The Effect Of Preeclampsia On Fetal HPA Axis And Its Mechanisms

It is a burgeoning theory that metabolic disorders such as coronary heart disease, stroke, hypertension, Type2diabetes and osteoporosis originate through unbalanced nutrition in utero. Preeclampsia is a pregnancy-specific disease characterized by de-novo development of concurrent hypertension and proteinuria and sometimes pro-gresses into a multiorgan cluster of varying clinical features, remaining a leading cause of maternal and perinatal mortality and morbidity. Preeclampsia (PE) is such a disease that deteriorates the condition of fetal nutrition. Furthermore, offspring of preeclamptic pregnancies are at the high risk of metabolic diseases in late life.Glucocorticoids (GCs) affect the prenatal period and adjust the expression of genes that control the development and function of vascular structure. The GC levels in fetal circulation should be maintained normal for the successful growth and development of fetal organs. Excessive GC exposure in utero will inhibit fetal growth and may predis-pose the fetus to hypertension and/or diabetes in the adulthood. The type2isoform of11β-hydroxysteroid dehydrogenase (11(3-HSD2) is a critical oxidase during the conver-sion from cortisol to cortisone in a unidirectional.11β-HSD2highly expressed in the placenta may regulate the transplacental passage of GCs and protect the developing fe- tus from the side effects of the physiological increase of maternal GC levels during pregnancy. Currently, there is no literature that focuses on the relationship between PE and subsequent adult metabolic disorders of their offsprings.No evidences neither on the long term effects, such as the changes in function or sturcture of hypotha-lamic-pituitary-adrenal axis (HPA) nor on the related mechanisms.In this study, we plan to set up animal model in rats with PE and to detect impor-tant parameters of their offspring, such as the level of hormones that are closely ass-cioated with the function of HPA axis in order to investigate the11β-HSD2expression and the promoter methylation in placentas and fetal of PE patients, and to explore the effects of maldevelopment in utero caused by PE and the resulting metabolic disorders in their offspring. Finally, we would also study the long term effects of PE to the off-spring by BSID-Ⅱ. The aims of our investigation were to reveal the changes that may existed in11beta-HSD2mediated HPA axis and to discuss the possible role in pro-gramming of the risk of metabolic diseases in the adulthood of the offspring of pree-clampsia.Part I Establishment of rats model of preeclampsia and mechanisms underlying the effects of preeclampsia on HPA axis function of off-springObjective:To observe the effect of preeclampsia on blood pressure in adult offspring rats and investigate the pathophysiological changes of the hypothalamic pituitary adrenal axis and its correlation with11B-HSD2by the animal model of rat with preeclampsia. Methods:The animal model of preeclampsia which was established by nitric oxide synthase inhibitor-L-NAME injection during pregnancy, was divided into the model group and control group. The pathologic change of rat brain and kidney was observed, hippocam-pal neurons change was observed by electron microscope; adrenocorticotropic hormone (ACTH) and corticosterone concentration of offspring rats were detected by enzyme linked immunosorbent assay; renal11B-HSD2level was detected by immunohisto-chemistry and RT-pCR; the levels of11B-HSD2and GR in renal and hippocampus tis-sue were deteced by immunohistochemistry; hypothalamic expression of CRH protein was detected by immunofluorescence assay.Results:After L-NAME injection in pregnant rats, the blood pressure and proteinuria grad-ually increased in the model group. The birth weight of fetal rats in model group de-creased significantly (P<0.001), diastolic blood pressure of model group was signifi-cantly higher than the control group born after twentieth weeks (P<0.05); corticosterone, ACTH level in offspring plasma was significantly higher than that in normal control group; hippocampal neurons apoptosis were observed in the model group; the expres-sion of11B-HSD2protein in renal tissue is significantly lower than that of control.Conclusion:The pathological injury of HPA axis in preeclampsia offsprings suggests a link between glucocorticoid exposure, HPA axis regulation and adult offspring of pree-clampsia. Part Ⅱ Alteration in methylation level at11β-hydroxysteroid dehydro-genase type2gene promoter and cortisol concentration in infants born to preeclamptic womenObjective:To clarify fetal methylation status at CpG sites of the promoter of11β-HSD2in preeclampsia, and ACTH, cortisol in the umbilical-cord blood, in order to discuss the possible role of ACTH, cortisol and alteration in methylation at the promoter of11β-HSD2in programming of the risk of metabolic diseases in the adulthood of the offspring of preeclampsia.Methods:Umbilical cord blood samples were taken from infants born to women of pree-clampsia (n=43;25of mild and18of severe) and normal pregnancy (n=78) and ge-nomic DNA was isolated. ACTH, cortisol were measured in the samples of mixed arte-rial and venous umbilical-cord blood by ECLIA. DNA methylation levels at11β-HSD2promoter were determined by Massarray quantitative methylation analysis. Methylation status was detected at6CpG sites of11β-HSD2promoter. Linear mixed model analysis and multivariate linear regression were performed to evaluate the correlation between the methylation levels and clinical parameters in preeclampsia and normal pregnancy. Results:The mean umbilical-cord plasma cortisol in the fetuses from pregnancies compli-cated by pre-eclampsia,264.39±167.10nmol/L, was significantly higher than the plas-ma CRH in the fetuses from normotensive pregnancies,148.34±48.49nmol/L (P<0.001).Fetal plasma ACTH was also significantly higher in the fetuses from pregnancies complicated by pre-eclampsia than in the normally grown fetuses born after normoten-sive pregnancies (PE,26.55±18.03pmol/L; normally grown,14.35±11.03pmol/L (P<0.01).Methylation levels of HSD9-2, HSD9-3, HSD23-2and HSD23-3and the mean methylation level were significantly lower in preeclampsia than in normal pregnancy (P=0.002,0.031,0.047and0.001, respectively and P<0.001in mean). The mean methy-lation level was significantly correlated with preeclampsia after the adjustment of birth weight, maternal age, gestational age at delivery and fetal gender (r=0.325, P<0.001).Conclusions:The elevations of cortisol and ACTH are important mechanisms in the accelerated placental and fetal maturation that has been associated with hypertensive pregnancy. Preeclampsia reduced methylation level at fetal11β-HSD2promoter. A positive correla-tion existed between11β-HSD2promoter methylation and preeclampsia. Part Ⅲ Analysis of gene expression and preliminary study of methyla- tion about11β-HSD2gene in placentas of pre-eclampsia patientsObjective:To.determine.the.promoter.methylation.status.of.type.2.isoform.of.11β-hydroxysteroid dehydrogenase (11β-HSD2) and its regulatory relationship with11β-HSD2gene ex-pression in placentas of pre-eclampsia (PE) patients.Methods:Placental.tissues.were.studied.by.hematoxylin and eosin staining and immunohis-tochemical staining. The11β-HSD2mRNA and protein expressions were detected by real-time polymerase chain reaction (real-time PCR) and Western blotting. The methy-lation of the11β-HSD2promoter sequence was examined by bisulfite sequencing PCR (BSP).Results:Trophoblast hyperplasia and discontinuous syncytial layer were observed in the PE group, and the11β-HSD2was distributed irregularly and its immunoreactivity was weakened distinctly. The expressions of11β-HSD2mRNA and protein decreased sig-nificantly in the PE group compared with the control group. Unexpectedly, almost no11β-HSD2methylation was detected in PE placental tissue (two fragments,0.6%vs.250%) or normal placental tissue (1%vs.0.6%). No significant difference in11β-HSD2promoter methylation was found between the two groups. Conclusions:The11β-HSD2expression decreased in PE women, but was not interrelated with the promoter methylation status. Part Ⅳ.Developmental assessment of the offspring of mothers with preeclamsia by BSID-ⅡObjective:To explore the effect of preeclampsia on neuropsychological development of the offspring.Methods:78cases of the mother with preeclampsia whose correct age (CA) between2to5.9months were assessed by BSID-Ⅱ.Results:Of the infants with mothers with preeclampsia, the scores of gross motor move-ment, fine movement and social contact in the infants with BW<1500g,1500-1999g decreased significantly than those in the infants with BW>2500g,2000-2499g (P<0.05), and the scores of MDI and PDI are also lower than those of the controls. Conclusions:Preeclampsia impaired the neuropsychological development of the offsprings.