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.     

Fetal karyotypes from fetoscopy blood samples

The fetal karyotype was determined in 42 out of 45 cases from fetal blood obtained by fetoscopy for prenatal diagnosis of β-thalassemia. The procedure described is quick and reliable and it is recommended for women over 35 years of age undergoing prenatal diagnosis for haemoglobinopathies.     

The safety of fetoscopy: (I). Effect of umbilical vessel puncture on the fetal heart rate and cord histology

The fetal heart rate (FHR) was continuously monitored during 42 umbilical vessel punctures performed at the placental insertion of the cord in 24 diagnostic fetoscopies in which pure fetal blood was obtained. In only one patient did a deceleration first appear during puncture and aspiration of fetal blood. In two patients decelerations preceded fetoscopy and in two others they began during the fetoscopy but before puncture of an umbilical vessel. In 19 patients, the FHR did not change at all during the procedure. Fetal haemorrhage after sampling was either absent or minimal. Six pregnancies were terminated because a positive diagnosis had been made and 18 healthy babies were born. Umbilical cords were examined after 7 terminations of pregnancy and after 6 deliveries. In the former group the puncture could just be seen with the naked eye and the needle track was demonstrated histologically in 6. No traces of the puncture or other abnormalities were found in the cords after delivery. Fetal blood sampling from umbilical cord vessels, particularly at the placental insertion of the cord, is the technique of choice since pure fetal blood can be obtained without increasing the risk of fetoscopy.     

Routine chromosome analysis on fetal blood microaliquots obtained at fetoscopy

A routine study of the fetal karyotype was performed on samples obtained at 64 fetoscopic procedures. In 13 cases only pure amniotic fluid was available for the cultures, while in the remaining 51 cases the chromosome analysis was carried out on PHA-stimulated lymphocyte microcultures set up with any excess fetal blood above the requirements for globin-chain synthesis. Karyotype could be determined on fetal lymphocytes in 44 cases (86 per cent). All the fetuses were chromosomally normal. This experience shows that cytogenetic analysis using microaliquots of fetal blood is a relatively simple technique which should be introduced into routine prenatal diagnosis by fetoscopy.     

Creatine kinase estimation in pure fetal blood samples for the prenatal diagnosis of duchenne muscular dystrophy

This paper compares the results of a survey of plasma creatine kinase (CK) activity measured in fetuses at-risk for Duchenne muscular dystrophy (DMD) with a reliable control series. Only pure fetal blood samples obtained by fetoscopy at between 17–24 weeks gestational age were used. Of the at-risk group 19 male pregnancies, mostly at low risk for DMD, proceeded to term with a normal outcome; there was no significant difference between their fetal plasma CK activities and the control group. Another 21 male pregnancies were terminated. This group included the highest risk mothers and hence was expected to contain a significant proportion of affected fetuses. The fetal plasma CK activity range was overlapping but significantly higher than the control group. No grossly elevated CK value was obtained. We conclude that, on average, DMD fetuses at this gestational age have higher plasma CK activity than controls. The problems of applying this finding to the prenatal diagnosis of DMD are discussed.     

Biochemical examination of fetal skin biopsy specimens obtained by fetoscopy: Use of the method for analysis of keratins and filaggrin

This paper describes a method for biochemical analysis of proteins from fetal skin biopsy samples. The method has wide potential application for diagnosis of disorders with a known protein abnormality detectable by protein staining or a specific antibody. Analysis requires a single 1 mm biopsy, is rapid (2 days) and extremely sensitive. In the present study, fetal skin biopsies from normal fetuses and a fetus at risk for lamellar ichthyosis were obtained. The epidermis or hairs with attached follicular cells were dissected from the remaining skin. Proteins were extracted and separated by SDS-polyacrylamide gel electrophoresis. Proteins from duplicate gels were transferred to nitrocellulose and immunostained for the acidic and basic keratins and for the keratin filament associated protein, filaggrin, using monoclonal antibodies. All samples contained keratins typical of fetal epidermis at 20 weeks gestation. Presence of filaggrin is variable at this age and depends on the presence of keratinized cells of hair canals. No keratin abnormalities in the fetus at risk for lamellar ichthyosis were detected, however, in one presumably normal biopsy, an abnormally low proportion of the 67 kd keratin and the presence of follicular keratins were evident. These results demonstrate that biochemical analysis of fetal biopsies is possible, thus increasing the diagnostic potential of the fetal biopsy procedure for disorders in which a known protein or antigen is altered in utero.     

Fetal cystic hygromata: Insights gained from fetal blood sampling

Twelve second-trimester fetuses with cystic hygroma underwent fetal blood sampling for rapid karyotyping, haematologic evaluation, and blood gas analysis. An abnormal karyotype was found in seven cases: monosomy X in five, trisomy 21 in one, and trisomy 13 in the other. Eight often fetuses undergoing blood gas analysis showed hypoxaemia, five of which were growth-retarded. Nine pregnancies were terminated. Of the remaining three, only one fetus survived the perinatal period.     

Prenatal diagnosis of alpha-1-antitrypsin deficiency by fetal blood sampling

Fetal blood sampling for the diagnosis of alpha-1-antitrypsin deficiency using protein isoelectric focusing was carried out in the period 1980–1985. The results of 25 cases from 18 mothers are reported. All had a previous history of a PiZ child affected by liver disease. The method was found to be technically satisfactory and the fetal results were subsequently confirmed in all 18 cases where follow-up was possible. The fetus was found to be PiZ in nine cases and all these pregnancies were terminated. Of the remaining pregnancies three cases aborted or were delivered prematurely and 13 proceeded to term without complications.     

Normal blood cell values in the early mid-trimester fetus

Samples of pure fetal blood from 116 fetuses of 15–21 weeks' gestation were obtained by direct vision fetoscopy. Ninety nine of these fetuses, presumed to be haematologically normal, were suitable for analysis. The data obtained show that the erythropoietic system is evolving rapidly in this gestational age range. The myeloid series shows no significant increase or decrease in numbers apart from eosinophils and basophils which increase significantly with gestational age whereas the platelet count remains constant. The growing application of fetoscopic blood sampling to the prenatal diagnosis and management of fetal blood disorders renders mandatory a knowledge of normal fetal blood values.     

Prenatal diagnosis of X-linked mental retardation with fragile (X) using fetoscopy and fetal blood sampling

Pure fetal blood, (uncontaminated with maternal blood), was obtained from two male fetuses at risk for X-linked mental retardation with fragile(X) at Xq27–28 by direct vision fetoscopy and fetal blood sampling. Both were shown to have this fragile site on the X chromosome while nine other fetal blood samples from pregnancies at risk for other X-linked diseases, or haemoglobinopathies did not show fragile sites at Xq27–28, and a blood sample from an abortus showed only 1 fragile site in 95 mitoses. Both pregnancies were terminated, cultures established from fetal tissues, and the diagnosis confirmed in each case. The problems of demonstrating the fragile site in tissues other than fetal blood in these pregnancies (such as amniotic fluid cells or fibroblasts from fetal tissues) are discussed.     

Genetic amniocentesis in biamniotic twin pregnancies by a single transabdominal insertion of the needle

We present a technique to aspirate amniotic fluid from both sacs in biamniotic twin pregnancies using a single abdominal insertion with a spinal needle. It was successful in 48 out of 55 cases of biamniotic twin pregnancies referred to our perinatal unit between 1985 and 1994. The single insertion technique was used when the inter-amniotic membrane was clearly evident and two separate free amniotic fluid pools could be reached by the operator with a single puncture. An adequate amount of amniotic fluid was sampled from both sacs to make a cytogenetic diagnosis in all cases. There were four fetuses with trisomy 21 in three twin pregnancies. In two cases, only one twin was affected whilst the co-twin was normal, so that a selective feticide was performed. No miscarriages due to genetic amniocentesis were reported. After 1990, all genetic amniocenteses in biamniotic twin pregnancies (except for one case due to late booking) were performed between 14 and 15 weeks of gestation and with all cases except one, it was possible to sample both twins by a single puncture. We suggest that early amniocentesis (14–15 weeks) by a single abdominal puncture could be a reliable and safe alternative to first-trimester chorionic villus sampling in twin pregnancies.     

Selective feticide of the affected twin by fetoscopic air embolism

Three twin pregnancies, each with one affected fetus (by microcephaly, haemophilia A and spina bifida respectively) were encountered. Selective feticide was performed at the patient's request by injection of filtered air into an umbilical vessel by fetoscopy. The method was successful in all three pregnancies two of which resulted in a live, healthy baby.     

The fetal intrahepatic umbilical vein as an alternative to cord needling for prenatal diagnosis and therapy

Seventy-one fetal blood samplings (FBS) were attempted from the intrahepatic portion of the umbilical vein (IHV) at 18–34 weeks; 54 were attempted primarily and 17 secondarily after a failed attempt at the placental cord insertion. Fetal blood was obtained in 89 per cent of the cases. Intravascular transfusion (IVT) was attempted on 31 occasions and successful in 24 (77 per cent). In all cases of failed sampling or transfusion via the IHV, prenatal diagnosis and/or therapy was accomplished using alternative procedures. On only one occasion was the procedure postponed. There were no losses or neonatal morbidity attributable to the procedure. FBS from the IHV may be considered as an alternative approach to sampling the placental cord insertion. It is recommended in cases where the approach to the placental cord insertion is difficult or hazardous.     

Prenatal diagnosis of thalassemia major by fetal blood analysis: Experience with 1000 cases

In this report we have summarized our experience with the prenatal diagnosis of β-thalassemia in 1000 pregnancies followed at least until 12 months after birth. In the majority of these cases, the thalassemia lesion was the nonsense mutation at the codon corresponding to amino acid 39, which produces the hematological phenotype of β-thalassemia. Fetal blood sampling was carried out by placental aspiration, by which a sufficient amount of fetal blood for analysis was obtained in the majority of cases (99 per cent). The fetal mortality associated with fetal blood sampling was 6·3 per cent. Those placental samples contaminated by maternal cells were successfully purified by Ørskov lysis. Fetal blood was analysed by globin chain synthesis on CM–52 columns, which gave reliable results. Two misdiagnoses (0·2 per cent) have been made of which one was due to a non-globin protein co-migrating with the β-chains while the other resulted from a misclassification of the type of thalassemia segregating in the family.     

Prenatal exclusion of late infantile metachromatic leucodystrophy in a late-presenting pregnancy by assay of fetal leucocytes

Metachromatic leucodystrophy was excluded in a fetus at risk, by assay of fetal blood collected at fetoscopy. Isolated fetal leucocytes were shown to have activities of arylsulphatase A and cerebroside sulphatase in the heterozygous range. The prediction was confirmed in the newborn.     

Umbilical and uterine flow velocity waveforms in pregnancies complicated by major fetal anomalies

A continuous wave Doppler unit was used to obtain umbilical and uterine artery flow velocity waveforms in pregnancies complicated by a major fetal abnormality. A total of 139 examinations were performed on 32 women between 26 to 41 weeks' gestation, and the records were reviewed to determine the changes associated with fetal malformation. The systolic/diastolic (A/B) ratio was used as an index of blood flow resistance in the umbilical artery and the systolic minus diastolic divided by systolic (A–B)/A for the branches of the uterine artery. Seventeen out of 32 patients showed high systolic/diastolic ratio in waveforms taken from the umbilical artery. In 30 out of 32 patients the uterine artery waveform was normal (in two patients the results were equivocal). It appears that a fetal mechanism may determine the changes in the umbilical placental circulation resulting in an umbilical artery pattern of high flow resistance in more than half of the patients with congenital anomalies.     

Fetal blood sampling via the umbilical cord using a needle guided by ultrasound report of 66 cases

Pure fetal blood has been aspirated in utero from the umbilical vein near the placental insertion of the cord using a twenty gauge needle under ultrasound guidance. Sixty-six samples were taken on 63 pregnancies between 17 and 32 weeks of gestation. One to two millilitres of blood can be obtained easily without amniotic fluid dilution or contamination by maternal blood, as confirmed by the measurements of the mean corpuscular volume, the histogram distribution of the red blood cells and the hematocrit. In all cases the Kleihauer test and isoelectrofocusing of the hemoglobins were performed. Coagulation factors were also studied in 60 cases. In 17 cases a medical abortion was voluntarily induced after the procedure, and the follow-up was normal during the observation period after sampling. In the other cases, pregnancies have continued normally and twelve healthy babies have already been born.     

Mitomycin C induced chromosome damage in fetal blood cultures and prenatal diagnosis of fanconi's anaemia

We report the use of fetal blood for the prenatal diagnosis of Fanconi anaemia (FA). The clastogenic action of Mitomycin C (MMC) is compared in blood cultures from different fetuses, normal controls and FA heterozygotes. The fetus at risk is shown to suffer from FA on the grounds of excessive chromosome breakage, both spontaneous and MMC induced.     

Tumour-associated antigens in maternal and fetal blood

Normal levels of cancer-associated antigen (CA) 19-9, neurone-specific enolase (NSE), cancer-associated antigen (CA) 125, and mucin-like carcinoma-associated antigen (MCA) during pregnancy were determined in 87 mothers and fetuses, using a solid-phase sandwich enzyme immunoassay. CA 19-9 concentrations were higher in the fetuses, whereas the other three tumour-associated antigen levels were higher in the mothers. Only fetal NSE and MCA levels were positively correlated with those in maternal serum. Contrary to adult samples, no difference was demonstrated between male and female fetal levels of CA 125. MCA was the only maternal marker that increased significantly with gestational age between 20 and 34 weeks' pregnancy.