[Establishment of multiple quantitative fluorescent polymerase chain reaction assay and its application in rapid prenatal diagnosis of common chromosome aneuploidies].

OBJECTIVE: To establish the multiple quantitative fluorescent polymerase chain reaction (QF-PCR) assay and evaluate its clinical application in prenatal diagnosis. METHODS: Totally 170 samples were collected between May 2008 and July 2009 in prenatal center of Peking Union Medical College Hospital; 123 of them were amniotic fluid, 9 were chorionic villous samples, 20 were fetal blood and 18 were villi from aborted fetuses. All samples were from women of Han nationality, with mean age of (34.1 ± 4.6) years old, and with mean gestational age of (19.6 ± 1.0) weeks. Cytogenetic cultures and karyotyping were made to every sample. Genomic DNA was extracted from the samples. The sequences of twenty short tandem repeat (STR) markers were designed according to the GenBank and references, including 6 STR markers in chromosome 21, 4 in chromosome 18, 4 in chromosome 13, 4 in chromosome X, 1 in chromosome Y and 1 universal marker in both X and Y chromosome. Each sample was amplified by two sets of multiple QF-PCR, which included 4 STR markers in each of 21, 18, 13 and sex chromosomes. If the result was uninformative, the third set of another 4 STR markers was added. RESULTS: (1) Karyotyping. Cytogenetic analysis were made for all the 170 samples, 151 (89%) of which were normal, and 19 (11%) were abnormal. (2) QF-PCR assay. 167 (98%) samples were detected by QF-PCR. The results were obtained within 2 - 3 days after sampling. 134 samples were proved normal by QF-PCR, which was consistent with karyotyping. Among the 19 abnormal karyotype samples, 18 were detected as abnormal(eight were 21-trisomy, three were 18-trisomy)by QF-PCR. Among the 167 samples, 150 (90%) were detected using the first and second set of STR mixtures, and 3 (2%) were detected when the third set of STR was added. The remain 14 (8%) were uninformative. (3) The diagnostic efficiency of QF-PCR. The sensitivity of QF-PCR in prenatal diagnosis of common aneuploidities was 95%, the specificity, the false positive rate, the false negative rate, the positive predictive value and negative predictive value were 100%, 0, 5%, 100% and 99%, respectively. (4) Autosome and sex chromosome detection by QF-PCR. Among all the STR markers, D21S1270 and D21S1411 had the highest heterozygosities in chromosome 21, and DXS8377 had the highest in sex chromosome. The amplifications were stable. CONCLUSION: Multiple QF-PCR assay is a valid alternative in rapid prenatal diagnosis of common chromosome aneuploidies. With high accuracy, it can be used for numerous sample test in large-scale laboratories.

[Influence of dexamethasone on expression of 11beta-HSD2 in primary cultured cytotrophoblasts from human preterm placenta].

OBJECTIVE: To study the influence of dexamethasone (DEX) on the expression of 11beta-hydroxysteroid dehydrogenase type 2 (11beta-HSD2) in primary cultured cytotrophoblasts from human preterm placenta. METHODS: Placenta villous cytotrophoblasts from preterm birth were cultured with the protocol of enzymatic dissociation and tissue incubation method. After isolation and identification, cytotrophoblasts were treated with the repeated administration of DEX (100 nmol/L), or DEX-RU486 (1 micromol/L) for 7 days, in which DEX was not added into the culture at 4th day. Cytotrophoblasts were collected everyday, and the expression levels of 11beta-HSD2 mRNA and protein were determined by real-time fluorescence quantitative PCR and Western blot method. RESULTS: After the treatment of DEX (100 nmol/L), the expression of 11beta-HSD2 mRNA and protein in cytotrophoblast increased in the first three days (P < 0.05). At 4th day, 11beta-HSD2 mRNA and protein declined in the absence of DEX. In 5th-7th day, the increase of 11beta-HSD2 expression were resumed when cytotrophoblasts received DEX again (P < 0.05). With the treatment of DEX and RU486 (l micromol/L), both mRNA and protein level of 11beta-HSD2 in cytotrophoblasts were lower than those with DEX alone, but there was not significantly different (P > 0.05). CONCLUSION: Repeated administration of DEX can upregulate the expression level of 11beta-HSD2 in primary cultured cytotrophoblasts from preterm placenta. Cytotrophoblasts from preterm birth may have the ability to protect the infants by the mechanism of 11beta-HSD2 regulation.

[Effect of N-acetylcysteine on lipopolysaccharide induced preterm labor in mice].

OBJECTIVE: To test the effect of N-acetylcysteine (NAC) on the mice with infection associated preterm labor. METHODS: The pregnant C57BL/6 mice were randomly distributed into five groups, each with 20 mice and were given lipopolysaccharide (LPS), saline solution (NS), NAC, LPS+NAC (therapy), and NAC+ LPS (prevention) respectively. The LPS (20 microg) was injected intraperitoneally to the mice every 12 hours since day 16 of gestation to induce preterm labor. The NAC was orally administered (0.5 g/kg) every 12 hours since day 15 or day 16 for the prevention and therapy groups respectively. The latency interval (from LPS injection to delivery of the first pup) and fetal survival rates were recorded in eight mice from each group. The remaining mice were killed at 4, 8, 12, and 24 hours after the LPS injection (three for a group each time). The NF-kappaB activity and IL-8 mRNA in uterine and maternal and fetal hepatic GSH-PX and serum IL-8, MDA, and SOD were examined. RESULTS: The average latency interval of LPS-treated mice was (15.1 +/- 1.9) hours, with a fetal survival rate of 4.3%. The NAC therapy extended the latency interval of LPS-treated mice to (35.4 +/- 2.1) hours, with a fetal survival rate of 69.0%. The NAC prevention extended the latency interval of LPS-treated mice to (44.8 +/- 2.6) hours, with a fetal survival rate of 84.3%. The expression of NF-KB P65 was activated and reached the peak 4 hours after the LPS injection. The uterine IL-8 mRNA and serum IL-8 and MDA reached the peak whereas the maternal and fetal hepatic GSH and SOD declined to the lowest 8 hours after the LPS injection. The NAC significantly inhibited the effect induced by the LPS (P < 0.01). CONCLUSION: The NAC has a therapeutic effect on the LPS-induced preterm labor in mice. It is even better to be used in preventing preterm labor. The mechanism of the protective effect of NAC may include the deactivation of the NF-kappaB/IL-8 and the reduce of the production of ROS.