体酮相比,前者更能改善小鼠子宫内膜胞饮突、LIF蛋白和LIFmRNA在小鼠子宫内膜的表达,且纠正了COS使内膜窗口期前移的负面影响,从而增加子宫内膜容受性。
5.4长方案COS及IVF-ET中,ET日E_2水平较HCG日相比有不同程度的下降,当其下降幅度≥30%时引起胚胎种植率和妊娠率的降低。
5.5长方案COS及IVF-ET中,ET日E_2水平下降幅度≥30%者占总周期数的74.23%。
5.6年龄大的患者其ET日E_2下降幅度偏大。
5.7长方案COS及IVF-EF中,当移植日血清E_2水平下降幅度≥30%时黄体期支持给予雌激素补充可以改善胚胎种植率和临床妊娠率。
5.8雌激素补充的剂量要根据E_2下降幅度而定,E_2下降幅度越大补充雌激素的剂量也应增大。
1.Backgroud
The disease incidence of infertility is about 10～15%in women of reproductive age,and it does harm to the harmony of family and society.There are four factors that are essential to the success getting pregnancy,they are sperm and ovum of high-quality, unobstructed fallopian tubes,receptive endometrium.Endometrium is the "soil" for embryo implantation,so it is very important to successful implantation.
The human endometrium becomes receptive to the embryo only for a limited period during the luteal phase of the menstrual cycle,the period during which endometrium is receptive to implantation,termed the implantation window,begins approximately 5-10 days after ovulation and is believed to encompass cycle days 19-24.Numbers of morphological and molecular markers specific to the implantation window has been identified,including endometrial morphological features,pinopodes and a large number of identified molecular mediators under the influence of ovarian hormones.The recent discovery of molecules crucial for successful embryo implantation has offered researchers precious insight into this field,for example, leukaemia inhibitory factor(LIF),integrins,Interleukin(IL)-1,epidermal growth factor(EGF),CSF-1,estradiol receptor,progesterone receptor,et al.
Nowadays,with the development of in vitro fertilization-embryo transfer (IVF-ET)used in infertility,people can achieve high ovulation rates by choosing different super-ovulation protocols.However,implantation rates per embryo transfer is still very low.There are many factors affecting successful implantation,but the endometrial receptivity and embryo quality are two of the crucial factors for successful implantation.A large number of identified molecular mediators,under the influence of ovarian hormones,have been postulated to be involved in this early stage of endometrial receptivity.In IVF-ET cycles,patients undergoing COS became a common practice.Use of GnRH agonists in ovarian stimulation protocols to suppress premature LH surges increased the number of mature oocytes and embryos available for transfer and subsequently improved pregnancy rates.GnRH agonist use,however, has been associated with several undesired effects.Premature luteolysis and inadequate endometrial priming because of oversuppression of pituitary function are some of the unwanted consequences.It is generally accepted that those reduce uterine receptivity and,thus,the chances of conception.
The necessity of luteal-phase support after GnRH agonists has been generally accepted.Luteal-phase support was routinely either in the form of progesterone, despite limited information about the quality of the luteal phase.Thus far,studies comparing different luteal support protocols in women undergoing ovarian stimulation used implantation and pregnancy rates as endpoints.Recently,there are data to support an improvement in implantation and pregnancy rates in the groups that had received a combination of estrogens and progesterone for luteal-phase support, but this is not a common practice by all.Whether this contributes to an improvement in the endometrial receptivity and subsequent implantation and pregnancy rates is unclear.Untill now,there are no study on molecular factors,endometrial morphology and clinical data to find whether estrogen supplementation for luteal-phase support will improve the endometrial receptivity.
2.Objective
In the present study,we evaluate the impact of different types of luteal-phase support after ovarian stimulation with a GnRH agonists protocol on the endometrial receptivity.In view of this,the objectives of the current research were to test the expression regulation of LIF and LIFmRNA、pinopod in peri-implantation mouse endometrium.Then study the effects on their expression as well as human implantation rate by setting up animal model based on different treatment protocols. The patients underwent long protocols for controlled ovarian stimulation for IVF treatment with GnRH agonist suppression were studied retrospectively.To study the effect of the serum estradiol level down-range on the day of embryo transfer in In-Vitro fertilization out- comes and the necessity and the effect of individual estradiol supplementation during the luteal phase in In-vitro fertilization outcome.
In a serial study,we try to evaluate the relationship of LIF,pinopode and implantation,the molecular mechanism of negative effects based on different treatment protocols.These studies provide a new theory basis for optimizing the treatment protocols to improve the In-vitro fertilization outcomes.
These studies are divided into three parts as listed below:
3.Material and methods
3.1 Material and methods of animal experiments
3.1.1 Grouping
Female mice were randomly divided into four groups:group A,group B and group C were controlled ovarian stimulation(COS) groups;Group D was nature cycle group. Female mice in COS groups were rendered pregnant by mating with male mice after HCG.The next morning,the vaginal plugs were examined.Pregnancy day 1 was determined on the vaginal plug day.
3.1.2 Medication program
Female mice in COS groups were divided into three groups according the different types of luteal-phase support.Group A:without hormonal support of the luteal phase in female mice undergoing COS,Group B:with progesterone for luteal-phase-support;Group C:with progesterone and estradiol supplementation for luteal-phase-support.
3.1.3 Tissue samples collection
Tissue samples were collected from pregnant mice on day3,day4,day5 of vaginal plugs day.
3.1.4 scanning electronic microscopy
Pinopodes were investigated by scanning electronic microscopy(SEM) in the uterine endometrial of pregnant mice.
3.1.5 LIF protein was determined by immunohistochemistry in the uterine endometrial of pregnant mice.
3.1.6 The LIF mRNA expression was measured by reverse transcriptional-polymerase chain reaction(RT-PCR) in the uterine endometrial of pregnant mice.
The levels of LIF protein and LIFmRNA in the endometrium were analyzed by semi-quantitative assay.
3.2 Clinieal Material and methods
3.2.1 In this study,97 IVF-ET cycles in long protocol were evaluated retrospectively. Serum estradiol was measured on the day of HCG administration and the day of embryo transfer(ET) by chemiluminescence technique.They were divided into six groups according to the down-range of serum estradiol level at the ET day,group one (＜20%),group two(20%-29%),group three(30%-39%),group four(40%-49%), group five(50%-59%),group six(≥60%).To study the effect of the serum estradiol level down-range on the day of embryo transfer in In-Vitro fertilization outcomes.
3.2.2 To study the necessity of estradiol supplementation,216 long protocol COS and IVF-ET cycles were evaluated retrospectively.They were divided into two groups according the methods of the luteal phase surport.Group A_1 97 cycles received only progesterone as the luteal phase surport.Group B_1 119 cycles received the same dosage of progesterone,combined with oral E_2 3mg/d to the patients who the down-range of serum E_2 level at the ET day were≥30%.
3.2.3 To study the effect of individual estradiol supplementation,104 long protocol COS and IVF-ET cycles were evaluated retrospectively.They were divided into four groups according the methods of the luteal phase surport.Group A_2 12 cycles received only progesterone as the luteal phase surport.Group B_2 18 cycles received the same dosage of progesterone,combined with oral E_2 3mg/d to the patients who the down-range of serum E_2 level at the ET day were 30%-39%.Group C 16 cycles received the same dosage of progesterone combined with oral E_2 4mg/d to the patients who the down-range of serum E_2 level at the ET day were 40%-49%.Group D 58 cycles received the same dosage of progesterone combined with oral E_2 4mg/d to the patients who the down-range of serum E_2 level at the ET day was≥50%.
3.2.4 To compare the embryo implantation rate and clinical pregnancy rate in estradiol supplementation luteal-phase support protocol with classical luteal-phase support protocol.
3.2.5 To compare the embryo implantation rate and clinical pregnancy rate in different estradiol supplementation protocols.
3.3 Statistical analysis
Statistical analyses were performed by running the SPSS11.0 software packages. Student's two-tailed t test was then used to compare the two study groups on normally distributed variables,P values less than 0.05 were used to define statistical significance.
4.Results
4.1 Results of animal experiments
4.1.1 Results of pinopodes in endometrial
In group B、C、D,there were small developing pinopodes in pregnant mouse endometrial surface on day 3;there were large fully developed pinopodes in endometrial surface,which was smooth with well defined borders resembling a mushroom on day 4 of these group;the regressing pinopodes were observed on day 5 of these group.Pinopodes in group D and group C were better than in group B.In group A,there were small developing pinopodes in pregnant mouse endometrial surface on day 3;the regressing pinopodes were seen on day 4 of group A.
4.1.2 Qualitation assay the expression of LIF protein
In the pregnant mouse of group C and D,weak immunostaining of LIF protein in endometrial epithelial cell and glandular epithelial cell was found on day 3 of gestation,strong immunostaining(++) peaks on day 4,and then immunostaining (+)peaks on day 5;in the pregnant mouse of group B,weak immunostaining of LIF protein in endometrial epithelial cell and glandular epithelial cell was found on day 3 of gestation,immunostaining(+)peaks on day 4,and then weak immunostaining on day 5;in the pregnant mouse of group A,weak immunostaining of LIF protein in endometrial epithelial cell and glandular epithelial cell was found on day 3 of gestation,weaker immunostaining on day 4 and day 5.
4.1.3 Semi-quantitative assay of LIF protein
The results of statistical analyses showed that:in the pregnant mouse of group C and D,the level of LIF protein on day 3-5 was significantly higher than group A(P＜0.05);in group B,the level of LIF protein on day 3-5 was significantly higher than group A(P＜0.05),but significantly lower than group C and D(P＜0.05).The expression of LIF was no different between group C and D.
4.1.4 Comparing the expression of LIF mRNA between different days in every group
In group B、C、D,the expression of LIF mRNA in pregnant mice endometrium were positive;the expression of LIF mRNA in pregnant mouse endometrium of group A were very weak.The results of semi-quantitative assay showed:the LIF protein surges of pregnant mouse in group B、C、D wereon day 4;but in pregnant mouse endometrium of group A,the expression of LIF protein was stronger immunostaining on day 3,and weaker immunostaining on day 4 and day 5.
4.1.5 Comparing the expression of LIF mRNA between different groups
The results of semi-quantitative assay of LIF mRNA in pregnant mouse endometrium showed:compared with group A,the level of LIF mRNA was significantly higher in the pregnant mouse of group B、C and D(P＜0.05);compared with group B,the level of LIF mRNA was significantly higher in the pregnant mouse of group C and D(P＜0.05);there were no significantly different between group C and D.
4.1.6 The peak expression of LIF mRNA
The results showed that:the expression ofpinopodes、LIF protein and LIF mRNA in group D and group C were better than in group B on day 4;and compared with COS group the expression of those were the weakest in group A on day 3.The results of semi-quantitative assay and statistical analysis showed:compared with group A,the levels of LIF mRNA and LIF protein were significantly higher in the pregnant mouse of group C and group D(P＜0.05);in group B,the levels of LIF mRNA and LIF protein were higher than in group A(P＜0.05),but were significantly lower than in group C and group D(P＜0.05);there were no significantly different between group C and D.
4.2 Clinical results
4.2.1 The estradiol concentration had the different down level on the day of embryo transfer.
4.2.2 The higher of the down-range of serum E_2 level,the lower of the embryo implantation and pregnancy rate.When the down-range of the estradiol on ET day was more than 30%,the embryo implantation rate and the clinical pregnancy rate were significantly lower(P＜0.05).
4.2.3 The down-range of the estradiol on ET day was more than 30%In 74.23%of all cycles.
4.2.4 The older patients had higher down-range of serum E_2 level,but there were no significantly different.
4.2.5 Comparing the parameters between different groups
There were no differences on average age,endometrium thickness,fertiliz -ation rates,embryo cleavage rates,and quantity or quality of embryos transferred between Group A_1 and Group B_1;there were also no differences among Group A_2、Group B_2、Group C and Group D.
4.2.6 Comparison between group A_1 and group B_1
In Group A_1 when the down-range of E_2 on ET day was more than 30%,the embryo implantation rate and the clinical pregnancy rate were significantly lower (P＜0.05).In Group B_1 when the down-range of E_2 was more than 40%,the embryo implantation rate and the clinical pregnancy rate were also significantly lower (p＜0.05).
4.2.7 Comparison between different doses of estradiol supplementation
Different doses of estradiol supplementation were given according to different down-range of the serum E_2 level,when the down-range of the estradiol on ET day was more than 40%,there were no significant differences on the embryo implantation rate and the clinical pregnancy rate(P＞0.05);when the down-range of the estradiol on ET day was more than 50%,the embryo implantation rate and the clinical pregnancy rate were significantly lower(P＜0.05).
5.Conclusions
5.1 The expression of pinopodes,LIF protein and LIF mRNA are weaker in COS group,and the implantation window opens earlier.
5.2 Luteal-phase support with progesterone could improve the endometrial receptivity greatly in COS group.
5.3 Compared with luteal-phase support with progesterone only,estradiol supplementation could improve the expression of pinopodes,LIF protein and LIF mRNA in mouse endometrium in COS group,and then improve the endometrial receptivity.
5.4 In long protocol COS and IVF-ET cycles,the rapidly decline of the serum estradiol has the important effect to the IVF-ET outcome.When the down-range of the serum estradiol is more than 30%from HCG day to ET day,the embryo implantation rate and clinical pregnancy rate are lower.
5.5 There are 74.23%of all cycles with the down-range of the serum E_2 level more than 30%.
5.6 The older patients have higher down-range of serum E_2 level.
5.7 In long protocol COS and IVF-ET cycles,the estradiol supplementation was necessary during the luteal phase surport to the patients whose down-range of E_2 level on ET day is more than 30%.
5.8 The dose of the estradiol supplementation should be individual based on the down-range of the serum E_2 level.
不孕症(Infertility)发病率约占育龄妇女的10～15%,是影响家庭乃至社会和谐与稳定的重要因素之一。受孕成功的四大因素是:高质量的卵子和精子、通畅的输卵管和良好的子宫内膜,四者缺一不可。其中子宫内膜是胚胎种植的“土壤”,良好的子宫内膜环境是受孕成功的关键。所以,子宫内膜容受性(Endometrialreceptivity)是目前生殖领域研究的热点。
正常子宫内膜仅在一特定时期允许着床,这一时期子宫内膜容受性最高,称为着床窗口期(Window of implantation),一般为排卵后6～10d,即正常月经周期的19～24d。子宫内膜容受性的评价标准包括子宫内膜厚度、形态、母体内分泌状况和子宫内膜的生殖调控因子,包括细胞形态学、分子生物学和基因水平的研究,如:胞饮突(pinopode,pp)、白血病抑制因子(Leukaemia inhibitory factor,LIF)、整合素家族(Integrins)、白细胞介素1(IL-1)、表皮生长因子(EGF)、集落刺激因子(CSF-1)、雌孕激素受体等。
控制性促排卵(Contral Ovarian Stimulation,COS)体外受精-胚胎移植(InVitro Fertilization and Embryo Transfer,IVF-ET)技术已较广泛地用于治疗难治性不孕症,其临床妊娠成功率受诸多因素的影响。如何提高其胚胎种植率和临床妊娠率是一个亟待解决的难题。子宫内膜容受性与胚胎质量是影响IVF-ET成功率的两大主要因素。而胚胎着床前子宫内膜的容受状态受雌、孕激素及生殖调控因子的协同调节。在IVF-ET控制性促排卵中大多数应用促性腺激素释放激素协同剂(Gonadotropicreleasingllormoneagonist,GnRHa)进行降调节,通过抑制内源性的促黄体生成素(Lutenizing Hormone,LH)峰的出现,使多个卵泡募集,获得多个高质量的卵子,与精子结合形成多个胚胎,选择优秀的胚胎进行移植,从而提高妊娠成功率,改善妊娠结局。但是,这种抑制作用可使排卵后黄体期的LH分泌也受到抑制,导致黄体功能不足,胚胎种植率和妊娠率的降低。
对接受控制性促排卵WF-ET患者进行黄体支持已得到大家的公认。传统的黄体支持方法是黄体期应用黄体酮(Progesterone,p),如何改进黄体期支持方案增加子宫内膜容受性以提高胚胎种植率和WF-ET妊娠成功率是近年来生殖领域研究的热点。近来有学者试着在传统方案的基础上添加小剂量雌激素,但只是在初步探索阶段,尚无确切的定论。到目前为止,有关黄体期支持添加雌激素与子宫内膜容受性关系的研究,报道甚少,尚未见运用细胞形态学、免疫组化、分子生物学手段及临床资料统计分析联合证实黄体期添加雌激素对子宫内膜容受性影响的研究。
2.研究目的
为探讨COS黄体期支持添加小剂量雌激素对子宫内膜容受性的影响,本课题首先通过建立动物模型,采用细胞形态学、免疫组化和分子生物学技术,观察不同COS黄体期支持方案小鼠子宫内膜胞饮突、LIF和LIFmRNA的表达,从而总结添加雌激素对小鼠子宫内膜容受性的影响。在此基础上,对临床进行控制性促排卵IVF-ET的患者进行回顾性分析,观察其胚胎移植(Embryo transfer,ET)日血清雌二醇(Estradiol,E_2)水平较注射绒毛膜促性腺激素(hCG)日下降幅度对人胚胎种植率和临床妊娠率的影响,进而对临床接受控制性促排卵IVF-ET患者采用不同方案进行黄体期支持,对各组患者IVF-ET实验室和临床资料进行研究分析,探讨ET日E_2下降幅度对人胚胎种植率和临床妊娠率的影响及黄体期支持添加雌激素的必要性。通过系列研究,深入探讨雌激素与子宫内膜容受性关系的内在分子机制,从而揭示控制性促排卵黄体期支持添加雌激素对子宫内膜容受性的影响,为改进临床治疗方案、提高IVF-ET技术妊娠成功率提供一定的理论依据。
3.材料与方法
3.1动物实验材料与方法
3.1.1分组方法:将正常雌性小鼠随机分为四组:A组、B组和C组均采用控制性促排卵,用hCG后分别与正常雄性小鼠2:1合笼,次日清晨检查阴栓,有阴栓者为受孕第1d;D组:自然周期组,与正常雄性小鼠2:1合笼,次日清晨检查阴栓,有阴栓者为受孕第1d。
3.1.2用药方法:控制性促排卵的雌性小鼠从受孕第1d开始接受不同的黄体期支持方法:A组:不采用黄体期药物支持;B组:单用黄体酮进行黄体期支持;C组:在应用黄体酮的基础上添加雌激素;D组:同时应用同等量的生理盐水。
3.1.3取材:A、B、C、D组小鼠分别于孕第3、4、5d剖腹取子宫。
3.1.4扫描电镜(scanning electronic microscopy,SEM)观察各组受孕小鼠围植入期胞饮突的表达。
3.1.5采用免疫组化(Immunohistochemistry,SP)法检测LIF蛋白在各组受孕小鼠围植入期子宫内膜的表达情况。
3.1.6采用逆转录聚合酶链反应(Reverse transcriptional-polymerise chain reaction,RT-PCR)法检测LIFmRNA在各组受孕小鼠围植入期子宫内膜的表达情况。
3.2临床材料与方法
3.2.1回顾性分析97个接受长方案控制性超排卵(COS)及IVF-ET治疗周期。应用电化学发光技术分别测定hCG日和ET日血清雌二醇(E_2)水平。根据ET日血清雌二醇(E_2)下降幅度分为6组:组1:12个治疗周期,E_2下降幅度＜20%;组2:13个,E_2下降幅度20%～29%;组3:11个周期,E_2下降幅度30%～39%;组4:16个周期,E_2下降幅度40%～49%;组5:24个周期,E_2下降幅度50%～59%;组6:21个周期,E_2下降幅度≥60%。观察ET日血清雌二醇(E_2)下降幅度对胚胎种植率和临床妊娠率的影响。
3.2.2为探讨雌激素添加是否可以改善IVF-ET的结局,回顾性分析216个长方案控制性超排卵IVF-ET周期的治疗结果,根据其黄体期支持用药情况分为两组:A_1组:97个周期,单用黄体酮进行黄体期支持;B_1组:119个周期,对移植日血清雌二醇(E_2)水平下降幅度≥30%的89个周期采用黄体酮加小剂量雌激素进行黄体期支持。
3.2.3为了探讨雌激素添加的剂量及方案,回顾性分析104个长方案控制性促排卵IVF-ET周期的治疗结果,根据其移植日血清E_2下降幅度黄体期支持添加雌激素的剂量分为四组:A_2组:12个周期,单用黄体酮进行黄体期支持;B_2组:18个周期,移植日血清E_2水平较hCG日下降幅度在30%～390,其黄体期支持采用黄体酮加3mg雌激素;C组:16个周期,移植日血清E_2水平下降幅度40%～49%;D组:58个周期,移植日血清E_2水平下降幅度≥50%;对于C组、D组患者采用黄体酮加4mg雌激素进行黄体期支持。
3.2.4对比雌激素添加与传统黄体期支持方案胚胎种植率和临床妊娠率的差异。
3.2.5对比添加不同剂量的雌激素其胚胎种植率和临床妊娠率的差异。
3.3数据处理与统计学分析
采用SPSS11.0统计软件进行分析,数值变量采用t检验或方差分析,分类变量采用卡方检验或精确概率法检验,P＜0.05为差异有统计学意义。
4.结果
4.1动物实验结果
4.1.1子宫内膜胞饮突表达结果:B、C、D组小鼠子宫内膜孕第3d为发育中的胞饮突,孕第4d子宫内膜表面胞饮突完全发育,边界清楚,大小一致,呈蘑菇样外观,5d子宫内膜可见退化的胞饮突;C和D组较好,B组稍次之。A组于孕3d即可见少量完全发育的胞饮突,但凸起较小,大小不均,第4d胞饮突开始退化。
4.1.2子宫内膜LIF蛋白定性表达结果:在C、D组受孕小鼠,孕3d腺上皮和腔上皮LIF蛋白表达水平呈弱阳性着色,孕4d时LIF蛋白表达水平显著上升达峰值,呈强阳性着色(++),孕5d时呈阳性着色(+)。B组孕3d腺上皮和腔上皮呈弱阳性着色,孕4d时,LIF蛋白表达水平呈阳性着色(+),孕5d时呈弱阳性着色。A组孕3d腺上皮和腔上皮LIF蛋白表达水平呈阳性着色(+),孕4d和5d时呈弱阳性着色。
4.1.3子宫内膜LIF蛋白半定量表达结果:图像分析和统计学处理结果显示小鼠C、D组受孕第3、4、5d子宫内膜LIF蛋白的表达水平均明显高于A组(P＜0.05);B组第3、4、5d子宫内膜LIF蛋白的表达水平高于A组(P＜0.05),但明显低于C、D组,差异有统计学意义(P＜0.05)。D组与C组相比无统计学差异。
4.1.4子宫内膜LIF mRNA表达组内比较结果:B、C、D组孕鼠子宫内膜LIFmRNA表达均为阳性,A组孕鼠LIF mRNA的表达极弱。半定量分析表明:组内比:B、C、D组受孕小鼠均于孕4d时LIF蛋白表达水平达峰值,而A组于孕3d腺上皮和腔上皮LIF蛋白表达水平最高,孕4d和5d时较低。
4.1.5 LIF mRNA表达各组间比较结果:各组小鼠子宫内膜LIF mRNA表达半定量分析组间比结果:小鼠C、D组明显高于A组;B组高于A组(P＜0.05),但明显低于C、D组,差异有统计学意义(P＜0.05)。C组与D组相比无统计学差异。
4.1.6 LIF mRNA表达高峰期比较结果:B、C、D组小鼠孕4d与A组孕3天相比,其子宫内膜胞饮突表达、腺上皮和腔上皮LIF蛋白表达、LIF mRNA表达结果:C和D组最好,B组稍次之,A组最差。LIF蛋白图像分析、LIF mRNA半定量分析和统计学处理结果显示:小鼠C、D组明显高于A组,差异有统计学意义(P＜0.05);B组高于A组(P＜0.05),但明显低于C、D组,差异有统计学意义P＜0.05);C组与D组相比无统计学差异。
4.2临床实验结果
4.2.1 ET日血清E_2水平均呈不同程度的下降。
4.2.2当ET日血清E_2水平下降幅度≥30%时胚胎种植率和妊娠率明显降低,差异出现有统计学意义(P＜0.05)。
4.2.3其中下降幅度≥30%者占总周期数的74.23%。
4.2.4年龄大的患者其下降幅度偏大,但无统计学差异。
4.2.5各组参数比较结果:A_1组与B_1组的取卵数目、受精率、卵裂率、人绒毛膜促性腺激素(hCG)日E_2水平、优质胚胎数、内膜厚度和移植胚胎数目相比差异无统计学意义(P＞0.05);A_2组、B_2组、C组、D组的取卵数目、受精率、卵裂率、人绒毛膜促性腺激素(HCG)日E_2水平、优质胚胎数、内膜厚度和移植胚胎数目相比差异无统计学意义(P＞0.05)
4.2.6 A_1组与B_1组结果比较:A_1组当胚胎移植日血清E_2下降幅度≥30%时,胚胎种植率和临床妊娠率下降,差异有统计学意义(P＜0.05);B_1组当E_2下降幅度≥40%时,胚胎种植率和临床妊娠率差异有统计学意义(P＜0.05);说明给予3mg/d戊酸雌二醇补充改善了E_2下降幅度在30～40%的患者,而下降幅度≥40%的患者需较大量的雌激素补充。两组整体相比,B_1组胚胎种植率明显高于A_1组(P＜0.05),妊娠率亦高于A_1组,但无统计学差异(P=0.15)。
4.2.8添加不同剂量雌激素结果比较:根据E_2下降幅度添加不同剂量的雌激素,C组当E_2下降幅度≥40%时,IVF的妊娠率和种植率的差异无统计学意义(P＞0.05):D组当E_2下降幅度≥50%时,IVF的妊娠率和种植率的差异有统计学意义(P＜0.05)。添加戊酸雌二醇3mg/d改善了E_2下降幅度在30～39%的患者胚胎种植率和临床妊娠率;添加戊酸雌二醇4mg/d改善了E_2下降幅度在40～49%的患者的胚胎种植率和临床妊娠率;而添加戊酸雌二醇4mg/d对于E_2下降幅度在≥50%的患者其胚胎种植率和临床妊娠率仍无明显的改善。
5.结论
5.1控制性促排卵使胞饮突、LIF蛋白和LIFmRNA在小鼠子宫内膜的表达减弱,且使子宫内膜着床窗口期提前,从而对子宫内膜容受性有负面影响。
5.2控制性促排卵黄体期给与黄体酮能明显改善小鼠子宫内膜容受性。
5.3控制性促排黄体期支持添加雌激素和单用黄体酮相比,前者更能改善小鼠子宫内膜胞饮突、LIF蛋白和LIFmRNA在小鼠子宫内膜的表达,且纠正了COS使内膜窗口期前移的负面影响,从而增加子宫内膜容受性。
5.4长方案COS及IVF-ET中,ET日E_2水平较HCG日相比有不同程度的下降,当其下降幅度≥30%时引起胚胎种植率和妊娠率的降低。
5.5长方案COS及IVF-ET中,ET日E_2水平下降幅度≥30%者占总周期数的74.23%。
5.6年龄大的患者其ET日E_2下降幅度偏大。
5.7长方案COS及IVF-EF中,当移植日血清E_2水平下降幅度≥30%时黄体期支持给予雌激素补充可以改善胚胎种植率和临床妊娠率。
5.8雌激素补充的剂量要根据E_2下降幅度而定,E_2下降幅度越大补充雌激素的剂量也应增大。
1.Backgroud
The disease incidence of infertility is about 10～15%in women of reproductive age,and it does harm to the harmony of family and society.There are four factors that are essential to the success getting pregnancy,they are sperm and ovum of high-quality, unobstructed fallopian tubes,receptive endometrium.Endometrium is the "soil" for embryo implantation,so it is very important to successful implantation.
The human endometrium becomes receptive to the embryo only for a limited period during the luteal phase of the menstrual cycle,the period during which endometrium is receptive to implantation,termed the implantation window,begins approximately 5-10 days after ovulation and is believed to encompass cycle days 19-24.Numbers of morphological and molecular markers specific to the implantation window has been identified,including endometrial morphological features,pinopodes and a large number of identified molecular mediators under the influence of ovarian hormones.The recent discovery of molecules crucial for successful embryo implantation has offered researchers precious insight into this field,for example, leukaemia inhibitory factor(LIF),integrins,Interleukin(IL)-1,epidermal growth factor(EGF),CSF-1,estradiol receptor,progesterone receptor,et al.
Nowadays,with the development of in vitro fertilization-embryo transfer (IVF-ET)used in infertility,people can achieve high ovulation rates by choosing different super-ovulation protocols.However,implantation rates per embryo transfer is still very low.There are many factors affecting successful implantation,but the endometrial receptivity and embryo quality are two of the crucial factors for successful implantation.A large number of identified molecular mediators,under the influence of ovarian hormones,have been postulated to be involved in this early stage of endometrial receptivity.In IVF-ET cycles,patients undergoing COS became a common practice.Use of GnRH agonists in ovarian stimulation protocols to suppress premature LH surges increased the number of mature oocytes and embryos available for transfer and subsequently improved pregnancy rates.GnRH agonist use,however, has been associated with several undesired effects.Premature luteolysis and inadequate endometrial priming because of oversuppression of pituitary function are some of the unwanted consequences.It is generally accepted that those reduce uterine receptivity and,thus,the chances of conception.
The necessity of luteal-phase support after GnRH agonists has been generally accepted.Luteal-phase support was routinely either in the form of progesterone, despite limited information about the quality of the luteal phase.Thus far,studies comparing different luteal support protocols in women undergoing ovarian stimulation used implantation and pregnancy rates as endpoints.Recently,there are data to support an improvement in implantation and pregnancy rates in the groups that had received a combination of estrogens and progesterone for luteal-phase support, but this is not a common practice by all.Whether this contributes to an improvement in the endometrial receptivity and subsequent implantation and pregnancy rates is unclear.Untill now,there are no study on molecular factors,endometrial morphology and clinical data to find whether estrogen supplementation for luteal-phase support will improve the endometrial receptivity.
2.Objective
In the present study,we evaluate the impact of different types of luteal-phase support after ovarian stimulation with a GnRH agonists protocol on the endometrial receptivity.In view of this,the objectives of the current research were to test the expression regulation of LIF and LIFmRNA、pinopod in peri-implantation mouse endometrium.Then study the effects on their expression as well as human implantation rate by setting up animal model based on different treatment protocols. The patients underwent long protocols for controlled ovarian stimulation for IVF treatment with GnRH agonist suppression were studied retrospectively.To study the effect of the serum estradiol level down-range on the day of embryo transfer in In-Vitro fertilization out- comes and the necessity and the effect of individual estradiol supplementation during the luteal phase in In-vitro fertilization outcome.
In a serial study,we try to evaluate the relationship of LIF,pinopode and implantation,the molecular mechanism of negative effects based on different treatment protocols.These studies provide a new theory basis for optimizing the treatment protocols to improve the In-vitro fertilization outcomes.
These studies are divided into three parts as listed below:
3.Material and methods
3.1 Material and methods of animal experiments
3.1.1 Grouping
Female mice were randomly divided into four groups:group A,group B and group C were controlled ovarian stimulation(COS) groups;Group D was nature cycle group. Female mice in COS groups were rendered pregnant by mating with male mice after HCG.The next morning,the vaginal plugs were examined.Pregnancy day 1 was determined on the vaginal plug day.
3.1.2 Medication program
Female mice in COS groups were divided into three groups according the different types of luteal-phase support.Group A:without hormonal support of the luteal phase in female mice undergoing COS,Group B:with progesterone for luteal-phase-support;Group C:with progesterone and estradiol supplementation for luteal-phase-support.
3.1.3 Tissue samples collection
Tissue samples were collected from pregnant mice on day3,day4,day5 of vaginal plugs day.
3.1.4 scanning electronic microscopy
Pinopodes were investigated by scanning electronic microscopy(SEM) in the uterine endometrial of pregnant mice.
3.1.5 LIF protein was determined by immunohistochemistry in the uterine endometrial of pregnant mice.
3.1.6 The LIF mRNA expression was measured by reverse transcriptional-polymerase chain reaction(RT-PCR) in the uterine endometrial of pregnant mice.
The levels of LIF protein and LIFmRNA in the endometrium were analyzed by semi-quantitative assay.
3.2 Clinieal Material and methods
3.2.1 In this study,97 IVF-ET cycles in long protocol were evaluated retrospectively. Serum estradiol was measured on the day of HCG administration and the day of embryo transfer(ET) by chemiluminescence technique.They were divided into six groups according to the down-range of serum estradiol level at the ET day,group one (＜20%),group two(20%-29%),group three(30%-39%),group four(40%-49%), group five(50%-59%),group six(≥60%).To study the effect of the serum estradiol level down-range on the day of embryo transfer in In-Vitro fertilization outcomes.
3.2.2 To study the necessity of estradiol supplementation,216 long protocol COS and IVF-ET cycles were evaluated retrospectively.They were divided into two groups according the methods of the luteal phase surport.Group A_1 97 cycles received only progesterone as the luteal phase surport.Group B_1 119 cycles received the same dosage of progesterone,combined with oral E_2 3mg/d to the patients who the down-range of serum E_2 level at the ET day were≥30%.
3.2.3 To study the effect of individual estradiol supplementation,104 long protocol COS and IVF-ET cycles were evaluated retrospectively.They were divided into four groups according the methods of the luteal phase surport.Group A_2 12 cycles received only progesterone as the luteal phase surport.Group B_2 18 cycles received the same dosage of progesterone,combined with oral E_2 3mg/d to the patients who the down-range of serum E_2 level at the ET day were 30%-39%.Group C 16 cycles received the same dosage of progesterone combined with oral E_2 4mg/d to the patients who the down-range of serum E_2 level at the ET day were 40%-49%.Group D 58 cycles received the same dosage of progesterone combined with oral E_2 4mg/d to the patients who the down-range of serum E_2 level at the ET day was≥50%.
3.2.4 To compare the embryo implantation rate and clinical pregnancy rate in estradiol supplementation luteal-phase support protocol with classical luteal-phase support protocol.
3.2.5 To compare the embryo implantation rate and clinical pregnancy rate in different estradiol supplementation protocols.
3.3 Statistical analysis
Statistical analyses were performed by running the SPSS11.0 software packages. Student's two-tailed t test was then used to compare the two study groups on normally distributed variables,P values less than 0.05 were used to define statistical significance.
4.Results
4.1 Results of animal experiments
4.1.1 Results of pinopodes in endometrial
In group B、C、D,there were small developing pinopodes in pregnant mouse endometrial surface on day 3;there were large fully developed pinopodes in endometrial surface,which was smooth with well defined borders resembling a mushroom on day 4 of these group;the regressing pinopodes were observed on day 5 of these group.Pinopodes in group D and group C were better than in group B.In group A,there were small developing pinopodes in pregnant mouse endometrial surface on day 3;the regressing pinopodes were seen on day 4 of group A.
4.1.2 Qualitation assay the expression of LIF protein
In the pregnant mouse of group C and D,weak immunostaining of LIF protein in endometrial epithelial cell and glandular epithelial cell was found on day 3 of gestation,strong immunostaining(++) peaks on day 4,and then immunostaining (+)peaks on day 5;in the pregnant mouse of group B,weak immunostaining of LIF protein in endometrial epithelial cell and glandular epithelial cell was found on day 3 of gestation,immunostaining(+)peaks on day 4,and then weak immunostaining on day 5;in the pregnant mouse of group A,weak immunostaining of LIF protein in endometrial epithelial cell and glandular epithelial cell was found on day 3 of gestation,weaker immunostaining on day 4 and day 5.
4.1.3 Semi-quantitative assay of LIF protein
The results of statistical analyses showed that:in the pregnant mouse of group C and D,the level of LIF protein on day 3-5 was significantly higher than group A(P＜0.05);in group B,the level of LIF protein on day 3-5 was significantly higher than group A(P＜0.05),but significantly lower than group C and D(P＜0.05).The expression of LIF was no different between group C and D.
4.1.4 Comparing the expression of LIF mRNA between different days in every group
In group B、C、D,the expression of LIF mRNA in pregnant mice endometrium were positive;the expression of LIF mRNA in pregnant mouse endometrium of group A were very weak.The results of semi-quantitative assay showed:the LIF protein surges of pregnant mouse in group B、C、D wereon day 4;but in pregnant mouse endometrium of group A,the expression of LIF protein was stronger immunostaining on day 3,and weaker immunostaining on day 4 and day 5.
4.1.5 Comparing the expression of LIF mRNA between different groups
The results of semi-quantitative assay of LIF mRNA in pregnant mouse endometrium showed:compared with group A,the level of LIF mRNA was significantly higher in the pregnant mouse of group B、C and D(P＜0.05);compared with group B,the level of LIF mRNA was significantly higher in the pregnant mouse of group C and D(P＜0.05);there were no significantly different between group C and D.
4.1.6 The peak expression of LIF mRNA
The results showed that:the expression ofpinopodes、LIF protein and LIF mRNA in group D and group C were better than in group B on day 4;and compared with COS group the expression of those were the weakest in group A on day 3.The results of semi-quantitative assay and statistical analysis showed:compared with group A,the levels of LIF mRNA and LIF protein were significantly higher in the pregnant mouse of group C and group D(P＜0.05);in group B,the levels of LIF mRNA and LIF protein were higher than in group A(P＜0.05),but were significantly lower than in group C and group D(P＜0.05);there were no significantly different between group C and D.
4.2 Clinical results
4.2.1 The estradiol concentration had the different down level on the day of embryo transfer.
4.2.2 The higher of the down-range of serum E_2 level,the lower of the embryo implantation and pregnancy rate.When the down-range of the estradiol on ET day was more than 30%,the embryo implantation rate and the clinical pregnancy rate were significantly lower(P＜0.05).
4.2.3 The down-range of the estradiol on ET day was more than 30%In 74.23%of all cycles.
4.2.4 The older patients had higher down-range of serum E_2 level,but there were no significantly different.
4.2.5 Comparing the parameters between different groups
There were no differences on average age,endometrium thickness,fertiliz -ation rates,embryo cleavage rates,and quantity or quality of embryos transferred between Group A_1 and Group B_1;there were also no differences among Group A_2、Group B_2、Group C and Group D.
4.2.6 Comparison between group A_1 and group B_1
In Group A_1 when the down-range of E_2 on ET day was more than 30%,the embryo implantation rate and the clinical pregnancy rate were significantly lower (P＜0.05).In Group B_1 when the down-range of E_2 was more than 40%,the embryo implantation rate and the clinical pregnancy rate were also significantly lower (p＜0.05).
4.2.7 Comparison between different doses of estradiol supplementation
Different doses of estradiol supplementation were given according to different down-range of the serum E_2 level,when the down-range of the estradiol on ET day was more than 40%,there were no significant differences on the embryo implantation rate and the clinical pregnancy rate(P＞0.05);when the down-range of the estradiol on ET day was more than 50%,the embryo implantation rate and the clinical pregnancy rate were significantly lower(P＜0.05).
5.Conclusions
5.1 The expression of pinopodes,LIF protein and LIF mRNA are weaker in COS group,and the implantation window opens earlier.
5.2 Luteal-phase support with progesterone could improve the endometrial receptivity greatly in COS group.
5.3 Compared with luteal-phase support with progesterone only,estradiol supplementation could improve the expression of pinopodes,LIF protein and LIF mRNA in mouse endometrium in COS group,and then improve the endometrial receptivity.
5.4 In long protocol COS and IVF-ET cycles,the rapidly decline of the serum estradiol has the important effect to the IVF-ET outcome.When the down-range of the serum estradiol is more than 30%from HCG day to ET day,the embryo implantation rate and clinical pregnancy rate are lower.
5.5 There are 74.23%of all cycles with the down-range of the serum E_2 level more than 30%.
5.6 The older patients have higher down-range of serum E_2 level.
5.7 In long protocol COS and IVF-ET cycles,the estradiol supplementation was necessary during the luteal phase surport to the patients whose down-range of E_2 level on ET day is more than 30%.
5.8 The dose of the estradiol supplementation should be individual based on the down-range of the serum E_2 level.
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