Genetic amniocentesis in twin pregnancy

Among 1041 pregnancies 13 twin gestations were detected by routine ultrasonography prior to genetic amniocentesis at the Department of Prenatal Physiopathology of the University of Bologna. Clear amniotic fluid from both sacs was obtained in 12 of 13 sets of twins. All 12 sets were cytogenetically normal with normal levels of α-fetoprotein. Only one patient spontaneously aborted liveborn immature twins 30 days after the procedure. The technique used to obtain samples of fluid is described in detail and the need for additional counselling prior to amniocentesis in twins is stressed.     

Increased risk of abortion after genetic amniocentesis in twin pregnancies

Forty-seven twin pregnancies among 3676 patients who had a genetic amniocentesis between 1973 and 1979, are reported. The detection rate of twins at the time of amniocentesis was 62 per cent. Five (17 per cent) of the 29 women with detected twin pregnancy aborted spontaneously, these are compared with 1 (6 per cent) of 18 women with undetected twin pregnancies and with 3 (3 per cent) of 93 singleton pregnancies, selected as controls as they had amniocentesis performed immediately before and after each of the twin mothers. Two of 9 (22 per cent) twin pregnancies, who had at least two punctures in at least one sac aborted, while 3 of 20 twin pregnancies with one puncture in each sac aborted (15 per cent). One of 18 (6 per cent) twin pregnancies, where only one sac was punctured, because the twin pregnancies were undetected, aborted. Amniocentesis of both sacs in twin pregnancies seems associated with an increased risk of spontaneous abortion. The indications for amniocentesis in twin pregnancies should be critically evaluated.     

Risk of amniocentesis and laboratory findings in a series of 1500 prenatal diagnoses

The experiences with 1500 midtrimester prenatal diagnoses are reported. Abnormal findings of amniotic fluid investigations led to 43 therapeutic abortions. In ± 30 percent of the chromosome anomalies diagnosed, the significance of the effect on fetal development was inconclusive. The outcomes of all pregnancies except one are known. Fetal loss and perinatal mortality involved 69 cases, 23 (33 percent) of which occurred within three weeks after amniocentesis. In these 23 cases there appeared to be a relationship between the degree of experience of the gynaecologist and fetal loss: 3·7 percent when this experience was limited to a maximum of 10 punctures diminishing to 0·3 percent with an experience of more than 50 punctures. It is concluded that the risk of an abortion due to amniocentesis decreases as the gynaecologist becomes more experienced with the puncture technique.     

Prenatal diagnosis of hemoglobinopathies in twin pregnancies by double simultaneous fetoscopy

A new technique for sampling fetal blood in twin pregnancies using two fetoscopes simultaneously is described. Two fetoscopes were inserted, one after the other, into both amniotic cavities and fetal blood samples were obtained from either the chorionic plate vessels or the umbilical cord insertion area. The observation of the bright tip of the second fetoscope behind the septum using the first fetoscope assured the successful entry of the two fetoscopes into the two different amniotic sacs. This technique was performed on 15 out of 17 patients. In all patients the fetuses were at risk of β-thalassemia major. Sampling was successful in all cases. Double simultaneous fetoscopy seems to be a safe and accurate technique without technical problems or complications. The simultaneous use of two fetoscopes opens new possibilities in intrauterine fetal surgery and research.     

Alkaline phosphatase activity in amniotic fluid in pregnancies with fetal disorders

Alkaline phosphatase (ALP) activity was determined in 255 amniotic fluid samples collected by amniocentesis between 15 and 39 weeks of gestation. The samples were originally used for chromosomal analysis and/or alpha-fetoprotein measurements. The mean ALP activity in early amniotic fluid from pregnancies with fetal trisomy 18 and 21 syndromes was half of that found in the controls. Highly elevated ALP activity (over 10 times the median level) was found in 14 samples. Two of these pregnancies had normal outcome. Three samples were from pregnancies with intrauterine fetal death. Fetal disorders, including abdominal wall defect (four cases), Meckel's syndrome (two), hydrops fetalis syndrome (two) and genital anomaly (one), were observed in nine cases. Moderately elevated ALP activity (over three times the median) was found in 10 cases, including five pregnancies with a preterm labour shortly after the sample collection. The results indicate that elevated ALP activity in the third trimester amniotic fluid is often associated with fetal disorders.     

Prenatal diagnosis of spina bifida aperta after first-trimester valproate exposure

In the context of a prospective study on the adverse effects of anti-epileptic drugs on fetal outcome, we evaluated our experience with prenatal diagnosis by ultrasonography and α- fetoprotein (AFP) determination in amniotic fluid. We compared these results with AFP values in maternal serum obtained prior to amniocentesis. From November 1985 to July 1990, amniocentesis at 16–18 weeks of gestation was performed in 267 pregnancies of 237 different women using anti-epileptic drugs. Among 92 pregnancies with maternal valproic acid use, five (including one concordantly affected monozygotic twin-pair) were terminated because of a spina bifida aperta, all prenatally diagnosed by AFP determination and acetylcholinesterase electrophoresis in amniotic fluid. The maternal serum AFP level was raised (⩾ 2.5 multiples of the median (MOM) for singleton pregnancies and ⩾ 4.5 MOM for twin pregnancies) in only two of these five affected pregnancies. We emphasize that maternal serum AFP levels may be unreliable for prenatal screening for fetal neural tube defects in women taking valproate and recommend that amniocentesis and fetal ultrasound examination should be offered directly.     

Prenatal diagnosis of cystic fibrosis: I. Prospective study of 51 pregnancies

A prenatal diagnosis was performed in 51 pregnancies with a 1-in-4 risk of having a child with cystic fibrosis. The criteria for determining an affected fetus were based on the results of alkaline phosphatase (ALP) residual activity after inhibition by phenylalanine and by homoarginine, of total ALP activity, and of gamma-glutamyltranspeptidase (GGTP) activity in the amniotic fluid taken between 16 and 19 weeks of pregnancy. The chromosomal analysis of amniotic fluid cells showed trisomy 13 in one case which was excluded from the analysis of biochemical assays. The biochemical assays were in the normal ranges in the amniotic fluid of 35 pregnancies: 26 have reached term and a normal infant has been born, 9 are still in progress. A deficiency of the ALP phenylalanine-inhibitable form, depressed values of total ALP and GGTP were observed in the amniotic fluid of 15 pregnancies: one pregnancy went to term and the infant had CF, in 14 cases the pregnancy was terminated, and meconium ileus was observed in ten of these cases. It was observed that the changes towards abnormal values became more significant with advancing gestational age and that 18 weeks appeared to be the optimum time for diagnostic amniocentesis.     

Short communications normal fetal growth despite persistent amniotic fluid leakage after genetic amniocentesis

Two women not only lost relatively large amounts of amniotic fluid immediately following genetic amniocentesis, but continued to lose fluid for the remainder of their pregnancies. Periodic ultrasonographic assessment confirmed normal fetal growth and presence of some amniotic fluid. Both women were delivered at term of normal offspring who showed no evidence of fetal deformations. Although amnionitis is a risk, cautious surveillance may permit continuation of pregnancies complicated by copious or persistent amniotic fluid leakage following genetic amniocentesis.     

Cytogenetic studies of amniotic fluid taken before the 15th week of pregnancy for earlier prenatal diagnosis: A report of 114 consecutive cases

One hundred and fourteen samples of amniotic fluid taken before 15 weeks of gestation were cultured for cytogenetic studies. The results of culturing these early amniotic fluid (EAF) samples were compared with the results of culturing 114 standard amniotic fluid (SAP) samples taken after 15 weeks of gestation matched for maternal age and received in the laboratory within the same week. Cell culture was successful in all 114 of the EAF samples and in 111 SAP samples. There was no significant difference in the days to harvesting and days to reporting in the two groups. Three samples of SAP failed to grow and two EAF samples produced tetraploid karyotypes, so that in these five cases amniocentesis had to be repeated. These problems were attributed to toxicity of a fungicide used in the culture medium. Pseudomosaicism was noted in two EAF samples and one SAP sample; and maternal cell contamination was noted in one EAF and one SAP sample. Thus, culturing and karyotyping cells harvested from EAF and SAP are similar, indicating that EAF samples from 12–14-week pregnancies could be used for prenatal diagnosis.     

Prenatal diagnosis of omphalocele and gastroschisis by ultrasonography

As the use of ultrasonography has become a routine procedure in the care of pregnant women, fetal congenital malformations have been frequently diagnosed prenatally. During a 4-year period abdominal wall defects (AWD) were diagnosed in 14 cases, five were gastroschises and nine were omphaloceles, seven females and seven males. Concomitant malformations were present in one case of gastroschisis and in five cases of omphalocele. Eleven AWD's were diagnosed in the second trimester and six pregnancies were interrupted. Eight children with AWD were born; two boys with omphalocele and one girl and one boy with gastroschisis are still alive. Alpha-fetoprotein was determined in amniotic fluid in 11 cases and also in maternal serum prior to the amniocentesis in eight cases. Chromosomal investigations were performed in 13 cases, 11 on amniotic fluid cells and two on lymphocytes from a blood sample made postnatally. Two abnormal karyotypes, 47,XX + 18 were found.     

Sampling pure fetal blood in twin pregnancies by fetoscopy using a single uterine puncture

A technique for sampling pure fetal blood in twin pregnancies using a single uterine entry with a fetoscope is described. The fetoscope was inserted into one sac and after blood had been obtained from that, twin, the fetus in the other sac was sampled by trans-septal passage of the blood-sampling needle. This was done in six out of seven patients, the first in the series having two separate insertions of the fetoscope, one into each sac. Pure fetal blood was taken from all 14 fetuses, either from the placental insertion of the umbilical cord or the umbilicus, and the volume of the samples ranged from 200 μl to 1200 μl. In six patients the fetuses were at risk of β-thalassaemia and in one of haemophilia A. Some observations are made relating zygosity to the ultrasonic and fetoscopic appearance of the septum between the sacs.     

Alpha-fetoprotein and acetylcholinesterase activity in first-and early second-trimester amniotic fluid

Normal ranges of amniotic fluid alpha-fetoprotein (AFP) and acetylcholinesterase activity (AChE) are described for gestational weeks 11–14 using rocket gel immunoelectrophoresis for AFP quantitation and a monoclonal antibody (4F19) enzyme antigen immunoassay for AChE activity measurement. The normal ranges were established by the examination of 281 amniotic fluid samples from 281 normal pregnancies. AFP was found to increase from a median level of 14.0 MIU/1 at 11 weeks to a maximum at 13 weeks (median=18.0 MIU/l) (P<0.05), thereafter falling (not significant). No AChE test result exceeded 4.8 nkat/l. In addition, AFP and AChE values for three cases of fetal malformation, identified by the biochemical analyses of amniotic fluid, are given. These cases included two fetuses with a neural tube defect and one fetus with an abdominal wall defect. Amniocentesis was performed at 10, 11, and 14 weeks, respectively. The AFP and AChE values were all high.     

Microvillar enzyme analysis in amniotic fluid and the prenatal diagnosis of cystic fibrosis

The activities of two microvillar enzymes, gamma-glutamyltranspeptidase (GGTP) and alkaline phosphatase (ALP) have been determined in amniotic fluid (AF) samples from 39 pregnancies with a 1-in-4 risk of cystic fibrosis. Seventeen of these were investigated prospectively. A reduced proportion of the fetal specific intestinal ALP isoenzyme was found in 7 of a total of 13 pregnancies with cystic fibrosis and in one pregnancy of confirmed normal outcome. Eight of the affected pregnancies were tested for AF GGTP activity and depressed levels were found in 15. None of the 3 liveborn cystic fibrosis cases in the prospective series was identified by the ALP assay although 2 had significantly reduced GGTP activity. There were several amniotic fluid samples from cases of cystic fibrosis, trisomy 18 and normal outcome which had discordant GGTP and ALP results. Four of the 6 cases of cystic fibrosis misclassified by the ALP assay had amniocentesis at 15 or 16 weeks gestation. Evidence is presented which confirms a previous suggestion that amniocentesis after 17 weeks gestation improves the predictability of the ALP isoenzyme assay for the prenatal diagnosis of cystic fibrosis.     

Transplacental amniocentesis: Is it really a higher-risk procedure?

The aim of this study was to compare transplacental with non-transplacental amniocentesis in terms of related complications. Between January 1991 and December 1992, 4564 genetic amniocenteses were performed in 4527 patients (4491 singleton, 35 twin, and one triplet pregnancy) at 15–16 weeks of gestation. All the procedures were ultrasound-guided and performed by the same operator. In 1487 cases, an anterior placenta was traversed with the needle, whereas in 3077 cases, the needle was inserted directly into the amniotic cavity without traversing the placenta. After the exclusion of patients in whom amniotic cell culture failed or in whom an abnormal karyotype was obtained, and of patients lost to follow-up, a total of 4454 patients (98 per cent) were followed for 30 days after amniocentesis. Two spontaneous abortions occurred after a transplacental procedure, and five after a non-transplacental procedure (P = not significant). There were no episodes of amniotic fluid leakage in the first group, whereas 16 ruptures of the membranes that resolved spontaneously occurred in the second group (P<0·01). Our data suggest that transplacental amniocentesis carries a similar abortive risk to and a lower risk of transient rupture of the membranes than non-transplacental amniocentesis and may therefore be preferred at the gestational period examined (15–16 weeks). However, the risk of feto-maternal haemorrhage, which is reported to be higher for a transplacental procedure, must be considered in the case of an anterior placenta.     

Short communication kazal type trypsin inhibitor in amniotic fluid in fetal developmental disorders

The amniotic fluid concentrations of the Kazal type trypsin inhibitor were studied in pregnancies with fetal developmental disorders. The samples were obtained by amniocentesis between 14 and 19 weeks of gestation. In cases with fetal malformations, the level was below the normal 10th centile in 15 out of 28 cases (54 per cent, P<0.05) and above the normal 90th centile in 2 cases (7.1 per cent). Low values were common in cases with intrauterine fetal death or congenital nephrosis. The levels were normal in fetal chromosomal aberrations.     

Extraction of DNA from amniotic fluid cells for the early prenatal diagnosis of genetic disease

Ten-ml samples of amniotic fluid were taken from pregnancies being terminated at 8–14 weeks' gestation. DNA was extracted from the amniotic cells by sequential centrifugation and analysed using the polymerase chain reaction (PCR). Fifteen samples were analysed for evidence of maternal contamination using Mfd5 oligo-nucleotide primers for repeat polymorphisms. Ten amniotic fluid samples were tested for the Delta-F₅₀₈ deletion characteristic of cystic fibrosis to demonstrate a diagnostic application for the technique. In each case, DNA extracted from fetal tissue from the same pregnancy was included in the controls. In 14 of the 15 cases tested with the Mfd5 primers, both the amniotic fluid DNA and the fetal DNA showed no evidence of contaminating DNA. In one case, neither the amniotic fluid cells nor the fetal cells yielded results. In nine of the ten cases tested with the Delta-F₅₀₈ primers, the amniotic fluid cell DNA provided accurate information about the genetic status of the fetus; in the tenth, the fetal DNA failed to amplify. The results indicate that adequate DNA can be extracted from amniotic fluid from 8 weeks' gestation onward and these samples are suitable for prenatal diagnosis using PCR.     

Prenatal diagnosis of Fabry's disease by direct analysis of chorionic villi

Six pregnancies of three carriers for X-linked Fabry's disease, were monitored by chromosome and enzyme analysis. Two affected male fetuses were detected by the demonstration of α-galactosidase deficiency in amniotic fluid cells and chorionic villi respectively. The use of chorionic villi enabled a diagnosis within a few hours after sampling in the ninth week of pregnancy whereas the use of amniotic fluid cells in the earlier case required two weeks of culturing after amniocentesis in the 16th week. Four female fetuses were found; heterozygosity was demonstrated in one by analysis of clones in the primary amniotic fluid cell culture.     

Incidence of abortion after genetic amniocentesis in twin pregnancies

Fetal outcome after genetic amniocentesis (AC) in viable twin pregnancies was analysed in a retrospective study at three centres in order to estimate the rate of fetal loss after AC. The maternal age ranged from 33 to 45 years (mean 36.7 years). The gestational age varied between 15 and 20 weeks of gestation (mean 17.1). In 98 viable twin pregnancies with complete follow-up, spontaneous abortion of both fetuses occurred within 28 completed weeks of gestation in eight pregnancies and six women aborted within 20 completed weeks of gestation after AC, corresponding to a rate of fetal loss of 8.1 and 6.1 per cent, respectively (excluding the loss of five twins with viable outcome of the co-twin in five pregnancies).     

First-trimester prenatal diagnosis in twin pregnancies

Two twin pregnancies at risk for a sex-linked disorder are described. Both pregnancies were dichorionic. Transabdominal sampling was chosen for prenatal diagnosis. Molecular genetic techniques raised suspicion with regard to the accuracy of the samples in one case. Second-trimester amniocentesis confirmed the error. Selective feticide of the affected fetus was performed. When first-trimester prenatal diagnosis is offered in dichorionic twin pregnancies, confirmation through molecular genetic testing can confirm that villi have been obtained from different fetuses. All parties must be aware that additional invasive diagnostic procedures in the second trimester may be required in cases of doubt.     

Prenatal diagnosis in monozygotic twins with Down syndrome who had different phenotypes

We report the case of monozygotic (MZ) male twin fetuses with different Down syndrome (DS) phenotypes. Prenatal fetal sonography showed a bichorial biamniotic pregnancy with increased nuchal translucency in twin A and a cervical cystic hygroma and heart defect in twin B. Cytogenetic analysis performed after double amniocentesis showed free and homogeneous trisomy 21 in both twins. Monozygosity was confirmed by molecular analysis. The pregnancy was terminated at 17 weeks of gestation (WG). Postmortem analysis confirmed the phenotypic discordance. To our knowledge, this is the first reported prenatal diagnosis of MZ male twins with different Down syndrome phenotypes but identical karyotypes. We discuss the mechanisms involved in phenotypic discordance of monozygotic twins and particularly the role of environmental factors. Copyright © 2007 John Wiley & Sons, Ltd.