Invasive Prenatal Diagnosis in Multiple Pregnancy
Ranjit Akolekar
The Facts
Introduction
Chorionic villus sampling (CVS) and amniocentesis are commonly performed invasive procedures for prenatal invasive diagnosis. Prior to any invasive procedure in pregnancy, there should be a discussion with women regarding the potential risks associated with the procedure.
There are various indications for carrying out invasive procedures in pregnancy. The main one remains the confirmation or exclusion of fetal aneuploidies following abnormal screening test results. Screening for fetal aneuploidies has evolved considerably in the past few decades from being based primarily on maternal age in the 1970s to the widespread implementation of routine first-trimester combined screening based on the assessment of fetal nuchal translucency thickness and maternal serum biochemistry.Screening in twins is based on similar principles and carried out in a similar way as screening in singletons. An important aspect assessing the risk of aneuploidies as well as undertaking invasive procedures is knowledge of the chorionicity of a multiple pregnancy as the risk of complications is associated with chorionicity rather than zygosity. At the first- trimester scan at 11-13 weeks' gestation, demonstration of a triangular echogenic placental chorionic tissue between the amniotic sacs at the junction with the placenta (λ-sign) is diagnostic of a dichorionic pregnancy, whereas absence of this echogenic chorionic tissue with the amniotic membranes joining the placenta (T-sign) is suggestive of a monochorionic pregnancy. In a dichorionic pregnancy, the risk of fetal aneuploidies is assessed independently for each of the fetuses. In monochorionic pregnancies, however, the risk is based on an average of likelihood ratios derived from assessment of maternal characteristics, nuchal translucency (NT) in both twins and maternal biochemistry.
Detection rates (DR) and false positive rates (FPR) of the combined screening for fetal aneuploidies between 11 and 13 weeks in twin pregnancies are comparable with singleton pregnancies with DR of 90% and FPR of about 6%. In monochorionic twin pregnancies, the false positive rate is higher compared to dichorionic pregnancies as the increased NT may be present in a higher proportion of chromosomally normal fetuses as a consequence of early presentation of twin-twin transfusion syndrome or due to higher incidence of fetal cardiac defects. The prevalence of multiple pregnancies is higher in mothers with advanced maternal age and those conceived by assisted reproductive techniques. Chromosomal abnormalities and structural defects are more common in multiple pregnancies, monochorionic pregnancies in particular. Therefore, accurate prenatal diagnosis is of crucial importance in management of these high-risk pregnancies.Invasive Diagnostic Procedures
Chorionic Villus Sampling (CVS)
Several cohort and case-control studies, systematic reviews and meta-analyses demonstrate that CVS in singleton pregnancies is safe with a procedure-related risk of pregnancy loss of 0.3-0.5%. The advantages of CVS in twins are similar to those in singleton pregnancies, but in multiple pregnancies, it has additional benefits as well. First, CVS allows for a definitive prenatal diagnosis at an earlier gestation and therefore provides reassurance if the results are normal, but if the results are abnormal, it facilitates earlier decision-making regarding termination of pregnancy. Second, early cytogenetic testing with CVS allows selective fetal reduction of the affected fetus with a potentially lower risk of complications. It is also likely to have a reduced psychological impact on parents if the selective fetal terminations are performed early in pregnancy.
Amniocentesis
Amniocentesis is a safe and accurate method for prenatal diagnosis in singletons. Similar to CVS, there is extensive evidence from cohort studies, systematic reviews and meta-analysis in singleton pregnancies suggesting that the procedure-related risk of miscarriage is not significantly different from controls and is 0.1-0.2%.
The advantage of amniocentesis compared to CVS is that the results are based on culture and analysis of amniocytes and therefore there is no chance of placental mosaicism as is noted in some pregnancies that undergo a CVS. The potential disadvantage of amniocentesis is that it can only safely be carried out at or after 15 weeks of gestation, and therefore the results of prenatal diagnosis are available to parents at a later gestation, which results in a management plan being made at a more advanced gestation compared to CVS. Scholars have carried out studies of amniocentesis in twins prior to 15 weeks' gestation, but similar to singleton pregnancies, early amniocentesis is associated with increased rate of fetal loss, failed procedures, multiple needle insertions, failed culture and fetal talipes equinovarus when compared to samplings carried out at gestation after 15 weeks. Early amniocentesis is therefore not recommended.Fetal Blood Sampling (FBS)
Fetal blood sampling (FBS) has been previously suggested for rapid karyotyping in twin pregnancies. With the widely accessible quantitative fluorescent polymerase chain reaction (QF-PCR) analysis with the results available within potentially 24-48 working hours, indications for FBS are limited. A study by Antsaklis et al. reported the results of FBS carried out in 84 twin pregnancies, mainly for prenatal diagnosis of hemoglobinopathies with an overall 8.2% procedure-related fetal loss up to 2 weeks post procedure. This is around four times higher than the risk of pregnancy loss associated with FBS in singleton pregnancy. Fetal blood sampling is still of clinical importance in multiple pregnancies, but this should be reserved for pregnancies with fetal anaemia, thrombocytopenia, fetal infection and fetal hydrops.
The Issues
Technical Considerations
Chorionic Villus Sampling
The main issues concerning CVS in multiple pregnancies include the technical challenges of sampling and higher procedure-related risks and sampling errors compared to singleton pregnancies.
Due to technical challenges associated with obtaining adequate samples in multiple pregnancies whilst ensuring low procedure-related loss rates, it is advisable that these procedures should only be carried out by experienced fetal medicine operators to ensure that accurate ‘mapping’ of the fetuses and placentae is done and correct ultrasound- guided placement of the instruments into each specific placenta is carried out. An important concern in multifetal CVS is sampling error and cross-contamination, mainly in cases when there are difficulties in identifying the placental margins by ultrasound or in pregnancies with fused dichorionic placentae. As with ultrasound assessment of fetal anatomy and the risk of chromosomal abnormalities, it is important to ensure that chorionicity, location of the amniotic sacs in relation to the cervix and the uterine fundus, their related placentas and the location of the cord insertions is accurately checked and documented prior to undertaking invasive testing. This is especially important as not only it will allow accurate sampling for prenatal diagnosis, but it is also important if a selective termination of pregnancy is required.Different techniques have been described for CVS in twin pregnancies, including transabdominal, transcervical or even a combined approach, especially in pregnancies with both anterior high and posterior low placentae. Transabdominal CVS can be performed with a special CVS needle which can be used for a single-puncture procedure or, alternatively, a double-needle approach can be used. If the latter is used, it is prudent to use different needles to ensure there is no contamination, especially when sampling dichorionic placentae. It is always good practice to sample the affected fetus that has either a structural defect or is at high risk of chromosomal abnormality first and then proceed to sample the placenta of the other fetus. Transcervical CVS can be performed using aspiration catheter or biopsy forceps.
As in the transabdominal approach, it is advisable to use a different instrument for each placenta. Systematic review of the literature has showed no superior CVS technique - that is, transabdominal, transcervical, a single-needle system, a double-needled system or a single uterine entry compared to double uterine entry are associated with similar risks and safety profiles.Amniocentesis
Similar to CVS procedures, the issues relating to amniocentesis are technical challenges of undertaking the procedure, higher procedure-related risks and sampling errors compared to singleton pregnancies. Therefore, amniocentesis in multifetal pregnancies should be undertaken by experienced operators as with other procedures. Amniocentesis in twin pregnancies requires accurate sampling of different amniotic sacs, and it is therefore important to identify the inter-fetal amniotic membrane to ensure sampling is done on either side of the membrane or across it from different sacs.
Essentially two different techniques are described for carrying out amniocentesis in multiple pregnancies. The first technique which is commonly used is two different needles inserted separately and sequentially into each amniotic cavity under ultrasound guidance. After the insertion of the first needle, about 20 mL of fluid is aspirated for cytogenetic evaluation and the needle is withdrawn. Then the second needle is inserted from a different or the same entry point and the second amniotic sac is sampled. In order to reduce the risk of resampling the same amniotic sac from a different angle, it is reasonable to choose two different entry points as far from each other as possible. The second technique, which avoids the potential for sampling error, is the use of a single-needle technique. An optimal view of the different amniotic sacs with an intervening amniotic membrane should be visualised and the needle is inserted into the first sac which is proximal to the insertion site. After aspiration of the amniotic fluid from the first sac, the stilette is reintroduced into the needle, which is then advanced into the second sac across the amniotic membrane into the distal sac. An initial 1 mL of fluid is aspirated and discarded (to lower the risk of contamination from the first sac) and then 20 mL is collected from the distal sac.
Some operators suggest the use of dye as a marker in one of the amniotic cavities to ensure accurate sampling; but in current practice, it is rarely used as it is feasible to carry out the procedure by ensuring sampling across different sides of the amniotic membrane.Procedure-Related Pregnancy Loss
A systematic review in 2013 reported that procedure-related pregnancy loss in twin pregnancies following CVS was about 1.0% higher than the background risk in controls. Similarly, in pregnancies undergoing amniocentesis, the procedure-related risk of miscarriage in twins was 1.1% higher compared to twins that did not undergo invasive testing. The authors did not find any statistically significant differences in pregnancy loss, regardless of the technique.
A recently published systematic review and meta-analysis by Mascio et al. also demonstrated that the procedure-related risk of pregnancy loss in twins following invasive procedures is not significantly different compared to control twin pregnancies that did not have an invasive procedure. In twin pregnancies undergoing CVS, the background risk of fetal loss was 2.0% compared with 1.8% in controls with a prevalence of procedure-related fetal loss of 0.5% (95%CI 0.0-2.2), which is similar to data from singleton pregnancies. Similarly, in twins undergoing amniocentesis, the incidence of fetal loss was 2.4% (95% CI 1.4-3.6) compared with 2.4% (95% CI 0.9-4.6) for twin pregnancies not undergoing amniocentesis. In addition to the total rate of fetal loss, the authors examined the fetal loss rate prior to 24 weeks and within 4 weeks of the procedure and found no significant difference in overall fetal loss in those that had an amniocentesis or CVS compared to those that did not. In twin pregnancies undergoing amniocentesis, there was no significant difference in the risk of fetal loss < 24 weeks of gestation (p = 0.11) or within 4 weeks of the procedure (p = 0.80) compared to those that did not have an invasive procedure. They reported similar results for fetal loss < 24 weeks in twin pregnancies undergoing CVS with no statistically significant difference in rate of fetal loss. Regarding different techniques, results from systematic reviews showed no statistically significant difference in fetal loss between the single uterine entry techniques versus the double uterine entry technique.
Special Considerations
Delayed Cytogenetic Testing
In some pregnancies, such as those that are discordant for fetal defects or chromosomal abnormalities, the management option of delayed cytogenetic testing in the third trimester can be discussed. In these cases, the choice between management options depends on weighing the pros and cons of the risk assessment of chromosomal abnormalities and the risks of procedure-related loss, particularly of the unaffected fetus. The decision to discuss delayed invasive testing option should take into account the implications of prenatal diagnostic testing on further management options in the pregnancy such as the potential for termination of pregnancy at that gestational age as the gestational limit for termination will vary depending on guidelines in different countries. A potential advantage of delayed or late cytogenetic testing is the reduction in risk of miscarriage or morbidity and morbidity secondary to extreme prematurity in case of preterm delivery of the unaffected co-twin. The disadvantage of delayed or late testing in the third trimester is the possibility of preterm delivery before invasive testing can be undertaken and, in such circumstances, the impact of delivery of a fetus with a structural or chromosomal abnormality on the perinatal outcome.
Testing in Monochorionic Pregnancies
One of the questions regarding cytogenetic testing in monochorionic pregnancies is whether invasive testing should be done for one or both of the monochorionic fetuses. Essentially, all monochorionic pregnancies are monozygotic, therefore by implication they have identical genotypes. There are, however, reports of monochorionic twins that are discordant for chromosomal abnormalities, and these are called heterokaryotypic pregnancies. The exact prevalence of these heterokaryotypic pregnancies is not known, but they are reportedly caused by mosaicism, skewed X inactivation, differential gene imprinting and micro deletions. Examples of such monozygotic twins with discordant karyotypes include mosaicism for Turner syndrome, Down syndrome, Patau syndrome, trisomy 1 and 22q11 deletion syndrome.
Caution should be exercised in twin pregnancies when the results of the QF-PCR suggest monozygotic pregnancy due to identical genotype. This is expected in a monochorionic pregnancy, but in cases of a dichorionic pregnancy with a similar gender, an identical genotype could represent a monozygotic pregnancy, but it could also be due to sampling error because of sampling the same placenta. The latter should be borne in mind and this possibility should be discussed with the parents. A further management option in this situation is an amniocentesis ensuring that sampling is from separate sacs.
Higher-Order Pregnancies
Data are limited regarding the risks of invasive procedures in higher-order multiple pregnancies. Similar to twin pregnancies, these are technically challenging procedures and should be carried out in tertiary fetal medicine units by experienced operators.
The Management Options
Chorionic villus sampling and amniocentesis are both relatively safe procedures for invasive prenatal diagnosis in twin pregnancies, and women and their families can be reassured that the overall rate of fetal loss is low. Although the overall rate of fetal loss in monochorionic twin pregnancies is higher compared to dichorionic twin pregnancies, the procedure-related risk of fetal loss, especially prior to 24 weeks or within 4 weeks of the procedure, is not significantly different in both when compared to those not undergoing invasive procedures. Although there is a paucity of data comparing different techniques such as single-needle versus double-needle insertion for carrying out procedures, there is evidence that use of a double-needle technique is not associated with higher risks of fetal loss and therefore can be used safely for carrying out these procedures. As in the case of singleton pregnancies, CVS should be carried out after 11 weeks of gestation and amniocentesis can be carried out after 15 weeks of gestation. It is critical that chorionicity of the pregnancy is determined prior to undertaking assessment of fetal anatomy and combined screening, but also to ensure that invasive procedures are carried out safely. Invasive procedures in twin pregnancies are technically challenging and they should be undertaken by experienced operators in specialist fetal medicine centres.
Key Points
• The prevalence of multiple pregnancies is increasing worldwide due to the use of assisted reproductive techniques
• Combined screening for aneuploidies has a high detection rate of chromosomal abnormalities as in singleton pregnancies.
• Assessment of chorionicity is not just an essential prerequisite for undertaking
a comprehensive assessment of fetal anatomy and combined screening for chromosomal abnormalities, but is an equally important assessment prior to undertaking invasive prenatal diagnosis.
• Pre-procedure planning with detailed ultrasound survey including assessment of chorionicity, location of placentae, amniotic sacs and labelling of twins is essential as it reduces the risk of sampling error.
• Chorionic villus sampling should not be carried out prior to 11 weeks of gestation and amniocentesis should not be carried out prior to 15 weeks of gestation.
• Women and their families should be reassured that the risk of procedure-related fetal loss before 24 weeks of gestation and within 4 weeks of the invasive procedure in twin pregnancies undergoing CVS and amniocentesis is low and not significantly different compared to the background rate of fetal loss in pregnancies not undergoing invasive procedures.
• There is no evidence of the superiority of any specific technique for performing invasive procedures, and although there is a lack of robust evidence comparing these techniques, both double-needle and single-needle entry appear to be safe.
• Invasive prenatal diagnostic procedures should be carried out by experienced operators in specialist fetal medicine centres.
References
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