摘要
目的：体外受精-胚胎移植（IVF-ET）是二十世纪七十年代发展起来的一项辅助生育技术（ART），其一般程序包括：控制促超排卵（COH）、取卵、体外受精（IVF）、胚胎培养、胚胎移植(ET)和黄体支持等步骤。COH是指在进行IVF或卵胞浆内单精子注射（ICSI）中以药物为手段在可控制的范围内诱发多卵泡发育，目的是期望获得较多高质量的卵子，以提高妊娠率。但由于：1、COH方案所产生卵泡期高雌二醇（E2）水平将有可能导致黄体期缩短；2、在取卵时卵泡抽吸或冲洗使部分颗粒细胞层丢失，亦可能影响黄体功能；3、当采用GnRH-a与FSH／HMG联合方案超排卵时，由于垂体受抑制，在短期内未能恢复促性腺激素（Gn）的分泌，所以移植后需要黄体支持。由于IVF-ET过程中大量外源性促性腺激素和性激素的使用可直接或间接影响体内的激素平衡，改变子宫内膜环境，干扰子宫内膜正常发育和与胚胎发育的同步性，从而可能影响胚泡着床和进一步发育。
表皮生长因子（EGF）是近年来发现的一种重要细胞生长因子，体内许多细胞，均可产生EGF，但胎盘滋养层细胞不能分泌EGF而存在EGF受体（EGFR），EGF可通过与靶细胞上的EGFR结合，刺激受体自身酪氨酸激酶并引发自身和底物磷酸化反应，从而促进胎盘细胞分化增殖、增加绒毛的营养摄入、促进滋养细胞合体化及增加胎盘的分泌功能，
并在胚胎和胎盘的植入中发挥作用。
孕妇、胎盘、胎儿三方面间功能有着复杂的关系，导致胎儿发育异常的母体或胎儿因素往往可反映在胎盘的形态、胎盘组织光镜与电镜的体视学上。随着这项辅助生育技术的迅速发展，人们对IVF-ET围产结局日益关注。但目前对IVF-ET足月妊娠胎盘形态学及免疫组织学的研究尚未见报道。本研究旨在通过比较IVF-ET足月妊娠胎盘与正常足月妊娠胎盘中EGFR的表达及胎盘形态学的差异，为IVF-ET围产结局的研究提供理论依据并拓宽新思路。
方法：
1 标本来源：取IVF-ET足月分娩的胎盘组织标本30例为研究组，并以30例相同孕周自然妊娠分娩的胎盘组织标本为对照组，两组患者年龄、孕周、身高、体重、胎数、分娩方式均无统计学差异。除外妊娠高血压综合症、妊娠合并贫血、双胎、三胎及其他慢性疾病者。
2 标本采集与处理：
2.1 于胎盘娩出后，立即在胎盘母体面底板区的中间带和中央带不同部位随机取出3块组织，在4%戊二醛固定液中切成1mm3的组织块，经固定→脱水→渗透→包埋后用Leica超薄切片机切片，经醋酸双氧铀、硝酸铅双重电子染色后在日立H-7500型透射电镜下观察及拍照；
2.2 同时在胎盘母体面垂直取下约1cm3大小的3块组织，于4%中性福尔马林液中固定24~48小时后，常规石蜡包埋。5μm厚度连续切片，分别进行HE染色和免疫组织化学（IHC）染色；
2.3 胎盘取材后，自胎盘脐带根部剪除脐带后 称取胎
盘重量，并记录新生儿体重。
3 应用免疫组织化学SP法检测各组胎盘中EGFR的表达强度，通过真彩色病理图像分析系统对各组HE染色结果进行定量检测，在电镜下观察各组胎盘的超微结构。
结果：
1 免疫组化结果：EGFR主要表达于胎盘的合体滋养细胞(ST)的胞膜和胞浆中，染色强度以刷状缘最强，IVF-ET组胎盘EGFR染色强度较正常对照组增强，有统计学差异（P<0.05）。
2 真彩色病理图像分析结果: IVF-ET组与对照组比较，不仅胎盘末梢绒毛血管数减少，血管占绒毛横切面积比减小，有统计学差异（P<0.05），而且IVF-ET组胎盘绒毛面密度降低，有显著性差异（P<0.01）。
3 电镜结果：
正常胎盘绒毛的超微结构：绒毛各层结构清晰，合体滋养层细胞位于绒毛浅层，具有多核，核的异染色质粗大，胞质中有很多大小不等大空泡，呈泡沫状，可见较丰富的粗面内质网、游离核糖体。合体细胞的游离面有排列整齐的微绒毛，尖端为球形膨大，并有分支，在微绒毛之间有胞膜内陷形成的小凹，合体细胞与细胞滋养细胞之间有桥粒连接。细胞滋养细胞呈立方形或多边形，位于绒毛深层，胞质内线粒体大而多，核内异染色质少而分散，电子密度低，绒毛间质可见丰富的毛细血管，壁薄，腔内可见胎儿红细胞。
IVF-ET组胎盘绒毛的超微结构：合体滋养细胞游离面微绒毛疏密不均，排列紊乱、融合、变形甚至缺失。细胞滋养细胞胞质内线粒体减少，体积小，游离核糖体减少。毛细血
管壁增厚，腔窄，血管内皮细胞胞质内线粒体肿胀，嵴排列紊乱，甚至部分或全部嵴消失，吞饮小泡数量减少。间质内可见胶原纤维增生，成纤维细胞内粗面内质网减少，高尔基复合体不发达，线粒体小而少。
4 IVF-ET组胎盘重量和新生儿体重与正常对照组比较无显著性差异(P>0.05)，新生儿出生体重与胎盘重量呈高度相关性（r=0.709, P<0.01）。
结论：
本文通过采用免疫组织化学SP法，病理真彩色图像分析系统，电镜对IVF-ET妊娠胎盘和正常妊娠胎盘进行比较，发现：
1 IVF-ET组胎盘中EGFR染色强度较正常对照组增强，差异有显著性（P<0.05）。原因可能与COH药物作用或黄体支持中母体血中超生理剂量孕酮的作用有关；
2 IVF-ET组胎盘绒毛较正常组胎盘绒毛发育不良、迟缓。由于IVF-ET过程中体内各种激素、细胞因子之间平衡紊乱，所以即使IVF-ET胎盘中EGFR表达增加，但?
Objectives: In vitro fertilization-embryo transfer (IVF-ET) was a kind of assisted reproductive technology (ART) developed from 1970s. Its general process included controlled ovarian hyperstimulation (COH), oocyte retrieval, in vitro fertilization (IVF), embryo culture, embryo transfer, luetal phase support and et al. COH was to evoke multifollicular development under the controllable range through drug when IVF or intracytoplasmic sperm injection (ICSI). The objective was to obtain more high quality ovum and to increase the pregnancy rate. But the reason of luetal phase support after ET may be as followings: 1. High level of estradiol in follicular phase might shorten luteal phase. 2. Follicle suction or clusis made granulosa luteal cells lose. 3. GnRH-a restrained the function of hypophysis, and gonadotropic hormone excretion couldn’t recover in short-term. Thus the progesterone supplement was necessary after ET. Ectogenesis gonadotropic hormone and sex hormone influenced the hormone balance inside the body directly or indirectly, which altered the environment of endometrium, confused the synchronism
between endometrium and embryogenesis, and influenced the imbed and growth of the blastocyst.
Epidermal growth factor (EGF) had been identified as an important growth factor recently. Lots of cells could excrete EGF inside the body except blastocyst trophoblastic cell, but on which existed epidermal growth factor receptor (EGFR). EGF combined EGFR on its target cells, which could accelerate placental cell to differentiate, proliferate, intake nutrition, enhance placental excretion function, and produce a significant effect on embryo and placent implantation.
There was a complicated relation among gravida, placent and fetus. With the fast development of ART, the perinatal outcome of pregnancy after IVF was paid more and more attention. The aim of this study was to compare the expression of EGFR on placenta and reveal the change of placental morphology between the term pregnancy after IVF-ET and the normal term pregnancy, through which to provide theory basement and broaden a new idea for the perinatal outcome of pregnancy after IVF-ET.
Methods:
1 Sample’s source: 30 cases of term labour after IVF-ET and 30 cases of natural labour as control group with identical weeks of gestation were observed in this study. There was no statistical difference among age, height, body weigh, foetus number and delivery mod, and all the cases should be excluded pregnancy-induced hypertension syndrome, anemia, twins,
triplets and other chronic diseases.
2 Sample’s collection and disposal:
2.1 After placent expulsed, take out 3 blocks of constitution immediately and randomly from intermediate zone and central zone of the floor plate of placental maternal surface in 4% glutaral fixation fluid; through fixation→desiccation→osmos→embed, slice with Leica ultramicrotome; double electron stain with acetic acid uranyl and lead nitrate; observe and take photographs under Hitachi-7500 transmission electron microscope.
2.2 Simultaneously cut 3 blocks constitution vertically from placental maternal surface, after fixed in 4% neutrality formalin for 24~48 hours, embedded in paraffin, 5μm serial section, stained by hematoxylin-eosin (HE) and immunohistochemistry (IHC).
2.3 After shearing umbilical cord from its root, weigh the placent and neonate.
3 Detect the expression and distribution of EGFR on the placenta in each group through immunohistochemistry SP method. Quantitate the result of HE stain through the true color patholoty image analysis system. Observe the placental ultrastructure of each group under the electron microscope.
Results:
1 The expression of EGFR was predominantly in syncy- tiotrophoblast cell’s membrane and plasm, and the intensity of stain was stronger on brush border. Compared with the control
group: the expression of EGFR in the group of IVF-ET was stronger, and the difference was significant (P<0.05).
2 Compared with the control group, the number of blood vessels in the end
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