体外受精—胚胎移植过程中卵泡液血小板激活因子的研究

发布时间：2018-05-25 07:45

本文选题：PAF + 卵泡液　；参考：《河北医科大学》2014年硕士论文

【摘要】：研究目的：通过体外受精-胚胎移植(In Vitro Fertilization embryoTransfer, IVF-ET)助孕的患者卵泡液中血小板激活因子(Platelet ActivatingFactor, PAF)水平及其与年龄、体重指数(Body mass index, BMI)、双侧卵巢窦卵泡数(Antral follicle count, AFC)、性激素及实验室参数的关系研究，以此来评价PAF在卵子发育、受精、胚胎发育中的作用及对妊娠结局的影响，为寻找一种能够客观、准确评价卵子质量及胚胎发育潜能的新方法提供理论依据。材料和方法：选择2013年7月1日~2013年8月31日于河北医科大学第四医院生殖中心首次接受IVF-ET（包括常规IVF和卵泡浆内单精子注射Intracytoplasmicsperm injection, ICSI）并在本周期移植患者共162例，作为研究对象，并排除女性既往患有结核、子宫内膜异位症及宫腔病变，近3个月未应用过激素类药物，既往无自然流产史，夫妻双方染色体正常，未发生卵巢过度刺激综合征（Ovarian hyperstimulation syndrome, OHSS）等。根据患者的情况选择标准长方案或微刺激方案进行控制性卵巢刺激。根据患者采集的精液情况选择常规IVF或ICSI方法授精。取卵后48h、72h观察胚胎发育情况。优质胚胎标准：取卵后72h胚胎细胞数≥7且≤12个，卵裂球大小均匀或略不均匀，碎片≤20%的胚胎为优质胚胎。为了研究卵泡液PAF水平与年龄、BMI、基础卵泡刺激素（Basalfollicle-Stimulating Hormone, bFSH）、基础卵泡刺激素/基础黄体生成素（Basal luteinizing hormone, bLH）、AFC、注射绒毛膜促性腺激素(Humanchorionic gonadotropin, HCG)日血清雌二醇(Estradiol, E2)和孕酮(Progesterone, P)、获卵数的关系，将162例患者以35岁为临界，分析年龄≤35岁和年龄＞35岁患者卵泡液中PAF水平的差异；以BMI=25kg/m2为临界，分析BMI＜25kg/m2和BMI≥25kg/m2患者卵泡液中PAF水平的差异；以bFSH=10IU/L为临界，分析bFSH≤10IU/L和bFSH＞10IU/L患者卵泡液中PAF水平的差异；以bFSH/bLH=2为临界，分析bFSH/bLH≤2和bFSH/bLH＞2患者卵泡液中PAF水平的差异；以AFC=7个为临界，分析AFC≤7个和AFC＞7个患者卵泡液中PAF水平的差异；以HCG日血清E2=1000pg/ml为临界，分析HCG日血清E2≤1000pg/ml和HCG日血清E2＞1000pg/ml患者卵泡液中PAF水平的差异；以HCG日血清P=2.6ng/ml为临界，分析HCG日血清P≤2.6ng/ml和HCG日血清P＞2.6ng/ml患者卵泡液中PAF水平的差异；以获卵数=4个为临界，分析获卵数≤4个和获卵数＞4个患者卵泡液中PAF水平的差异。为了进一步研究卵泡液中PAF水平对相同条件人群卵子质量及胚胎发育的影响，将同时满足非多囊卵巢综合症（Polycystic ovary syndrome,PCOS)、年龄≤35岁、BMI＜25kg/m2、AFC＞7个及应用标准长方案的68例移植患者，按卵泡液中PAF水平分为三组：①P AF≤5.9ng/ml组（17例），②5.9＜PAF＜11ng/ml组（34例），③P AF≥11ng/ml组（17例），在三组的年龄、不孕年限、BMI等基础情况及Gn天数、Gn用量、获卵数及移植日子宫内膜厚度等COS情况均处于同一水平的前提下比较三组患者受精率、双原核(Pronuclear,2PN)形成率、优胚率、植入率及妊娠率的差异。应用电化学发光法(Electro-Chemiluminescence Immuno assay, ECLIA)检测上述患者血清中的E2、P、LH、FSH及HCG水平；应用酶联免疫吸附法(Enzyme-linked immunosorbent assay, ELISA)检测患者取卵日第一个穿刺的成熟卵泡液的PAF水平。统计学处理：数据统计分析采用SPSS13.0版软件包，计量资料结果以均数±标准差(x±s)表示，比较采用t检验、方差分析、秩和检验、x2检验，相关性分析采用pearson及spearman相关分析。P＜0.05为差异有统计学意义。结果: 1年龄＞35岁患者卵泡液中PAF水平明显低于年龄≤35岁患者（P＜0.05）。 2BMI＜25kg/m2和BMI≥25kg/m2患者之间的卵泡液中PAF水平，无统计学差异（P＞0.05）。 3bFSH≤10IU/L和bFSH＞10IU/L患者之间的卵泡液PAF水平无统计学差异（P＞0.05）。 4bFSH/bLH≤2患者卵泡液中PAF水平高于bFSH/bLH＞2患者，但无统计学差异（P＞0.05）。 5AFC≤7个和AFC＞7个患者卵泡液中PAF水平无统计学差异（P＞0.05）。 6HCG日血清E2水平＞1000pg/ml患者卵泡液PAF水平高于HCG日血清E2≤1000pg/ml，但无统计学差异（P＞0.05）。 7HCG日血清P≤2.6ng/ml和HCG日血清P＞2.6ng/ml患者卵泡液中PAF水平无统计学差异(P＞0.05)。 8获卵数≤4个患者卵泡液中PAF水平低于获卵数＞4个患者，但无统计学差异(P＞0.05)。 9卵泡液中PAF水平与年龄、BMI、bFSH、bFSH/bLH、AFC、HCG日血清E2和P水平、获卵数的相关性分析：卵泡液中PAF水平与年龄呈明显负相关（r=-0.197，P＜0.05）；PAF与BMI(r=0.006，P＞0.05)、bFSH(r=0.097，P＞0.05)、bFSH/bLH(r=0.039，P＞0.05)、AFC(r=0.098，P＞0.05)、HCG日血清E2水平(r=-0.101，P＞0.05)、HCG日血清P水平(r=0.066，P＞0.05)、获卵数(r=0.015，P＞0.05)无明显相关性，但发现HCG日血清E2以1000pg/ml为界，PAF与之有不同相关性，当HCG日血清E2≤1000pg/ml时，卵泡液中PAF水平与之呈明显正相关（r=0.567，P＜0.05）；而HCG日血清E2水平＞1000pg/ml时，卵泡液中PAF与之呈明显负相关（r=-0.242，P＜0.05）。 10年龄、不孕年限、BMI等基础情况及应用促性腺激（Gonadotrophin,Gn)天数、Gn用量、获卵数及移植日子宫内膜厚度等COS情况均无统计学差异的68例移植患者按卵泡液中PAF水平分为PAF≤5.9ng/ml、5.9＜PAF＜11ng/ml和PAF≥11ng/ml三组。三组的受精率和植入率随着PAF水平的升高而升高，但无统计学差异（P＞0.05）。 11三组的2PN形成率有统计学差异，PAF≥11ng/ml组患者的2PN形成率在三组中最高，为71.16%（P＜0.05），且明显高于PAF≤5.9ng/ml组的59.81%，两组比较有统计学差异（P＜0.05），而PAF≥11ng/ml组和5.9＜PAF＜11ng/ml组、PAF≤5.9ng/ml组和5.9＜PAF＜11ng/ml组比较无统计学差异（P＞0.05）。 12三组的优胚率有统计学差异，PAF≥11ng/ml组患者的优胚率在三组中最高，为64.96%（P＜0.05），明显高于PAF≤5.9ng/ml组的46.09%，两组比较有统计学差异（P＜0.05），也明显高于5.9＜PAF＜11ng/ml组的50.63%，两组比较有统计学差异（P＜0.05），但PAF≤5.9ng/ml组和5.9＜PAF＜11ng/ml组比较无统计学差异（P＞0.05）。 13三组的妊娠率有统计学差异，PAF≥11ng/ml组患者的妊娠率在三组中最高，为70.59%（P＜0.05），且明显高于PAF≤5.9ng/ml组的23.53%，两组比较有统计学差异（P＜0.05），而PAF≥11ng/ml组和5.9＜PAF＜11ng/ml组、PAF≤5.9ng/ml组和5.9＜PAF＜11ng/ml组比较无统计学差异（P＞0.05）。结论：PAF是可以促进卵子发育的细胞因子，卵泡液中PAF水平受年龄影响。卵巢功能正常的患者取卵日卵泡液PAF水平较高。HCG日血清不同的E2水平对卵泡液PAF水平有不同影响，PAF不仅可以促进颗粒细胞E2的分泌，E2过高会导致PAF水平的下降，在IVF助孕过程中，，要适度控制COS程度，减少过高E2产生及获卵数的过多，从而改善IVF结局。取卵日卵泡液PAF水平可以作为预测妊娠结局的指标，也可以作为一种评价卵子质量及胚胎发育潜能的新方法。
[Abstract]:Objective: the level of platelet activating factor (Platelet ActivatingFactor, PAF) in follicular fluid of patients with In Vitro Fertilization embryoTransfer (IVF-ET) and its age, body mass index (Body mass index, BMI), double ovarian follicles and ovarian follicles, sex hormones and reality In order to evaluate the effect of PAF on the development of egg, fertilization, embryo development and the effect on the outcome of pregnancy, this study provides a theoretical basis for finding a new method that can objectively and accurately evaluate the quality of egg and the potential of embryonic development.
Materials and methods:
For the first time in the reproductive center of the fourth hospital of Hebei Medical University, July 1, 2013 ~2013, IVF-ET (including Intracytoplasmicsperm injection, ICSI) in the routine IVF and follicle syrup were given for the first time, and 162 cases were transplanted in this period. In the last 3 months, no hormone drugs have been used. There was no history of spontaneous abortion. The chromosomes of both husband and wife were normal, and no ovarian hyperstimulation syndrome (Ovarian hyperstimulation syndrome, OHSS) occurred.
According to the patient's condition, the control ovarian stimulation was selected by the standard long prescription or microstimulus. According to the condition of the semen collected by the patient, the conventional IVF or ICSI method was selected. The embryo development was observed by 48h and 72h after the egg. The standard of high quality embryos: the number of 72h embryos after the oocyte was more than 7 and less than 12, and the size of the blastomere was uniform or slightly uneven. The embryo of less than 20% of the fragment is a good embryo.
In order to study the PAF level and age of follicular fluid, BMI, basal follicle stimulating hormone (Basalfollicle-Stimulating Hormone, bFSH), basal follicle stimulating hormone / basal luteinizing hormone (Basal luteinizing hormone, bLH), AFC, and serum estradiol (Humanchorionic gonadotropin) and progesterone (Humanchorionic gonadotropin). Sterone, P), the relationship between the number of eggs was obtained. The difference in the level of PAF in follicular fluid of age < 35 years and age > 35 years old was analyzed in 162 patients at the age of 35 years. The difference of PAF levels in the follicle fluid of BMI < 25kg/m2 and BMI > 25kg/m2 patients was analyzed with BMI=25kg/m2 as critical. The difference in the level of PAF in the follicular fluid of the patients and the difference in the level of PAF in the follicular fluid of bFSH/bLH < 2 and bFSH/bLH > 2 were analyzed with bFSH/bLH=2 as critical. The difference of PAF levels in the follicular fluid of AFC less than 7 and AFC > 7 were analyzed with AFC=7 as critical. The difference in the level of PAF in follicular fluid of patients with E2 > 1000pg/ml in daily serum; the difference between HCG day serum P < 2.6ng/ml and HCG day serum P > 2.6ng/ml patient's PAF level was analyzed with HCG day serum P=2.6ng/ml, and the difference between the number of ovum number less than 4 and the number of ovum more than 4 patients was analyzed.
In order to further study the effect of PAF level in follicular fluid on oocyte quality and embryo development in the same condition population, it will meet non polycystic ovarian syndrome (Polycystic ovary syndrome, PCOS), age less than 35 years old, BMI < 25kg/m2, AFC > 7 and 68 cases of applied standard long prescription, divided into three groups according to the level of PAF in the follicular fluid: (1) P AF < 5.9ng/ml group (17 cases), 5.9 < PAF < 11ng/ml group (34 cases), P AF > 11ng/ml group (17 cases). In the three groups of age, infertile years, BMI and other basic conditions and Gn days, Gn dosage, the number of eggs and the thickness of the endometrium of the transplant day were at the same level and compared the fertilization rate of the three groups. The difference in the rate of adult, the rate of optimal embryo, the rate of implantation and the rate of pregnancy.
Electro-Chemiluminescence Immuno assay (ECLIA) was used to detect the levels of E2, P, LH, FSH and HCG in the serum of the above patients. The level of the mature follicle fluid was detected by enzyme linked immunosorbent assay (Enzyme-linked immunosorbent assay).
Statistical analysis: SPSS13.0 software package was used for statistical analysis of data, and the results of measurement data were expressed with mean standard deviation (x + s), and t test, variance analysis, rank sum test, x2 test, and correlation analysis with Pearson and Spearman correlation analysis.P < 0.05 were statistically significant.
Result:
1 the level of PAF in follicular fluid of patients aged 35 to 35 was significantly lower than that of patients younger than 35 years old (P < 0.05).
PAF levels in follicular fluid between patients with 2BMI < 25kg/m2 and BMI > 25kg/m2 were not significantly different (P > 0.05).
There was no significant difference in PAF level of follicular fluid between patients with 3bFSH < 10IU/L and bFSH > 10IU/L (P > 0.05).
The level of PAF in follicular fluid of patients with 4bFSH/bLH < 2 was higher than that of bFSH/bLH > 2, but there was no significant difference (P > 0.05).
PAF levels in follicular fluid of patients with 5AFC < 7 and AFC > 7 patients were not significantly different (P > 0.05).
The level of serum E2 in patients with 6HCG > 1000pg/ml > PAF level was higher than that in HCG day serum E2 < 1000pg/ml, but there was no significant difference (P > 0.05).
There was no significant difference in PAF level in follicular fluid of patients with serum P < 2.6ng/ml and HCG days in serum 7HCG > 2.6ng/ml on day 7HCG (P > 0.05).
8 the level of PAF in follicular fluid was lower than that in 4 patients, but there was no significant difference between the 4 patients (P > 0.05).
9 the level of PAF in follicular fluid and age, BMI, bFSH, bFSH/bLH, AFC, HCG day levels of serum E2 and P, and the correlation analysis of the number of eggs obtained: the level of PAF in the follicle fluid was negatively correlated with age (r=-0.197, P < 0.05); (r=-0.101, P > 0.05), the level of serum P (r=0.066, P > 0.05) on HCG day, the number of acquired eggs (r=0.015, P > 0.05) was not significantly correlated, but it was found that HCG day serum E2 was bounded by 1000pg/ml, and there was a different correlation between PAF and it. 1000pg/ml showed a negative correlation with PAF in follicular fluid (r=-0.242, P < 0.05).
The 10 age, infertile years, BMI and other basic conditions as well as the use of gonadotropin (Gonadotrophin, Gn) days, the amount of Gn, the number of eggs and the thickness of the endometrium in the transplantation day were not statistically different in the 68 cases of transplant patients, which were divided into PAF < 5.9ng/ml, 5.9 < PAF < 11ng/ml and PAF > 11ng/ml three. Three groups of fertilization rate and planting. The intake rate increased with the increase of PAF level, but there was no significant difference (P > 0.05).
11 the formation rate of 2PN in the three groups was statistically different. The 2PN formation rate in the group of PAF > 11ng/ml was the highest in the three groups, 71.16% (P < 0.05), and significantly higher than that in the PAF < 5.9ng/ml group. The two groups were statistically different (P < 0.05), and PAF > 11ng/ml group and 5.9 < PAF < 11ng/ml group. 5.9 Statistical difference (P > 0.05).
12 the optimal embryo rate in the three groups was statistically different. The best embryo rate in the PAF group was the highest in the three groups, 64.96% (P < 0.05), which was significantly higher than the 46.09% in the PAF < 5.9ng/ml group. The two groups were statistically different (P < 0.05), and were significantly higher than 50.63% in the 5.9 < PAF < 11ng/ml group. There was a statistically significant difference between the group two (P < 0.05), but PAF < < 5.9ng/ml. There was no significant difference between the group and 5.9 < PAF < 11ng/ml group (P > 0.05).
13 the pregnancy rate in the three groups was statistically different. The pregnancy rate in the group PAF > 11ng/ml was the highest in the three groups, 70.59% (P < 0.05), and significantly higher than that in the PAF group (23.53%). The two groups were statistically different (P < 0.05), while PAF > 11ng/ml and 5.9 PAF < 11ng/ml, PAF < 5.9ng/ml group and 5.9 < 5.9 Differences in learning (P > 0.05).
Conclusion: PAF is a cytokine that can promote the development of egg. The level of PAF in follicular fluid is influenced by age. The level of PAF in the follicle fluid of the ovarian follicle fluid is higher on.HCG day and the level of E2 has different effects on the level of PAF in follicular fluid. PAF can not only promote the secretion of granular E2, but the high E2 will lead to the decrease of PAF level. In the process of IVF pregnancy, it is necessary to control the degree of COS moderately, reduce the excessive E2 production and the excessive number of eggs, thus improve the IVF outcome. The PAF level of the egg day follicle can be used as an index for predicting the outcome of pregnancy, and can also be used as a new method to evaluate the quality of egg and the potential of embryonic development.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R714.8

【参考文献】