【作者】 李健；

【导师】 王自能；

【作者基本信息】 暨南大学 ， 妇产科学， 2012， 博士

【摘要】 第一章正常妊娠孕晚期糖皮质激素与胎儿发育的关系目的：研究正常妊娠孕晚期母胎循环糖皮质激素(glucocorticoid，GC)与胎儿出生体重及早、中及晚期B超下描记的胎儿各种人体测量学指标之间的关系。方法：随机选取432例正常妊娠为研究对象，用化学发光法测定脐血及母血血清皮质醇浓度，用B超描记早、中及晚期胎儿各种人体测量学指标。结果：妊娠时期B超测量时间：晚期：孕267.61±14.1天；中期：孕160.17±16.12天；早期：孕89.95±7.31天。母体血清皮质醇897.46±342.77nmol/L,胎儿血清皮质醇229.67±139.04nmol/L，两者比值为4.97±2.98。两因素相关分析显示母血血清皮质醇与下列B超下胎儿人体测量学指标呈负相关：晚期双顶径(R~2=0.028, p=0.001),晚期头围(R~2=0.045, p<0.001),晚期胸横径(R~2=0.03, p=0.001),晚期腹横径(R~2=0.016, p=0.013),晚期腹围(R~2=0.025, p=0.002)及晚期股骨长(R~2=0.027, p=0.001)。在考虑B超测量时间、孕前孕妇体重指数（body mass index，BMI）、孕妇分娩体重、胎儿性别、皮质醇测量时孕妇子宫收缩状态及皮质醇测量时间为混杂因素的情况下，多因素回归分析显示母血血清皮质醇与下列B超下胎儿人体测量学指标呈负相关：晚期双顶径(R~2=0.512, p=0.009),晚期头围(R~2=0.498, p=0.001),中期双顶径(R~2=0.819, p=0.013),中期小脑横径（R~2=0.76, p=0.014)及早期双顶径(R~2=0.789, p=0.008)。而其他的B超下胎儿人体测量学指标及胎儿出生体重与母血血清皮质醇不相关。胎儿血清皮质醇与B超下胎儿人体测量学指标及胎儿出生体重不相关。结论：研究显示正常妊娠生理浓度变化的母血皮质醇与胎儿头部发育呈负相关，而不与胎儿出生体重相关。这一现象提示母血皮质醇可能影响胎儿大脑发育，而这可能与胎儿成年期不良神经内分泌结局有关。第二章ABCB1基因多态性与胎儿发育及母胎界面糖皮质激素转运的关系第一节ABCB1基因多态性与胎儿发育的关系目的：探讨正常妊娠ABCB1基因多态性与胎儿发育的关系。方法：用聚合酶链式反应-限制性片段长度多态性（PCR-RFLP）方法检测正常妊娠262例脐血及142例母血ABCB1/C3435T位点基因多态性，B超测量方法同第一章。结果：ABCB1/C3435T基因多态性CC、CT和TT基因型频率脐血组分别为32.44%、53.82%和13.74%；母血组分别为35.91%、50.70%和13.38%，两组差异无显著性(P>0.05),两组研究群体的等位基因发生频率均符合Hardy-Weinberg平衡(P>0.05)。母体ABCB1/C3435T基因型与B超下胎儿人体测量学指标及胎儿出生体重不相关。然而胎儿TT型出生体重为3176.39±436.30g,胎儿CC+CT型出生体重为3345.04±404.61g(p=0.022)。经校正分娩孕周、孕前孕妇BMI、孕妇分娩体重及胎儿性别相关混杂因素后，差异仍然显著(p=0.009)。在考虑B超测量时间、孕前孕妇BMI、孕妇分娩体重及胎儿性别，多因素回归分析显示胎儿ABCB1/C3435T基因型与晚期B超下腹横径(R~2=0.538, p=0.010)及腹围(R~2=0.534, p=0.005)呈负相关。结论：低出生体重是成人心血管疾病的发病的重要危险因素，胎儿ABCB1/C3435T基因多态性可能是导致其联系得因素之一。ABCB1/C3435T位点TT基因型携带者胎盘P-糖蛋白介导的某种底物由母体向胎儿通过降低，有可能影响到孕晚期胎儿腹部器官的发育，进而导致新生儿出生体重降低。第二节ABCB1基因多态性与母胎界面糖皮质激素转运的关系目的:探讨正常妊娠ABCB1基因多态性与母胎界面皮质醇转运的关系。方法：ABCB1/C3435T基因分型方法同上一节。血清皮质醇测定方法同第一章。结果: TT基因型组胎儿血清皮质醇浓度小于CT及CC+CT基因型组，差异具有显著性（p<0.05）; TT基因型组母血与脐血血清皮质醇浓度比值大于CT及CC+CT基因型组，差异具有显著性（p<0.05）。多因素回归分析显示，胎儿ABCB1/C3435T基因型与胎儿血清皮质醇浓度呈负相关（R~2=0.399，B=-33.47，p=0.003），与母血与脐血血清皮质醇浓度比值呈正相关（R~2=0.260，B=0.589，p=0.021）。母体ABCB1/C3435T基因多态性与血清皮质醇浓度无相关关系。结论:正常妊娠胎儿ABCB1/C3435T基因多态性与母胎界面皮质醇转运相关。第三章HSD11B2基因多态性与胎儿发育及母胎界面糖皮质激素代谢的关系目的：探讨正常妊娠HSD11B2基因多态性与胎儿发育及母胎界面皮质醇代谢的关系。方法：用PCR-毛细管电泳方法检测１８７对正常妊娠母血及脐血HSD11B2基因1号内含子CA重复序列微卫星多态性。从研究人群随机抽取33例汉族人群，用基因测序方法检测HSD11B2启动子/G-209A、G-194C、G-151A及G-126A基因多态性。血清皮质醇测定方法同第一章。结果：本研究中33例汉族人群HSD11B2启动子/G-209A、G-194C、G-151A及G-126A位点均为野生型GG纯合子，未见突变型GA及GC杂合子或者AA及CC纯合子。在考虑分娩孕周、胎儿性别、血清皮质醇测量的时间及血清皮质醇测量时的子宫收缩状态为混杂因素的情况下，多因素回归分析显示脐血HSD11B2基因1号内含子重复序列微卫星多态性（LL组VS SS+SL组）与母血血清皮质醇呈正相关（R~2=0.26，B=96.27, p=0.007），胎儿HSD11B2基因1号内含子CA重复序列越短，母血皮质醇浓度越高。脐血HSD11B2基因1号内含子重复序列微卫星多态性与脐血皮质醇，母血HSD11B2基因1号内含子重复序列微卫星多态性与母血或者脐血皮质醇，以及母血或者脐血HSD11B2基因1号内含子CA重复序列长度与新生儿出生体重及孕晚期Ｂ超下胎儿人体测量学指标之间的关系，均未见相关性。结论：正常妊娠胎儿HSD11B2基因1号内含子CA重复序列微卫星多态性与母胎界面皮质醇代谢相关。第四章正常妊娠孕晚期糖皮质激素与胎盘体视学的关系目的：探讨正常妊娠孕晚期母胎循环GC与胎盘绒毛微血管体视学之间的关系。方法：随机选择30名未临产且行剖宫产的正常足月妊娠孕妇为研究对象，通过CD34标记胎盘绒毛血管内皮细胞，进行胎盘绒毛间质内微血管体视学研究，用Western-blot方法检测胎盘GC受体前调节蛋白11β-HSD2蛋白及P-糖蛋白表达情况，血清皮质醇测定方法同第一章。结果：母体血清皮质醇631.227±176.75nmol/L,胎儿血清皮质醇116.42±41.67nmol/L,两者比值为6.29±3.59。两因素相关性分析及多因素回归分析均显示：正常妊娠胎盘绒毛微血管长度密度(Lv)和体积密度(Vv)及胎盘重量未见与孕晚期母血及脐血皮质醇或者胎盘GC受体前调节蛋白11β-HSD2蛋白及P-糖蛋白表达水平相关。结论：正常妊娠生理浓度下孕晚期母胎循环GC对胎盘绒毛微血管体视学指标影响不明显。更多还原

【Abstract】 Chapter Ⅰ: Relationship between late gestational serum cortisol and fetalgrowth in normal pregnancyObjective：To analyze the association between late gestational maternal and fetal serum cortisoland fetal birth weight and ultrasound parameters describing fetal growth done in early, middleand late pregnancy in normal pregnancy.Methods：Blood cortisol was quantified at delivery in432mother/child pairs who were normalpregnancy. Differential ultrasound examination of the fetal body was done in early, middle andlate pregnancy.Results: Ultrasound examination was done in early (gestational day89.95±7.31), middle(gestational day160.17±16.12) and late pregnancy (gestational day268.89±12.42). Thematernal cortisol was897.46±342.77nmol/L, the newborn’s cortisol was229.67±139.04nmol/L and the ratio between maternal blood and newborn’s blood was4.97±2.98. Bivariatecorrelation analyses showed that maternal cortisol shows a significantly negative correlation withthe following ultrasound parameters: late biparietal diameter (R~2=0.028, p=0.001), late headcircumference (R~2=0.045, p<0.001), late pectoral diameter (R~2=0.03, p=0.001), late abdominaldiameter (R~2=0.016, p=0.013), late abdominal circumference (R~2=0.025, p=0.002) and late femurlength (R~2=0.027, p=0.001). Multivariable regression analysis, considering timing of theultrasound examination, the child’s sex, maternal BMI, maternal age, maternal body weight atdelivery, the timing of cortisol measurement and maternal uterine contractions states, revealedthat maternal serum cortisol was significantly negative correlated with ultrasound parametersdescribing the fetal brain: late biparietal diameter (R~2=0.512, p=0.009), late head circumference(R~2=0.498, p=0.001), middle biparietal diameter (R~2=0.819, p=0.013), middle cerebellum transverse diameter(R~2=0.76, p=0.014) and early biparietal diameter(R~2=0.819, p=0.013). Thesame analysis revealed that birth weight as well as other ultrasound parameters such asabdominal circumference and femur length were not correlated to maternal cortisol levels ornewborn’s cortisol.Conclusions: Our study demonstrated that maternal cortisol secretion within physiologicalranges may be inversely correlated to fetal brain growth but not to birth weight. It remains to bedemonstrated weather maternal cortisol secretion negatively influencing fetal brain growthtranslates to adverse neurological outcomes in later life. Chapter Ⅱ：Relationship between ABCB1polymorphism and fetal growthand the cortisol transportation in maternofetal interfacePart Ⅰ: Relationship between ABCB1polymorphism and fetal growthObjective：To explore the relationship between ABCB1polymorphisms and fetal growth duringnormal pregnancy.Methods：The ABCB1/C3435T genotype was examined in262fetal and142maternal bloodsamples during normal pregnancy by polymerase chain reaction-restriction fragment lengthpolymorphism(PCR-RFLP). Fetal growth was assessed by differential ultrasound examinationof the fetal body prior to birth and by measuring birth weight.Results: The frequency of the gene polymorphisms of ABCB1/C3435T: In the fetal group, thefrequency of CC, CT, TT genotype were32.44%,53.82%,13.74%, respectively, and in thematernal group, were35.91%,50.70%,13.38%, respectively. There was no statistic differencebetween the two groups (P>0.05). All allele frequencies of the two research groups were foundto be in Hardy–Weinberg equilibrium(P>0.05).The maternal ABCB1/C3435T polymorphismshowed even no trend for an association with birth weight or any ultrasound parameterdescribing late gestational fetal body shape. Genotyping the newborns, however, demonstratedthat newborns carrying two copies of the T allele had a birth weight of3176.39g, whereas CTand CC newborns had a birth weight of3345.04g (p=0.022). Adjusting for gestational age atdelivery, child’s sex, maternal BMI, maternal age and body weight at delivery confirmed thisfinding (p=0.009). Considering gestational day of late ultrasound examination, gestational ageat delivery, child’s sex, maternal BMI, maternal age and maternal body weight at delivery, the fetal ABCB1/C3435T genotype revealed likewise a significant negative correlation withabdominal diameter and abdominal circumference (R~2=0.538, p=0.010and R~2=0.534, p=0.005,respectively).Conclusions: Low birth weight maybe a risk factor for cardiovascular diseases in later life. Thefetal ABCB1/C3435T gene polymorphism may contribute to this risk. Since P-glycoproteincontrols transport of various biological agents, we suggest that P-glycoprotein is involved in thetransport of biological agents to the fetus that are important for normal fetal growth. Part Ⅱ: Relationship between ABCB1polymorphism and the cortisoltransportation in maternofetal interfaceObjective: To explore the relationship between ABCB1polymorphisms and the cortisoltransportation in maternofetal interface during normal pregnancy.Methods: The ABCB1/C3435T genotype was examined with the same method part I. Bloodserum cortisol levels were measured with the same method as chapter I.Results: The fetal blood serum cortisol in the TT genotype group was significantly lower thanthose in the CT and CC+CT genotype groups（p<0.05）; The blood serum maternal/fetal cortisolratio in the TT genotype group was significantly higher than those in the CT and CC+CTgenotype group (p<0.05). Multivariable regression analyses, considering gestational age at delivery,the child’s sex, the timing of cortisol measurement and maternal uterine contractions states, showed thatthe fetal ABCB1/C3435T genotype revealed a significantly negative correlation with fetalblood serum cortisol (R~2=0.399,B=-33.47，p=0.003) and a positive correlation with blood serummaternal/fetal cortisol ratio (R~2=0.260,B=0.589,p=0.021）.The maternal ABCB1/C3435Tpolymorphism assessed by comparable models showed no significant association with theblood serum cortisol level.Conclusions: The fetal ABCB1/C3435T polymorphism is related to the cortisol transportationin maternofetal interface during normal pregnancy. Chapter Ⅲ：Relationship between HSD11B2polymorphism and fetal growthand the cortisol metabolism in maternofetal interfaceObjective: To observe the relationship between HSD11B2polymorphism and the cortisolmetobolism in maternofetal interface and fetal growth during normal pregnancy.Methods: The first intron of HSD11B2(CA)nmicrosatellite polymorphism was at detected in187mother/child pairs during normal pregnancy by PCR-capillary electrophoresis. TheHSD11B2promoter/G-209A,G-194C,G-151A and G-126A genotype were examined in33Chinese han samples, which were randomly selected from the total study population, by genesequencing. Blood serum cortisol levels were measured with the same method as chapter I.Results: All of the HSD11B2promoter/G-209A、G-194C、G-151A and G-126A genotype wereGG, none of these sites were GC,CC,GA or AA in33Chinese han population. Multivariableregression analysis, considering gestational age at delivery, the child’s sex, the timing of cortisolmeasurement and maternal uterine contractions states, showed that the fetal HSD11B2(CA)nmicrosatellite polymorphism(LL group VS SS+SL group) revealed a positive correlation withmaternal serum cortisol(R~2=0.26，B=96.27, p=0.007). Assessed by comparable models, none ofthe significant relationships between the fetal HSD11B2(CA)nmicrosatellite polymorphismand fetal serum cortisol, between maternal HSD11B2(CA)nmicrosatellite polymorphism andfetal or maternal serum cortisol, between maternal or fetal HSD11B2(CA)nmicrosatellitepolymorphism and any of the late pregnancy ultrasound measurements or birth weight werefounded.Conclusions: The fetal HSD11B2(CA)nmicrosatellite polymorphism is related to the cortisolmetabolism in maternofetal interface during normal pregnancy. Chapter Ⅳ: Relationship between late gestational serum cortisol andplacental stereology in normal pregnancyObjective: To analyze the association between late gestational maternal and fetal serum cortisoland placental stereology. Methods：30normal full-term pregnancy women, who were not in labor and their babies weredelivered by cesarean section, were randomly recruited in this study. A sterological study on theplacental villous capillary was evaluated with anti-CD34as the labelled vascular endothelialcells. The protein expressions of11β-HSD2and p-glycoprotein in the placenta were detected bywestern-blotting. Blood serum cortisol levels were measured with the same method as chapter I.Results: The maternal cortisol was631.227±176.75nmol/L, the newborn’s cortisol was116.42±41.67nmol/L and the ratio between maternal blood and newborn’s blood was6.29±3.59.Both of bivariatecorrelation analysis and multivariable regression analysis showed that the length density (Lv),the volume density (Vv) and the weight of the placental villous capillary in normal pregnancywere not significantly related to maternal or fetal serum cortisol or the protein expressions of11β-HSD2and p-glycoprotein in the placenta.Conclusions: The impact of maternal or fetal serum cortisol in normal full-term pregnancywithin physiological ranges on placental stereological parameters is not significant.更多还原