Bax, Bak and mitochondrial oxidants are involved in hypoxia-reoxygenation-induced apoptosis in human placenta.

Although apoptosis is prominent in placental cells in pregnancy complications such as preeclampsia, the cause is unknown. We surmised that hypoxia-reoxygenation (HR) is the mechanism and hypothesized that mitochondrial oxidants and Bcl-2 proteins cause HR-induced placental apoptosis. Our goal was studying expression of five Bcl-2 proteins--Bcl-2, Bcl-xL, Bax, Bak, Bad--and testing effects of diazoxide and cyclosporine A on oxidative stress and apoptosis in villous tissues subjected to HR. Term human placentas were obtained from normal pregnancies following elective caesarean deliveries. Villous tissues were subjected to "repetitive HR" (one hour at 2% O(2) then one hour at 8% O(2), alternatively, for a total of 6h) or "prolonged HR" (3h at 2% O(2) then 3h of 8% O(2)). Samples maintained at 2% and 8% O(2) served as hypoxic and normoxic controls, respectively. Prolonged HR caused the most severe villous apoptotic changes, increased the expression of Bax and Bak mRNA and protein and reduced the expression of Bcl-2 mRNA. Pre-administration of diazoxide and cyclosporine A reduced TUNEL-positive nuclei and levels of nitrotyrosine and 4-hydroxy-2-nonenol after prolonged HR. Thus, duration of hypoxia and reoxygenation is important in determining severity of HR-induced apoptosis in placenta. These apoptotic changes are closely associated with Bax and Bak effects and oxidative stress in mitochondria.

Cytogenetic and molecular studies of a familial paracentric inversion of Y chromosome present in a patient with ambiguous genitalia.

Here we describe the first reported case of a patient with a familial paracentric inversion in the long arm of the Y chromosome and ambiguous genitalia. FISH analyses with Y chromosome YACs demonstrated that the inversion breakpoints of the patients and the father's Ys appear to be the same and lie within interval 5B of the Y chromosome. PCR and sequence analysis indicated that our patient carries a normal SRY gene. For an additional comparison of the patient's inv(Y) with the father, two other Y chromosome sequences were examined. Molecular studies of this familial inverted Y chromosome showed no differences in the ZFY and TSPY genes between the father and the patient suggesting that the short arm of our patient's inv(Y) is identical to that of the patient's father. Southern analysis using a probe of the DAX-1 gene indicated that a single copy of DSS (dosage sensitive sex reversal) locus was present in the patient. Our results suggest that the abnormal sexual development in our patient is likely attributable to (an)other mechanism(s) than mutation in the SRY gene and dosage alteration of the DAX-1 gene.

Presence of cells of fetal origin in maternal circulation of pregnant women.

Fetal cells can be identified by using the polymerase chain reaction to test for the presence of human Y-chromosome-specific ZFY and SRY gene DNA sequences in maternal peripheral blood of women who bear a male fetus. Thirty-one pregnant women were studied in the first trimester to determine when fetal cells become detectable in the maternal circulation. Among the 19 women whose peripheral blood samples were positive for Y-chromosome-specific DNA sequences, the presence of fetal cells was quite case-variable from the 6th to 12th gestational weeks. Twenty-eight women who had given birth to their first male babies were studied postpartum to determine when fetal cells disappear from the maternal circulation. Fetal cells can still be detected in maternal blood 10 months postpartum in some cases. These results suggest that identification of fetal cells in the maternal circulation is possible. Nevertheless, interpretation of fetal cells in maternal circulation should be handled very carefully with respect to when these fetal cells first became detectable and potential interference from previous pregnancies.

Amniotic fluid alpha-fetoprotein levels during midtrimester of trisomy pregnancies.

To investigate the association between low amniotic fluid alpha-fetoprotein (AFP) and trisomy pregnancies, we retrospectively reviewed 26 trisomy pregnancies including 18 fetuses with Down's syndrome and eight with trisomy 18. The amniotic fluid AFP median values of Down's syndrome, trisomy 18, and the study groups were 0.73 MoM, 1.15 MoM, and 0.85 MoM, respectively. There was a significant difference between the mean values of the Down's syndrome-affected fetuses (0.78 +/- 0.29 MoM) and that of the control group (p < 0.001), whereas no such difference was found for that of trisomy 18-affected fetuses (1.16 +/- 0.38 MoM). Only three patients in the study group (3/26, 11.5%) had an amniotic fluid AFP value below 0.5 MoM, including the two cases of Down's syndrome (2/18, 11.1%) and one case of trisomy 18 (1/8, 12.5%). Most of the values for the trisomy pregnancies were within the normal range, thereby precluding the possibility of using this measurement as an alternative to fetal karyotyping as a screening test for Down's syndrome or other trisomy pregnancies.

alpha-Fetoprotein levels of paired samples between the amniotic fluid and maternal serum from 16 to 18 weeks' gestation in Chinese women.

To assess the relationship between alpha-fetoprotein (AFP) concentrations of amniotic fluid and maternal serum from 16 to 18 weeks' gestation, 890 paired samples of maternal serum and amniotic fluid were collected from women with normal singleton pregnancies. The gestational age was determined by ultrasonographic dating before amniocentesis, and the AFP measurements were performed by a single reference laboratory. There was a significant rise in the maternal serum AFP (MSAFP) concentration from 16 to 18 weeks' gestation. Amniotic fluid AFP concentrations significantly declined from 16 to 18 weeks' gestation. This study failed to demonstrate any statistical relationship between the AFP concentration of maternal serum and amniotic fluid (r = 0.031). This finding indicates that amniotic fluid AFP levels cannot be predicted by MSAFP levels between 16 and 18 weeks' gestation. Simple diffusion may not be the only mechanism for the transfer of AFP through the fetal membrane to maternal circulation.

Analysis of birth weight and gestational age in Taiwan.

Appropriate interpretation of monitored fetal growth throughout pregnancy in individual patients and populations is dependent upon the availability of adequate standards. Previously published standards either were based on small samples, data decades old or were characteristic of foreign subpopulations. We have reviewed the data on a series of 46,575 singleton live births at Chang Gung Memorial Hospital from 1979 to 1989. The ratio of males to females was 1.08. The fetal growth pattern in the third trimester of pregnancy approximates a sigmoid curve. Fetal growth was most rapid from the 32nd to the 37th week of gestation with an average increase of 240 g per week. The birth weight declined beyond 42 weeks' gestation. There were significantly greater weight gains amongst male fetuses as compared to female fetuses from the 34th to the 42nd week of gestation. The mean birth weight recorded at 40 weeks' gestation in male and female newborns was 3,381 g and 3,262 g, respectively. Comparing the birth weight of term pregnancies using our data, with those of a previous, two-decade old report (1945-1967) by Chen, we found that birth weight were slightly higher in this study. The derived fetal growth curves are useful for clinical, public health, and investigational purposes.

Deep vein thrombosis during pregnancy.

Antepartum deep vein thrombosis is quite rare in Chinese women during pregnancy. An adequate diagnosis and early treatment are extremely important as the deep vein thrombosis can embolize, in particular, to the pulmonary vasculature. We present a case of pregnancy at 36 weeks gestation complicated by deep vein thrombosis. The objective diagnosis of deep vein thrombosis for obstetric patients is the noninvasive Doppler ultrasound and plethysmography. An early diagnosis is important to reduce maternal and fetal risks related to embolization. Continuous therapy of low dose intravenous heparin is safe and effective during pregnancy.

Fetal cells in the maternal circulation during first trimester in pregnancies.

To investigate the presence of fetal cells in the maternal circulation during early pregnancy, the polymerase chain reaction was used to test the presence of human Y chromosome-specific ZFY and SRY gene DNA sequences in maternal peripheral blood specimens from 19 women carrying male fetuses and 12 women carrying female fetuses. The presence of fetal cells was suggested as early as 6 weeks gestation in 1 of the 19 women bearing male fetuses. Fetal cells were present in the maternal circulation of 15 of the 19 women by 9 weeks gestation, and in only 1 of the 19 were fetal cells not detected until the 12th week after conception. These results suggest that identification of fetal cells in the maternal circulation is possible with a properly designed and executed polymerase chain reaction. However, there was considerable variation with respect to when these fetal cells first became detectable during pregnancy. These fetal cells are potentially a valuable source of material for biochemical and genetic studies of the fetuses.

[Incidental myomectomy in cesarean section].

In the retrospective study, from January 1984 to December 1987, incidental myomectomy was performed on 47 patients who received cesarean section. The control group consisted of 80 random samples who received cesarean section during the same period. The general information in both groups was similar. Postoperative infection rate was similar in both groups. Myomectomy added 11 minutes to the operation time, 112 milliliter to the operation blood loss and extended the hospital stay about one and a half day. Recovery of gastrointestinal tract function was one hour later in myomectomy group. There were no wound infection or serious morbidity in both groups. In order to estimate the value of incidental myomectomy in cesarean section, a further study of long term change of the myoma should be considered.

Maternal serum alpha-fetoprotein levels between 13 and 24 weeks' gestation.

Maternal serum alpha-fetoprotein (MSAFP) screening has been widely used and proven valuable in the prediction of a variety of fetal disorders. Any laboratory intending to provide MSAFP screening needs to establish its own reference data. In order to establish a normal MSAFP median value at our own laboratory, 5256 samples of MSAFP were collected from uncomplicated, singleton pregnant women between 13 and 24 weeks' gestation. The MSAFP median levels steadily rise with advancing gestation about 16% per gestational week in average. There were 0.17% and 4.4% of the pregnancies with serum AFP levels less than 0.25 multiple of the median (MoM) and 0.5 MoM respectively, and 4.22% and 1.66% with serum levels above 2.0 MoM and 2.5 MoM. Accurate and satisfactory interpretation of MSAFP screening should be emphasized in the establishment of a well-developed normal median value.

Management of the fetus with an abdominal wall defect: experience of 31 cases.

The diagnosis of ventral abdominal wall defect can now be made prior to birth. With this diagnosis, the family can make decisions and a planned optimal management can lead to a successful outcome. There were 31 cases of ventral wall defect identified at Chang Gung Memorial Hospital (CGMH) from January 1979 through March 1988. Twenty of them were classified as gastroschisis; among them, 17 (85%) were born in outside clinics and none of them had associated anomalies. In contrast, among 11 cases of omphalocele, there was a lower frequency of transferred cases (27% vs 85%), and 4 cases had additional defects, including two multiple anomalies and two bladder exstrophies. There were no significant differences between gastroschisis and omphalocele in the mortality rate (30% vs 36%), in the incidence of intrauterine growth retardation (IUGR) (30% vs 27%) and in the Cesarean section rate (15% vs 18%). All 4 cases of prematurity (less than 36 weeks of gestational age) expired after delivery and 2 of these had body weights of less than 1500 g. Three out of 5 cases delivered by Cesarean section expired; the mortality (60%) was higher than that of vaginal delivery (28%). All 3 cases were gastroschisis, 2 of them were transferred from outside clinics and all expired due to sepsis. The diagnosis of ventral wall defect should be made prenatally, with obstetric ultrasonography, maternal serum alpha-fetoprotein screening and fetal karyotyping. Therefore, fetal transport in utero to a referral center and optimal perinatal care for those fetuses with potentially correctable lesions can be well planned.

Maternal urine alpha-fetoprotein concentrations between 14 and 21 weeks of gestation.

BACKGROUND: The aim of this study was to ascertain the normal range of the midtrimester maternal urine alpha-fetoprotein (AFP) concentrations in Taiwanese pregnancies. METHODS: AFP was measured in the urine samples, obtained before genetic amniocentesis, from 268 women with normal singleton pregnancies between 14 and 21 weeks of gestation. Week-specific median values for urine AFP/creatinine (Cr) were calculated by weighted linear regression after log transformation and the data were converted to units in the multiple of the median (MoM). The gestational age in all cases was determined by ultrasound parameters. RESULTS: The levels of urine AFP and AFP/Cr increased gradually with advancing gestational age. The AFP/Cr MoM values of singleton pregnancies after log transformation showed a normal distribution with a mean (standard deviation) of 0.0071 (0.3228). The median, 10th and 90th centiles of AFP/Cr were 0.98, 0.43 and 3.61 MoM, respectively. Of the pregnant Taiwanese women studied, 4.9% (13/268) and 16% (43/268) had urine AFP/Cr MoM levels less than 0.31 MoM and 0.5 MoM respectively. CONCLUSION: The establishment of a reference range which allows for gestational differences in AFP/Cr levels is essential for further antenatal testing.

Elevated second-trimester maternal urine free beta-human chorionic gonadotropin levels in Asian pregnancies with fetal chromosomal abnormalities.

OBJECTIVES: To investigate the second trimester maternal urine free beta-human chorionic gonadotropin (hCG) levels of chromosomally abnormal pregnancies in Asians. METHODS: Free beta-hCG levels were analyzed from the urine samples of 110 control and 17 chromosomally abnormal pregnancies, including 11 cases of Down syndrome, 1 case of trisomy 18, and other chromosomal abnormalities (one mosaic deletion and 4 translocations) from the second trimester of pregnancy. Results were normalized to urine creatinine (Cr) concentration and converted to the multiple of the median (MOM) level for the appropriate gestation. Gestational age of all cases was determined by ultrasound parameters. RESULTS: The median free beta-hCG MOM levels of Down syndrome (4.02 MOM) and other chromosomally abnormal pregnancies (2.03 MOM) are significantly higher than that of normal pregnancies (0.99 MOM) (p = 0.002 and p = 0.024, respectively). Nine of 11 (81.8%) Down syndrome cases, one trisomy 18 case, and 2 of 5 (40%) other chromosomally abnormal cases would be expected to be above the 95th centile of the control values (2.95 MOM cut-off). CONCLUSION: Urine free beta-hCG could be a potential and useful marker in the detection of fetal Down syndrome and other chromosomal abnormalities in Asians.

Fetal blood sampling and cytogenetic abnormalities.

From September 1984 to April 1991, we performed cytogenetic analysis on fetal blood samples from 214 second- and third-trimester pregnancies. One hundred and thirty-four cases were referred to consider the possibility of chromosomal mosaicism following amniocyte studies. The confirmation rate of mosaicism is at 0 per cent (0/9), 1.4 per cent (1/70), and 40 per cent (22/55) for cases of level I, level II, and level III mosaicism, respectively. Four out of 17 cases were positive for the diagnosis of fragile X syndrome. Of 63 cases with abnormal ultrasound findings, blood disorders, or other genetically related clinical conditions, 11 were found to have a chromosome abnormality. Fetal blood sampling is a valuable adjunct to other methods in the prenatal diagnosis of chromosomal mosaicism or pseudomosaicism. It is also useful when rapid cytogenetic diagnosis is desired because of malformations detected in pregnancies at a late gestational age.

Expression and chromosomal localization of cDNA clones from an enriched human retinal pigment epithelial (RPE) cell line library: identification of two RPE-specific genes.

We have previously constructed an enriched cDNA library from a human retinal pigment epithelium (RPE) cell line and generated expressed sequence tags (ESTs) from novel clones. Here, we report the analysis of expression of 14 cDNAs and identify two clones, AA1 and AA28, that appear to be specifically expressed in RPE but not in any other tissue tested. We have also localized 15 novel cDNAs (including the two RPE-specific cDNAs) to human chromosomes using in situ hybridization or in conjunction with somatic cell hybrid analysis. The cDNAs were mapped to the following chromosomal regions: 1p35-->p33, 1q41-->q42 (two clones), 3q11.2-->q13.1, 3q24-->q25, 4q13-->q21, 6q22-->q23, 7q34-->q36, 10q23-->q24, 11q23-->q24, 15q25-->q26, 19p13.3, 20p13, 21q11.2-->q21, and 21q22.2-->q22.3. The genetic and functional analysis of the two RPE-specific genes should contribute to a better understanding of RPE function. Chromosomal localization of RPE cDNAs will be valuable in identifying candidate genes for inherited diseases involving RPE dysfunction and aid in establishing the expression map of the human genome.

Congenital malformation in newborns. Analysis of 501 cases.

Over a 14-year period in Chang Gung Memorial Hospital, 510 out of 44, 362 newborns were found to have birth defects. Maternal age, gestational age, parity, infant sex and birth weight were analyzed for each anomaly and compared to normal newborns. The average maternal age and parity for newborns with congenital anomalies were not significantly different from normal newborns. Mothers giving birth to babies with chromosomal aberrations, however, had a significantly older maternal age than the normal population. The gestational age at delivery was significantly shorter for all except craniofacial anomaly. In addition, there was a high percentage of intrauterine growth retardation in congenital anomalies. The central nervous system, the musculoskeletal system and craniofacial systems were the most commonly involved. The leading anomalies included cleft lip, cleft palate, anencephaly, polydactyly, hydrops fetalis, trisomy 21 and cystic hygroma. With improved ultrasound equipment and other prenatal diagnostic procedures, many defects of the fetus can now be identified. If the fetus is diagnosed with a surgically correctable lesion like cleft lip, it can be kept to term, delivered, then managed postnatally. If life-incompatible malformations have been detected before the 24th week, physicians are in a good position to counsel the parents. After the 24th week termination is proscribed by law. Therefore, physicians must take special care to detect fetal abnormalities early.

Prenatal detection of two different monosomic cell lines by chorionic villus sampling.

We present a prenatal case of mosaicism with at least two monosomy cell lines: one with monosomy 21 (45,XY,-21) and one missing the Y (45,X) and a possible third 46,XY in chorionic villus cell culture. Cytogenetic studies were initiated following the ultrasound detection at 11 weeks of a large cystic hygroma and in utero growth retardation. Spontaneous fetal demise occurred at 12 weeks and the pregnancy was terminated. To our knowledge, this is the first report of two different monosomic cell lines found in chorionic villus cells.

Perinatal outcome of chorionic villus sampling versus amniocentesis.

This study assesses the relative risks of first trimester transcervical chorionic villus sampling (CVS) versus midtrimester amniocentesis performed between April 1986 and March 1988. The most common indication for prenatal diagnosis was advanced maternal age. We discovered 5.1% chromosomal aberrations in CVS compared to 1.0% in amniocentesis. Bleeding was the most frequent early complication, and only 1 case had major hemorrhage with subsequent spontaneous abortion. The fetal loss rate (gestational age less than 28 weeks) was 4.5% in CVS versus 1.2% in amniocentesis, which was not significantly different from the background fetal loss rate reported in normal pregnancies after an 8-week gestational age. Three cases of fetal loss after CVS were probably procedure-related; 1 case had spontaneous abortion and 2 cases had chorioamnionitis. Therefore, we considered that the causal relationship between CVS and the infection was highly probable. The clinical pregnancy outcome indicated that there were no differences in overall perinatal mortality, Apgar score, body weight, body length, gestational age at delivery, intrauterine growth retardation, placenta weight and placental disorders between the CVS group and the amniocentesis group. The pregnancies did not reveal any specific effects of the prenatal diagnostic procedure, but a long-term pediatric follow-up is needed.

Real-time quantitative PCR analysis for alpha-thalassemia-1 of Southeast Asian type deletion in Taiwan.

Since homozygosity of the alpha-thalassemia-1 of Southeast Asian (SEA) type deletion results in hydrops fetalis, a novel protocol based on the real-time quantitating polymerase chain reaction (PCR) technique has been developed to quantify the intact and aberrant alpha-globin genes in adults. The ratio of the normal/SEA-bearing alpha-globin genes was expressed in cycle threshold (C(T)) values. Theoretically, a relative ratio of one to one was anticipated in individuals carrying the SEA type deletion. Twenty-five heterozygous and 20 normal cases were analyzed retrospectively with this protocol. Data showed that the CT values for the intact alpha-globin gene allele and the allele bearing the SEA type deletion in carriers were 28.74+/-1.49 and 26.46+/-2.05, respectively. Therefore, the ratio of normal/SEA type deletion-bearing alpha-globin genes in the carriers was 1.09+/-0.043. No ambiguous results were observed from other less common genotypes associated with alpha-thalassemia, such as the Philippine type deletion. Based on the results, we concluded that this protocol could provide a rapid method to mass screen carriers with alpha-thalassemia-1 of SEA type deletion in this region.