| Background Preterm birth (PB) is one of the most important reasons for the perinatal morbidity and mortality, and the effective treatment for PB can improve the perinatal outcomes and therefore enhance the population quality of whole country. Due to promoting the maturation of fetal lungs is the basic principle in the therapeutic schedule, antenatal glucocorticoids (GC) are administrated in the clinical practices. Single course of antenatal glucocorticoids has got the good results of therapy and little side effects, so the clinicians would like to accept it. As for the pregnant women with high risk of PB who are not deliveried beyond one week from the initial administration of GC, there has been not a sufficient proof to confirm the effect of multiple courses of antenatal GC, nor reach the consensus. The outcomes of the animal experiments for multiple courses of antenatal GC tend to induce restriction of fetal growth and neural development retardation, whereas the clinical observations seldom approve these conclusions. Recently, from the study of the effect of fetal programming, there has been found that placenta contains the most important rate-limiting enzyme of GC metabolism, i.e. 11β-hydroxysteroid dehydrogenase (11β-HSD). 11β-HSD2, as one of the two kinds of isoenzymes of 11β-HSD, plays an important role in protect the fetus from the GC though inactivating it. But there are not relevant studies about the changes of 11β-HSD2 in multiple courses of antenatal GC in placenta in preterm birth, and it is eager to know whether the expression of 11β-HSD2 is enhanced to act as a protector or is reduced to attenuate the defensive role during this process. Objective 1. To evaluate the short- and long-term outcomes of multiple courses of antenatal GC on preterm birth; 2. To determine the expression of 11β-HSD2 and glucocorticoid receptor (GR) in placenta after the clinical administrations of multiple courses of antenatal GC in preterm birth; 3. To study the influence of dexamethasone on expression of 11β-HSD2 and GR in primary cultured cytotrophoblasts from human preterm placenta in vitro.Methods 1. A Cohort study was performed for 716 cases of preterm birth in West China Second Hospital of Sichuan University during 6 years. According the courses of antenatal GC, all cases were divided into less than 1, 1, 2,≥3 courses of antenatal GC groups and control group. Clinical data such as neonatal birth weight, birth length, Apgar scores, and short-term complications were recorded carefully. Furthermore the infants were followed up by 1~6 years for their body and neural development. Logistic regression method was used to evaluate the maternal and infant outcomes. 2. Placental villous samples were collected at parturition from pregnant women with preterm birth. According the courses of antenatal GC, all cases were divided into 1, 2, 3 courses of antenatal GC groups and control group. 11β-HSD2 and GR mRNA were determined by real-time fluorescence quantitative polymerase chain reaction (PCR) method using SYBR Green I, and 11β-HSD2 and GR protein were detected by immunohistochemical method. 3,Placenta villous cytotrophoblasts from preterm birth were cultured by combination of enzymatic dissociation method and tissue incubation method. After purification and identification, acquired cytotrophoblasts were treated with dexamethasone (DEX) (100nM), or DEX+RU486 (1μM). After inoculation, the cells were collected each day from 1 to 7 days. 11β-HSD2 and GR mRNA were determined by real-time fluorescence quantitative PCR method using SYBR Green I, and 118-HSD2 and GR protein were detected by Western blot method.Results 1,The incidence of preterm birth was 4.6% during 6 years and the premature rapture of membrane was the first leading cause. There were no statistic significance in the Z-scores of birth weight and birth length of newborns among each GC treatment groups and the control group (P>0.05). Logistic analysis showed that the 2-course GC group had a lower risk of newborn asphyxia in 1-minute (OR,0.57;95% CI,0.35～0.92), and 3-course GC group also had a lower risk of newborn asphyxia in 1-minute (OR,0.10;95% CI,0.01～0.74). As for newborn asphyxia in 5-minute, 2-course GC group had a lower risk (OR,0.13;95% CI,0.03～0.43). The risk of infant and children mortality declined in 2-course GC group (OR,0.18;95% CI,0.04～0.87). There was no significant difference of maternal and perinatal infection between GC group and control group (P>0.05). 2,Immunohistochemical method showed that there were expressions of 11β-HSD2 and GR protein in preterm placenta. The 11β-HSD2 mRNA in control group was lower than GC group (P<0.05) detected by real time fluorescence quantitative PCR method, and each GC groups had higher 11β-HSD2 mRNA than control group (P<0.05). There were no significant difference of GR mRNA among control group and each GC groups (P>0.05). The results of expression of 11β-HSD2 and GR protein from imunohistochemical method were consistent with real time fluorescence quantitative PCR. 3,Combination of enzymatic dissociation method and tissue incubation method cultured high purification of placenta villous cytotrophoblasts from preterm birth. 11β-HSD2 and GR mRNA were detected in cultured cytotrophoblasts by real time fluorescence quantitative PCR method, and 11β-HSD2 and GR protein were also found in cultured cytotrophoblasts by Western blot method. After inoculation with DEX (100nM), 11β-HSD2 mRNA and protein began to rise until 3rd day (P<0.05); at 4th day, 11β-HSD2 mRNA and protein declined in the absence of DEX; from 5th～7th day, they resumed increasing when cytotrophoblasts retrieved DEX (P<0.05). Inoculated with DEX and RU486 (1μM), 11β-HSD2 mRNA and protein were less high than those with DEX alone, but there was not significantly different (P>0.05). The results of expression of 11β-HSD2 and GR protein from imunohistochemical method were consistent with real time fluorescence quantitative PCR. Conclusions 1,Multiple courses of antenatal GC have the function of accelerating fetal lung maturation due to reducing the incidence of lower Apgar scores of premature infants. Furthermore, multiple courses of antenatal GC can not decrease the birth weight and length of infants, as well as have less influence on the children development and are unable to increase the occurrence of maternal and perinatal infections. Therefore, the advantage of multiple courses of antenatal GC overweighs the disadvantage, which indicates that multiple courses of antenatal GC should be extended to the clinical use. 2,Antenatal glucocorticoids can upregulate the expression of 11β-HSD2 in preterm placenta instead of GR, and these effects still exist regardless of the different courses. Placental cytotrophoblasts have the ability to regulate the expression of 11β-HSD2, and repeated administration of GC can not impair this ability. Thus placental cytotrophoblasts are capable of protecting the infants. 3,Combination of enzymatic dissociation method and tissue incubation method can culture high purification of placenta villous cytotrophoblasts from preterai birth. Repeated administration of DEX can upregulate the 11β-HSD2 level of primary cultured cytotrophoblasts from preterai placenta, whereas can hardly downregulate the GR level. Co-existence of RU486 may have little influence on these upregulating effects for 11β-HSD2. Thus the cultured cytotrophoblasts from preterai birth have the ability to protect the infants though the mechanism of 11β-HSD2 regulation.
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