Ethical guidance on human embryonic and fetal tissue transplantation: a European overview.

This article presents an overview of regulations, guidelines and societal debates in eight member states of the EC about a) embryonic and fetal tissue transplantation (EFTT), and b) the use of human embryonic stem cells (hES cells) for research into cell therapy, including 'therapeutic' cloning. There appears to be a broad acceptance of EFTT in these countries. In most countries guidance has been developed. There is a 'strong' consensus about some of the central conditions for 'good clinical practice' regarding EFTT. International differences concern, amongst others, some of the informed consent issues involved, and the questions whether an intermediary organisation is necessary, whether the methods of abortion may be influenced by the possible use of EFT, and whether EFTT should only be used for the experimental treatment of rare disorders. The potential use of hES cells for research into cell therapy has given a new impetus to the debate about (human) embryo research. The therapeutic prospects with regard to the retrieval and research use of hES cells appear to function as a catalyst for the introduction of less restrictive regulations concerning research with spare embryos, at least in some European countries. It remains to be seen whether the prospect of treating patients suffering from serious disorders with transplants produced by therapeutic cloning will decrease the societal and moral resistance to allowing the generation of embryos for 'instrumental' use.

Outcome of first-trimester chorionic villus sampling for genetic investigation in multiple pregnancy.

OBJECTIVES: To evaluate the efficacy of and risk associated with chorionic villus sampling for genetic investigations in multiple pregnancies, and to evaluate the accuracy of the ultrasonographic detection of chorionicity during the first trimester. PATIENTS AND METHODS: A total of 198 sets of twins and nine sets of triplets from 10 087 consecutive first-trimester pregnancies undergoing chorionic villus sampling were considered. Gestational age ranged from 7 to 12.6 weeks. Assessment and confirmation of chorionicity was based on a multiplicity of features. Dichorionicity was established in 169 sets of twins (85.3%) and trichorionicity in all triplet cases, while 29 twins were considered monochorionic. Chorionic villus sampling was performed transabdominally in all but one case, and identification of the placental insertion of the umbilical cord was the main benchmark for sampling. Sampling risks were evaluated by comparing clinical outcome with that of a control population of 63 dichorionic twin pregnancies which underwent no invasive procedure. RESULTS: Determination of the presence or absence of the lambda sign led to a correct assignment of chorionicity in all cases, while the presence of a membrane thickness of 2 mm or more reflected a 100% specificity with a 22% false negative rate. Sampling was successfully performed in all cases and in only four cases (1.0%) were two needle insertions needed. At follow-up no evidence of incorrect sampling was reported. Karyotyping was provided to all patients, and in 94.1% of cases both short and long-term culture methods were carried out. No difference in fetal and perinatal losses between the study and control populations was found, but a higher rate of deliveries before 37 weeks and of low birth weight babies was noted amongst controls. CONCLUSIONS: Chorionicity in twin pregnancy can be determined with certainty between 7 and 12 weeks of gestation; in cases of confluent placentas reliability is provided by determining the presence or absence of the lambda sign. This study indicates that first-trimester transabdominal chorionic villus sampling is a highly efficient, reliable, and relatively safe approach for genetic diagnosis in twin pregnancies. Although a precise evaluation of the relative risks of chorionic villus sampling and mid-trimester amniocentesis in twins must await randomized control studies, the advantages of a first-trimester diagnosis to enable early decision-making about selective fetal reduction are obvious.

Improvement in outcomes of multifetal pregnancy reduction with increased experience.

OBJECTIVE: This study was undertaken to evaluate a decade of data on multifetal pregnancy reductions at centers with extensive experiences. STUDY DESIGN: A total of 3513 completed cases from 11 centers in 5 countries were analyzed according to year (before 1990, 1991-1994, and 1995-1998), starting and finishing numbers of embryos or fetuses, and outcomes. RESULTS: With increasing experience there has been a considerable improvement in outcomes, with decreases in rates of both pregnancy loss and prematurity. Overall loss rates in the last few years were correlated strongly with starting and finishing numbers (starting number > or =6, 15.4%; starting number 5, 11.4%; starting number 4, 7.3%; starting number 3, 4.5%; starting number 2, 6.2%: finishing number 3, 18.4%; finishing number 2, 6.0%; finishing number 1, 6.7%). Birth weight discordance between surviving twins was increased with greater starting number. The proportion of cases with starting number > or =5 diminished from 23.4% to 15.9% to 12.2%. The proportion of patients >40 years old increased in the last 6 years to 9.3%. Gestational age at delivery did not vary with increasing maternal age but was inversely correlated with starting number. CONCLUSION: Multifetal pregnancy reduction outcomes at our centers for both losses and early prematurity have improved considerably with experience. Reductions from triplets to twins and now from quadruplets to twins carry outcomes as good as those of unreduced twin gestations. Patient demographic characteristics continues to change as more older women use assisted reproductive technologies. In terms of losses, prematurity, and growth, higher starting numbers carry worse outcomes.

RhD genotyping by quantitative fluorescent polymerase chain reaction: a new approach.

OBJECTIVE: To develop a new method of RhD/d genotype determination using a quantitative fluorescent PCR (QF-PCR) assay. METHODS: Polymerase chain reaction amplification (PCR) of fragments of exon 7 of both the RHD and RHCE genes was performed from 32 amniotic fluid and 26 chorionic villus samples known to be heterozygous for the RHD gene, 74 peripheral blood samples of RhD-positive blood donors (homozygous or heterozygous) estimated by serologic typing and 24 RhD-negative fetal samples. The number of copies of the RHD gene in RhD-positive samples was determined by comparing the fluorescent intensities of the amplification products specific for the RHD and the RHCE genes. RESULTS: A ratio of fluorescent intensities of 1:1 clearly indicated D/D homozygous individuals whereas a ratio of 1:2 was demonstrated in samples from D/d heterozygous individuals. The mean fluorescent intensity ratio of the peak areas of homozygous samples was 1.12 (SD 0.128), the mean ratio of the peak areas of heterozygous samples was 0.51 (SD 0.060). Complete agreement was obtained between RhD/d typing by QF-PCR and RhD genotypes assessed by family studies and serological methods. CONCLUSIONS: The fluorescent PCR-based DNA test allows easy, rapid and accurate determination of the zygosity for the RHD gene. This new technique provides useful information for the clinical management of pregnancies of sensitised RhD-negative mothers.

Selective termination for structural, chromosomal, and mendelian anomalies: international experience.

OBJECTIVE: Our purpose was to evaluate the outcomes of selective termination for fetal anomalies at 8 centers with the largest known experiences worldwide. STUDY DESIGN: Outcomes in 402 cases of selective termination in pregnancies with dizygotic twins from 8 centers in 4 countries were analyzed by year, gestational age at procedure, and indication. Reductions of fetuses were as follows: 2 to 1, n = 345; 3 to 2, 39; >/=4 to 2 or 3, n = 18. Potassium chloride was used in all procedures. RESULTS: Selective termination resulted in delivery of a viable infant or infants in >90% of cases. Loss up to 24 weeks occurred in 7.1% of cases in which the final result was a singleton fetus and in 13.0% of cases in which the final result was twins. Loss was 6.6% as a result of structural abnormalities, 7.0% for chromosomal abnormalities, and 10% for mendelian abnormalities (difference not statistically significant). Loss rates for procedures were as follows: 9-12 weeks, 5.4%; 13-18 weeks, 8.7%; 19-24 weeks, 6.8%; and >/=25 weeks, 9.1% (difference not statistically significant). Mean gestational age at delivery was 35.7 weeks. No differences were seen in outcomes by maternal age. The rate of very early premature deliveries has fallen in recent years. There were no known cases of disseminated intravascular coagulation or serious maternal complications. CONCLUSION: (1) Selective termination, in the most experienced hands, can be technically performed in all 3 trimesters with good outcomes in >90% of cases. (2) The previously observed increase in second- versus first-trimester losses has diminished. (3) Third-trimester procedures, where legal, can be performed with a good outcome for the surviving fetus.

Molecular analysis of 4q35 rearrangements in fascioscapulohumeral muscular dystrophy (FSHD): application to family studies for a correct genetic advice and a reliable prenatal diagnosis of the disease.

In the majority of facioscapulohumeral muscular dystrophy (FSHD) families (about 95%) the genetic defect has been identified as a deletion of a variable number of KpnI repeats in the 4q35 region, although no specific transcripts from this locus have been isolated so far. Molecular diagnosis is based on the detection by probe p13E-11 of EcoRI small fragments, in the range 10-28 kb, that are resistant to BlnI digestion. In family studies this probe is used with other 4q35 polymorphic markers to assign the haplotype associated with the disease. So far, we performed DNA analysis in 145 FSHD families and identified the 4q35 DNA rearrangement not only in affected individuals, but also in healthy subjects at risk of transmitting the disease, such as non-penetrant gene carriers and somatic mosaics. In addition we applied prenatal tests to 19 fetuses, using DNA extracted from chorionic villi samples (CVS) at 10-11 weeks of gestation. The FSHD status, as determined by the presence of BlnI-resistant small fragments associated with the at risk haplotype, was assessed in nine fetuses; in the remaining 10 cases the disease was excluded. Our results show that molecular analysis of 4q35 rearrangements is a reliable indirect method to perform diagnostic, predictive and prenatal tests in FSHD.

Rapid detection of chromosome aneuploidies by quantitative fluorescence PCR: first application on 247 chorionic villus samples.

We report the results of the first major study of applying quantitative fluorescence polymerase chain reaction (QF-PCR) assays for the detection of major chromosome numerical disorders. The QF-PCR tests were performed on a total of 247 chorionic villus samples, which were analysed blind, without any knowledge of the results obtained using conventional cytogenetic analysis. The aims of this investigation were to evaluate the detection power and accuracy of this approach by testing a large number of fetal samples and to assess the diagnostic value of each of the chromosome specific small tandem repeat (STR) markers used. In addition, we introduced three more markers specific for chromosomes 13, 18, and X to allow an accurate analysis of samples homozygous for a particular STR. Fluorescent labelled primers were used to amplify 12 STRs specific for chromosomes 21, 18, 13, X, and the amylogenin-like DNA sequence AMXY, expressed on the X and Y chromosomes. In this blind study of 247 fetal samples, 222 were correctly diagnosed by QF-PCR as normal for each of the five chromosomes investigated; 20 were diagnosed by QF-PCR as trisomic for chromosomes 21, 18, or 13, in agreement with the cytogenetic tests. Only one false negative result was observed, probably owing to the mishandling of the sample, which had been transferred through three laboratories before being analysed by QF-PCR. The 247 samples also included four cases of mosaicism or translocation; one case of mosaic trisomy 21 was detected by QF-PCR and the other cases were not identified by QF-PCR. The results of this investigation provide clear evidence that the QF-PCR assays are powerful adjuncts to conventional cytogenetic techniques and can be applied for the rapid and accurate prenatal diagnosis of the most frequent aneuploidies.

Coping with serum screening for Down syndrome when the results is given as a numeric value.

Forty-six pregnant women undergoing second-trimester biochemical screening for Down syndrome were asked to fill in the State-Trait Anxiety Inventory (STAI) questionnaire to assess their anxiety level at two different moments: when recruited to the study (at 11-13 weeks' gestation), and after the test result was communicated. The test result was given as a numeric value of risk (1/x), rather than as positive/negative. There were 10 women in whom the risk after biochemical screening increased (median delta risk = +1/535, range = 1/69 to 1/1083), whereas in the remainder the risk decreased (median delta risk = -1/1576; range = -1/142 to -1/4947) compared with the baseline value calculated on maternal age alone. Although only in a minority of women the STAI score after biochemical screening exceeded the reference range, the change in the STAI score was significantly higher when the risk increased, and the change in the risk estimate correlated significantly with the change in this index of anxiety. Three out of seven women with a 'negative' test, but increased risk estimate and increased anxiety after biochemical screening chose to undergo amniocentesis. A policy of providing the result of biochemical screening for Down syndrome as a numeric value, even for 'negative' tests, may cause some women to experience anxiety and request amniocentesis.

A new non-radioactive method for the screening and prenatal diagnosis of myotonic dystrophy patients.

Myotonic dystrophy (DM) is an autosomal dominant neuromuscular disease with an estimated incidence of 1 in 8000 and is the most common form of muscular dystrophy affecting adults. An unstable, untranslated part of the myotonic dystrophy protein kinase gene on the long arm of chromosome 19, composed of CTG repeats, is a genetic marker for DM. We have developed a fast non-radioactive polymerase chain reaction (PCR) procedure to detect the (CTG)n repeat expansion in DM patients and their relatives. Genomic DNA extracted from peripheral blood lymphocytes was amplified by PCR using specific primers to flank the region containing the triplets. To improve the amplification of this CG-rich region, either 10% glycerol or rTth DNA polymerase XL (extra long) was added to the reaction mixture, allowing amplification of huge expansions otherwise not polymerized by PCR. The PCR products were Southern blotted and the expansion revealed using a fluorescein-labelled (CTG)10 probe. We compared our results with those obtained in 24 patients and relatives using genomic digestion followed by radioactive Southern blot; in all cases the results overlapped. The same technique was used for prenatal diagnosis in pregnant DM mothers. We conclude that this new method is reliable for the genetic testing of DM patients.

First 10,000 chorionic villus samplings performed on singleton pregnancies by a single operator.

Chorionic villus sampling (CVS) was performed in 10,000 consecutive singleton pregnancies by a single principal operator, working in two institutions. The procedure was performed between 8 and 32 gestational weeks: transabdominal (TA) sampling was carried out in 8479 cases and transcervical (TC) in 1521. Patients were referred for chromosomal risk in 89.1 per cent of cases, Mendelian disorders in 10.5 per cent, and DNA investigations for paternity or infectious agents in 0.4 per cent of cases. The sampling success rate for both TA and TC techniques by the second insertion was 99.8 and 99.2 per cent, respectively. TA sampling succeeded in a higher number of cases at the first insertion (98 per cent vs. 86.8 per cent) and was associated with smaller samples (< 10 mg) in fewer cases (3.2 per cent vs. 4.9 per cent). Cytogenetic analysis was highly successful (99.4 per cent) and accurate; however, in one case a de novo structural rearrangement of chromosome I was not recognized. Mosaicism or rare trisomies were reported in 1.30 per cent of cases. Five diagnostic errors in DNA investigation (0.51 per cent) ended with the birth of affected fetuses. Fetal loss through 28 weeks' gestation in the pregnancies intended to continue was 2.58 per cent; the rate increased with maternal age (1.22 per cent at less than 30 years to 3.8 per cent at 40 years or more), while gestational age affected the abortion rate only at 8 weeks (odds ratio=2.22, P<0.05). Rates of premature delivery, low birth weight, and perinatal mortality did not differ from the Italian standards. By comparison with the Italian Birth Defects Registry data, no differences were found for the major malformations, including transverse limb reduction defects (TLRDs) (4.34 vs. 3.28 x 10,000). Total malformations and TLRDs did not show any pattern relation to either maternal age or gestational age.

Prenatal cystic fibrosis screening in a low-risk population undergoing chorionic villus sampling for fetal karyotyping.

Screening for cystic fibrosis (CF) has been offered to pregnant women seeking chorionic villus sampling (CVS) for prenatal chromosomal abnormality investigation. The mutation panel has increased over the years to include 8 mutations and can detect 65% of abnormal CF genes in the Italian population. Testing was offered to a total of 2214 consecutive pregnant women; 45 of them declined screening (take up rate: 98%). In 1055 of the 2169 pregnancies screened, the test was at first done on the fetus, while in the remaining cases both parents were investigated. Among parents 46 carriers were identified (2.1%), in 41 of whom the mutation was delta F508. In two couples both parents were heterozygous, and in one the fetus was affected and the pregnancy was terminated. Although CF testing offered to pregnant women undergoing invasive investigation such as CVS may not be the model for a mass screening, its very high effectiveness can represent an advantageous component of more comprehensive strategies.

Prenatal diagnosis by chorionic villus sampling.

Chorionic villus sampling (CVS) retains its great advantage over mid-trimester amniocentesis by producing early results. Moreover, rapid analytical techniques reduce significantly the waiting time between sampling and diagnosis, while recombinant DNA technology and human gene mapping progress amplify enormously the spectrum of the indications. The recent inclusion in the prenatal diagnosis package of screening tests based on DNA analysis for the major genetic diseases (i.e. cystic fibrosis, fragile-X mental retardation syndrome) may efficiently contribute to prevent the genetic disease. The role of CVS in twin pregnancy has been investigated and compared to amniocentesis. Although these techniques are equally safe, CVS should be considered the approach of choice for a number of technical advantage and in relation to selective fetal reduction in discordant twins. Recent reports have substantially contributed on the hypothetical relationship between limb reduction defects (LRDs) and chorion biopsy. The analysis of LRDs among more than 130,000 CVS reported to WHO CVS-Registry has been unable to find out any relationship between sampling and fetal malformations, including LRDs. In conclusion, first trimester CVS should be considered the gold standard procedure for prenatal diagnosis of genetic diseases.

Chorionic villus sampling safety. Report of World Health Organization/EURO meeting in association with the Seventh International Conference on Early Prenatal Diagnosis of Genetic Diseases, Tel-Aviv, Israel, May 21, 1994.

Accumulated experience of 138,996 cases of chorionic villus sampling shows that chorionic villus sampling is a safe procedure with an associated fetal loss rate comparable to that of amniocentesis. The chorionic villus sampling registry shows that chorionic villus sampling is currently performed primarily between 9 and 12 weeks' gestation and carried no increased risk of limb reduction defects: the overall incidence of limb reduction defects after chorionic villus sampling is 5.2 to 5.7 per 10,000, compared with 4.8 to 5.97 per 10,000 in the general population. Analysis of the pattern distribution of limb defects after chorionic villus sampling revealed no difference from the pattern in the general population. This applies specifically to transverse limb defects. Together with the overall incidence of limb reduction defects, these data provide no evidence for any risk for congenital malformation determined by chorionic villus sampling. Because chorionic villus sampling is currently performed generally after 8 completed weeks of pregnancy, few data are available for analysis of complications related to earlier procedures. Avoiding early chorionic villus sampling also excludes sampling in cases of early fetal death, which can be diagnosed reliably by ultrasonography at 9 weeks of pregnancy.

International, collaborative experience of 1789 patients having multifetal pregnancy reduction: a plateauing of risks and outcomes.

OBJECTIVE: To develop the most up-to-date, complete data base of multifetal pregnancy reduction (MFPR) from cases, and to provide the best counseling for couples with multifetal pregnancies. METHODS: From nine centers in five countries, 1789 completed MFPR cases were collected and outcomes evaluated. Pregnancy losses were defined as through 24 weeks and deliveries categorized in groups of 25-28, 29-32, 33-36, and 37 or more weeks. RESULTS: Overall, the pregnancy loss rate was 11.7% but varied from a low of 7.6% for triplets to twins and increased with each additional starting number to 22.9% for sextuplets or higher. Early premature deliveries (25-28 weeks) were 4.5% and varied with starting number. Loss rates by finishing number were highest for triplets and lowest for twins, but gestational age at delivery was highest for singletons. CONCLUSIONS: Multifetal pregnancy reduction has been shown to be a safe and effective method to improve outcome in multifetal pregnancies. Outcomes are worse with higher-order gestations and support the need for continued vigilance of fertility therapy.

Developmental expression of dystrophin, dystrophin-associated glycoproteins and other membrane cytoskeletal proteins in human skeletal and heart muscle.

Dystrophin, utrophin and the dystrophin-associated glycoproteins, beta-dystroglycan and adhalin, were analyzed, together with the membrane cytoskeletal proteins beta-spectrin, vinculin and talin, and adult and fetal myosin heavy chains, in 25 normal human fetuses from 8 to 24 weeks of gestation. Dystrophin was present in heart and skeletal muscle from 8 weeks although in the latter was mainly in the cytoplasm at this stage. Utrophin expression increased until around gestational weeks 19/21, but by 24 weeks immunostaining and immunoblot band intensities had reduced. Beta-dystroglycan was scarce in skeletal muscle at 8 weeks, increased with maturation and was more abundant in heart of the same age. Adhalin appeared later than beta-dystroglycan on skeletal muscle fiber surfaces, positivity became more intense as the fibers matured. In heart adhalin was detectable only in groups of cells at 12-16 weeks. From 8 weeks all fetal myotubes expressed beta-spectrin on their surfaces, while vinculin and talin positivity was mainly at the periphery of the fascicles, increasing with age. Adult slow myosin was seen in most myotubes at 10 weeks. Secondary myotubes then formed which increasingly expressed adult fast myosin, while still retaining fetal myosin. By 24 weeks most fibers expressing adult slow myosin had lost fetal myosin and were more mature in the expression of most membrane proteins. Muscle membrane organization during human fetal development is a complex process and takes place earlier in heart than skeletal muscle.

Somatic stability in chorionic villi samples and other Huntington fetal tissues.

We have studied different tissues from two affected fetuses with Huntington's disease (HD). In the first case the analysis was performed at 11 weeks of pregnancy; CAG repeats from seven different tissues were compared with the results obtained in the chorionic villi sample (CVS). We found 42 CAG repeats in all samples. In the second case the study was done at 12 weeks; eight tissues (including brain) were studied and compared with the CVS; in all of them, 44 CAG repeats were obtained. Our results show a somatic stability in the different analyzed tissues and suggest that mitotic instability can be a secondary consequence of neuronal degeneration and gliosis. Likewise, our data show great viability in the prenatal diagnosis (PD) of Huntington's disease using samples from any tissue.

Crown-rump length in aneuploid fetuses: implications for first-trimester biochemical screening for aneuploidies.

This study examined the effect of estimation of gestational age from the menstrual history compared with that from crown-rump length (CRL) measurement on the detection rate of screening for aneuploidies in the first trimester. Pregnancy-associated plasma protein A (PAPP-A) was assayed in blood collected prior to chorionic villus sampling in 356 women with unaffected pregnancies and 28 women with an aneuploid pregnancy. There were 14 Down's syndrome (DS) pregnancies. All pregnancies were dated from menstrual history and CRL measurement. The average CRL gestation in the aneuploid population was 2.5 days less than that derived from the LMP (95 per cent confidence interval (CI) for LMP-CRL gestation: using the algorithm based on unaffected pregnancies 0-3.5 days; using the matched case-control approach 1-4.5 days). The average CRL gestation in the DS population was 2 days less but this did not reach statistical significance (95 per cent CI for LMP-CRL gestation: using the algorithm -1 to 4.5 days; using the matched case-control approach 0 to 5.5 days). The detection rate of aneuploidies in the first trimester using maternal serum PAPP-A was reduced by 7 per cent (and by 3 per cent for DS) for a 5 per cent false-positive rate when using CRL rather than LMP to date the pregnancy. This phenomenon is a consequence of an apparent reduction of gestational age when estimated by CRL in the aneuploid population. Further studies are required to evaluate whether CRL is an unbiased estimate of gestation for Down's syndrome pregnancies.

First trimester fetal reduction: its role in the management of twin and higher order multiple pregnancies.

Multiple pregnancy may be the result of stimulated or non-stimulated, and of assisted or natural conception. As observed in the past decade, assisted conception technologies have significantly increased the prevalence of multiple pregnancy. The increase has been much more marked for triplets and higher order births. Rates of perinatal mortality and fetal and maternal complications are higher in twins than in singletons, and the adverse outcome rises with increasing number of multiples. Unplanned multiple pregnancy may be felt to be emotionally and physically so stressful an experience as to drive patients to refusal of pregnancy itself, or to want to reduce the number of fetuses to an acceptable standard. Fetal reduction techniques have emerged as a very effective medical approach to improve pregnancy outcome and a key option of patients trying to carry a pregnancy to term. Multiple fetuses are most frequently heterozygotes; therefore the risk of each of them being affected by a Mendelian disease or sporadic chromosomal aberration is an independent probability. Thus, the incidence of genetic defects in at least one fetus is increased and directly related to the order of multiples, and this makes it worthwhile to offer karyotyping of the fetus(es) to be spared, before the reduction procedure takes place. When a multiple pregnancy is established, one may conclude selective reduction is the most effective therapeutic approach for reducing risks.