First-trimester amniotic fluid and extraembryonic coelomic fluid acetylcholinesterase electrophoresis

Acetylcholinesterase (AChE) gel electrophoresis was performed on samples of amniotic fluid and extraembryonic coelomic fluid obtained by high resolution transvaginal ultrasound-guided amniocentesis from 38 women between 8 and 12 weeks of pregnancy. AChE was positive in 33 per cent (12/36) of the amniotic fluid samples; the percentage of positive results decreased as gestation advanced. AChE was positive in 32 per cent (9/28) of the extraembryonic coelomic fluid samples. In 81 per cent (21/26) of matched samples, the AChE results were identical in the two fluids. Amniotic fluid and extraembryonic coelomic fluid AChE electrophoresis cannot be used to diagnose neural tube defects prior to 12 weeks of gestation.     

Persistently elevated AFP and AChE in amniotic fluid from a normal fetus following demise of its twin

Intrauterine fetal demise (IUFD) in one of twins at 12 weeks of gestation was accompanied by markedly elevated maternal serum alpha-fetoprotein (AFP) at 17 and 18 weeks. Amniotic fluid AFP from the healthy surviving twin's sac at 18·5 and 23 weeks was also greatly increased along with a positive acetylcholinesterase (AChE) band. Persistently elevated AFP and positive AChE so long after fetal demise–-6·5 and 11 weeks post IUFD–-has not, to our knowledge, been previously described. In similar cases, high level ultrasound and careful placental examination at birth should be utilized to search for fetal abnormalities or multiple pregnancy with IUFD.     

The use of amniotic fluid AFP action limits in diagnosing open neural tube defects

An analysis is made of alphafetoprotein (AFP) concentrations in 3630 amniotic fluids submitted for prospective prenatal diagnoses over a 7-year period. There were 89 cases of anencephaly, 74 of open spina bifida and 3467 with normal singleton outcomes. The AFP data were expressed in both standard deviations above the mean and multiples of the normal median for individual weeks of gestation. False positive and false negative rates were comparable in the two systems at selected cut-offs. It is concluded that either system may be used in setting action limits for the primary distinction of unaffected pregnancies from those in which an open neural tube defect is present.     

Microvillar enzyme assays in amniotic fluid and fetal tissues at different stages of development

A systematic study of microvillar enzyme activities in the amniotic fluid in correlation with their values in different fetal tissues during development has been undertaken. Microvillar enzymes appeared in the amniotic fluid at the time of disappearance of the anal membrane, 12–13 weeks, and declined from the 18th week until the 24th week. The study of fetal tissues and fluids has shown that gamma-glutamyltranspeptidase is mainly of liver origin. The significant decrease of the activities of these amniotic fluid enzymes has been the basis of prenatal diagnosis of cystic fibrosis. These assays may be useful for the diagnosis of certain digestive tract abnormalities at later stages of pregnancy.     

Transcervical (TC) and transabdominal (TA) CVS for prenatal diagnosis in rotterdam: Experience with 3611 cases

Data from 3611 consecutive CVS (TC, N= 1780; TA, N= 1831) were analysed with emphasis put on influence of maternal and gestational age at CVS on the fetal loss rate < 28 weeks. For TC-CVS the gestational age varied from 9.3–11.6 weeks, for TA-CVS from 9.3–20 weeks. Sampling efficacy at first attempt was 86.5 per cent and 95 per cent respectively. In 4.6 per cent an abnormal result was established. In older mothers (N=2362) the fetal loss rate was significantly higher (p = <0.05) when sampled before 12 weeks (TC-CVS 6.2 per cent, TA-CVS 5.8 per cent). When the CVS (TA) was performed after 12 weeks the fetal loss rate decreased to 2.4 per cent. In 1079 younger women the fetal loss rate remained low (TC 2.8 per cent; TA < 12 weeks 1.8 per cent; TA > 12 weeks 1.7 per cent) and was not influenced by gestational age at the time of sampling. We concluded both methods safe and reliable when the choice of application considers maternal age.     

Spontaneous abortion after amniocentesis in women with a history of spontaneous abortion

The incidence of spontaneous abortion after amniocentesis (19 to 28 weeks gestation) in women who have had previous spontaneous abortions is compared with the rate in women who have not had previous spontaneous abortions. The outcome of the pregnancy after amniocentesis and the previous history of spontaneous abortion is reported for 691 pregnancies. The rate of spontaneous abortion after amniocentesis was found to be significantly higher in women who had one or more previous spontaneous abortions, 12/238 (5 per cent), than in women who did not, 6/453 (1.3 per cent). In women who reported two or more previous spontaneous abortions, the rate was 7/81 (8.6 per cent). No statistically significant effect of maternal age or gravidity was detected. The incidence of spontaneous abortion after amniocentesis was greater in the three weeks following the procedure (three for each of the three weeks) than in the subsequent seven weeks (nine for seven weeks).     

Transabdominal villus sampling in early second trimester: A safe sampling method for women of advanced age

Transabdominal chorionic villus sampling (TA-CVS) was performed in 707 viable singleton pregnancies to exclude chromosomal abnormalities. Maternal age ranged between 36 and 49 years (mean 37·9 years); gestational age varied between 10·2 and 18·3 weeks (mean 13·3 weeks). In 639 women (90·4 per cent), a sufficient amount of chorionic tissue (⩾ 10 mg) was obtained after one needle insertion; in 66 women (9·3 per cent) two insertions were needed. An abnormal chromosome pattern was established in 19 cases (2·9 per cent). Vaginal bleeding or spotting within 28 days after TA-CVS occurred in 11 cases (1·5 per cent). The completed follow-up of 678 chromosomally normal pregnancies showed an overall fetal loss rate of 2·6 per cent before 28 weeks. The overall perinatal mortality was 0·9 per cent. When relating fetal loss to gestational age at TA-CVS, this was 6·6 per cent in women sampled before 12 weeks against only 1·8 per cent after 12 weeks. At the same time, the percentage of fetal loss occurring within 2 weeks following the procedure was 75 and 30 per cent, respectively. It is suggested that these data reflect the decline in spontaneous abortion rate during this particular period of pregnancy. It is concluded that TA-CVS is an effective procedure which, when performed after the natural decrease of fetal loss, appears to be a safe option for women of advanced maternal age.     

First-trimester diagnosis of hypophosphatasia. Importance of gestational age and of purity of CV samples

Prenatal diagnosis of hypophosphatasia was made by alkaline phosphatase (ALP) assay on a chorionic villous sample taken in the first trimester. Very low activities of the LBK isoenzyrnes indicated an affected fetus. Diagnosis was confirmed at 12 weeks of gestation by measurement of LBK isoenzyme activities in fetal bone tissue. In control chorionic villous samples an inverse relation was observed between LBK and placental ALP percentage during gestational age. High LBK ALP activities are observed in decidual tissue. Chorionic villous tissue must not be sampled after 12 weeks of gestation and decidual tissue must be excluded from the sample.     

Detection of rubella virus in fetal and placental tissues and in the throats of neonates after serologically confirmed rubella in pregnancy

From 35 therapeutic abortions performed because rubella had occurred at 2–19 weeks of pregnancy, 120 fetal organs, 12 specimens of mixed products of conception, and 15 placentae were tested for rubella virus. Virus was isolated from 10 out of 11 fetuses (91 per cent) from women infected at 2–8 weeks, from 5 out of 8 (63 per cent) infected at 9–10 weeks, and from 2 out of 16 (13 per cent) infected at 11–19 weeks. Hybridization tests for viral RNA on 39 fetal organs from eight cases revealed infection in four additional fetuses. Virus was isolated from only 3 out of 15 aborted placentae, but hybridization tests on six placentae revealed infection in three additional specimens. Hybridization was superior to virus isolation for detecting rubella infection in products of conception and is therefore potentially the better method for examining chorionic villus biopsies. Rubella virus was isolated from the throats of 4 out of 9 infants (44 per cent) infected during the first 12 weeks of gestation, but from none of 13 infected after 17 weeks. Infants in the latter group are unlikely to infect susceptible contacts.     

A further prenatal diagnosis of mosaic tetrasomy 12p (Pallister-Killian syndrome)

A case of mosaic tetrasomy 12p was detected in amniotic fluid cell cultures from a 28-year-old woman referred to us at 26 weeks' gestation because of hydramnios. The fetus was shown on ultrasonography to have an omphalocele and a short femur length. Labour was induced at 32 weeks. An infant with multiple congenital anomalies was delivered and died after 10 min. The diagnosis of i(12p) or Pallister-Killian syndrome was confirmed cytogenetically in fibroblast and lymphocyte cultures. Increased LDH-B activity was demonstrated in fibroblasts.     

Prenatal diagnosis of cystic fibrosis using a monoclonal antibody specific for intestinal alkaline phosphatase

A monoclonal antibody (AAP-1), specific for the intestinal isoenzyme of alkaline phosphatase (ALP), has been used to develop an immunoassay for amniotic fluid samples. Values in the immunoassay correlated closely with those obtained by direct determination of phenylalanine-inhibitable ALP. A panel of 124 control second-trimester amniotic fluids and 21 fluids with a 1 in 4 risk of a cystic fibrosis fetus were examined in the immunoassay. Eight of 10 affected cases had values below an arbitrary cut-off of one third median, while all the non-affected cases were above this level. Almost identical results were obtained by enzymatic determination of phenylalanine-inhibitable ALP. However, in both systems the false positive rate (control fluids with values below one third median), was unacceptably high. It is pointed out that at present the most effective system for the prenatal diagnosis of cystic fibrosis is achieved by measuring the ratio of intestinal to total ALP in amniotic fluid supernatants. This is probably best effected by enzymatic assay in the presence of phenylalanine and homoarginine inhibition.     

Evaluation of transcervical chorionic villus sampling with a completed follow-up of 1550 consecutive pregnancies

Data from 1550 consecutive pregnancies after first-trimester prenatal diagnosis by transcervical chorionic villus sampling (TC-CVS) are presented. The sampling efficacy was 97.8 per cent; the mean amount of collected villus tissue was 23 mg (range 5–100 mg). There were 97 affected fetuses, mainly (73.2 per cent) with a chromosomal abnormality or a male karyotype in carriers of X-linked disease. Pregnancy termination in these and four other women for social reasons resulted in 1449 continuing pregnancies. In these pregnancies, the fetal loss rate up to 28 weeks of gestation was 5.1 per cent with the highest loss rate (3.9 per cent) before 16 weeks. When relating this fetal loss rate to maternal age, this was 6.1 per cent in the advanced maternal age group (⩾36 years) against 3.1 per cent in the younger age group. In 1376 pregnancies continuing beyond 28 weeks, the perinatal mortality rate was 1.1 per cent; the percentage of non-genetic congenital anomalies was 0.9 per cent. The reproductive pattern of women at high genetic risk after CVS followed by pregnancy termination was evaluated. Within 12 months after the first CVS followed by pregnancy termination, 70 percent of women again requested CVS in a subsequent pregnancy.     

Variables influencing growth and morphology of colonies of cells from human amniotic fluid

Growth of cells from amniotic fluid was studied with respect to cell concentration in the inoculum, blood contamination of the fluid, fluid colour, fluid clarity, gestational age of the pregnancy, and growth factors. Dependent variables measured were colony formation, colony size, and colony morphology after 7, 11, and 14 days of culture. The following conclusions were established from these studies: small sample volumes are the most efficient for producing colonies; cells from very bloody or dark brown fluids have a slower rate of growth; growth of cells from cloudy (noncontaminated) fluids is better than growth of cells from clear fluids; the proportion of colonies that are epithelioid varies with gestational age; the stimulating effect of 100 ng/ml fibroblast growth factor on cells from amniotic fluid was confirmed.     

Prenatal diagnosis of tetrasomy 47,XY, + i(12p) confirmed by in situ hybridization

A case of tetrasomy i(12p) detected prenatally is reported. The patient, a black, 33-year-old G3P2002 at 24 weeks' gestation with an unremarkable family history presented herself for prenatal care. Ultrasound examination showed a fetus with diminished femoral and humeral lengths, and hydramnios. A level II scan confirmed the presence of an omphalocele. Amniocentesis at 31 weeks showed 47,XY, + i(12p) karyotype. An infant with multiple congenital anomalies was delivered at 34 weeks. The infant died after 5 h. Genetic and ultrasonographic examinations in the third trimester were helpful in the investigation of this fetus with multiple congenital anomalies. The careful, complete team counselling afforded by this approach enabled the mother and family to be well adjusted to the strong possibility (and subsequent reality) of an abnormal infant.     

Predictive value of amniotic acetylcholinesterase analysis in the diagnosis of fetal abnormality in 3700 pregnancies

The value of acetylcholinesterase (AChE) analysis as an adjunctive test to amniotic alpha fetoprotein (amAFP) for the diagnosis of fetal abnormality has been investigated in a series of 3785 amniotic fluid samples. Quantitative analysis of AChE performed retrospectively on a selected group of 541 amniotic fluid samples failed to discriminate between normal and open neural tube defect pregnancies. Qualitative analysis of AChE by polyacrylamide gel (PAG) electrophoresis in the same series of 541 fluids correctly identified 251 of the 255 pregnancies with open neural tube defect and 29 of the 31 pregnancies with false positive amAFP results. The failure of the test to diagnose 4 cases of open neural tube defect was probably attributable to the age and condition of the stored AF samples. Routine diagnostic testing of AChE isoenzymes in a further 3244 AF samples successfully identified all 170 cases of open neural tube defect and 20 cases with other fetal defects. Thirteen fluids gave false positive AChE results (0.4 per cent) compared to 59 of the series in which there were false positive amAFP results (1.8 per cent). Six of the 13 false positive AChE cases had AChE bands of low intensity which would not be regarded as diagnostic of fetal abnormality, and in five the AChE band may have been the result of significant blood contamination. False positive AChE results contributed to the decision to abort three apparently normal fetuses, but a normal AChE result undoubtedly helped to save a number of pregnancies with false positive amAFP results. Our experience suggests that repeating the amniocentesis may help in resolving the rare diagnostic difficulty of a positive AChE result with or without an elevated amAFP in the absence of ultrasound evidence of fetal abnormality, particularly where there is blood contamination of the amniotic fluid sample.     

Marker chromosomes in A series of 10000 prenatal diagnoses. Cytogenetic and follow-up studies

In a series of 10 000 prenatal diagnoses 15 marker chromosomes were detected in our centre. Six of these were familial whilst nine had originated de novo. They were analysed with various staining methods. DA-DAPI staining was positive in nine out of 12 pregnancies. Six pregnancies were continued. Five normal children were born, one ended in intrauterine fetal death of a normal fetus at 37 weeks. Nine pregnancies were terminated, showing six normal fetuses, one familial cat-eye syndrome, one fetus with Down syndrome caused by additional trisomy 21 and one fetus with cystic kidneys resp. It is concluded that it seems safe to continue the pregnancy in cases of a familial marker, identical to that of one parent, whilst a de novo DA-DAPI positive marker seems to present a low risk for fetal anomalies.     

Fetal ductus venosus flow velocity waveforms and maternal serum afp before and after first-trimester transabdominal chorionic villus sampling

Doppler flow velocity waveform recording in the fetal ductus venosus and umbilical artery as well as maternal blood sampling for serum alpha-fetoprotein (MSAFP) was performed before and after transabdominal chorion villus sampling (TACVS) in 36 women of advanced maternal age (≥ 36 years). Gestational age ranged between 11 and 13 weeks. No chromosomal anomaly was detected. No statistically significant difference was observed in ductus venosus velocity parameters or in the umbilical artery pulsatility index (PI) before and after CVS in 35 women with a normal pregnancy outcome. One case resulted in fetal loss. Post-CVS median MSAFP levels at 12 weeks (25 kIU/1) and 13 weeks (35 kIU/1) were significantly higher than pre-CVS levels. In three cases, post-CVS MSAFP levels were higher than 600 kIU/1, correlating with feto-maternal transfusions of approximately 1.0–1.4 ml, i.e., of around 40 per cent of feto-placental blood volume. One of these cases displayed absence of fetal peripheral blood flow velocities and fetal bradycardia following TACVS, resulting in fetal loss 1 week later. The remaining two cases had a normal pregnancy outcome, but showed a more than 50 per cent reduction in ductus venosus velocity after TACVS, whereas umbilical artery PI remained unchanged. However, similar velocity changes were associated with only small feto-maternal transfusions. Umbilical artery PI values remained unchanged.     

Amniotic fluid protease activity and the prenatal detection of cystic fibrosis

Changes in the protease activity in amniotic fluid has been proposed as a valid method for the prenatal detection of cystic fibrosis (CF). We have studied by quantitative and qualitative procedures, sixty four amniotic fluids: two of them from CF-affected fetuses. Interpretation of the benzoyl arginine ethyl ester (BAEE)-staining patterns after isoelectric focusing was often difficult, and repeated experiments gave variable results. In order to improve gel discrimination, we performed amniotic fluid electrofocusing in the presence of detergents: 0.1 per cent Triton X-100, 0.1 per cent DOC, or 0.1 per cent SDS. In these conditions, the pattern revealed by BAEE was modified, but no differences were observed between CF and normal amniotic fluids.     

Pregnancy outcome after transcervical cvs with a flexible biopsy forceps: Evaluation of risk factors

The pregnancy outcome of 1936 women who had transcervical chorionic villus sampling (CVS) with a flexible biopsy forceps was evaluated. Follow-up until 4 weeks after delivery was 99.4 per cent. Various patient- and procedure-related risk factors for spontaneous loss (fetal or neonatal death) were analysed using stepwise logistic regression analysis. The overall spontaneous loss rate was 4.5 per cent. Factors found to be significantly associated with spontaneous loss were quantity of villi ≤ 15 mg (relative risk (RR) 2.13), a history of first-trimester miscarriage (RR 1.87) or delivery between 16 and 27 weeks (RR 3.87), cervical culture positive for anaerobes (RR 4.52) or group B streptococcus (RR 3.62), post-procedural bleeding >3 days (RR 1.99), and multiple insertions (RR 2.64). Significant differences in loss rates between individual operators were found. A learning effect was not present. There were no infants born with terminal transversal limb anomalies in our series.     

Chorionic villus sampling by biopsy forceps. Results of 1580 procedures from a single centre

The results of a prospective series of 1580 chorionic villus sampling (CVS) procedures using biopsy forceps are presented. Most of the procedures (1442), including 11 sets of twins, were performed by the transcervical approach (TC-CVS), using a curved-shank thin forceps, and 138 by the transabdominal approach (TA-CVS), using a trocar-guided straight thin forceps. The mean gestational age for TC-CVS was 10.9 weeks, and in 233 cases (16 per cent) the procedure was carried out between the 12th and 14th weeks. The mean gestational age for TA-CVS was 16.7 weeks. The major indication for CVS was advanced maternal age (92.7 per cent in the TC and 91.8 per cent in the TA approach), and indications for abnormal ultrasound findings were more common in the TA approach (4.5 per cent) than in TC-CVS (0.07 per cent). Although sampling was apparently accomplished in all the procedures, in 3.1 per cent of the TC-CVS and 2.2 per cent of TA-CVS procedures, the samples were less than 1 mg after dissection. A cytogenic report was obtained in 96.1 per cent of the TC-CVS and 90.6 per cent of the TA-CVS. Maternal serum alpha-fetoprotein (MSAFP) was measured before and after TC-CVS and the post-CVS MSAFP was positively correlated with the sample weight. Second-trimester amniocentesis following CVS was required in 5.2 per cent (TC-CVS) and 6.5 per cent (TA-CVS), due to the failure to obtain a cytogenetic report or diagnostic confirmation. The follow-up to the 20th week was 100 per cent by ultrasound scan, and 88.6 per cent from the 21st week to 1 week after delivery. Fetal loss rates within 2 weeks of the procedure were 1.7 per cent (TC-CVS) and 0.8 per cent (TA-CVS) and total fetal loss accumulated to 1 week after delivery was 4.6 per cent (TC-CVS) and 5.9 per cent (TA-CVS). Factors found to increase significantly fetal loss in the TC-CVS series were maternal age and the collection of very small samples, but not the number of forceps insertions.