Diagnostic Procedures in Multiples


The number of multiple pregnancies has exponentially increased in the last several decades mainly as a result of women delaying childbearing until they are of advanced maternal age and the expanded use of assisted reproductive techniques. Studies have shown that there is an increased risk of structural and genetic abnormalities in twins compared with singleton gestations. This chapter discusses the indications, risks, techniques, and complications of invasive procedures that are used for prenatal diagnosis in multiple pregnancies, specifically chorionic villus sampling and amniocentesis.


multiple pregnancy, twins, genetic testing



The number of multiple pregnancies has exponentially increased in the last several decades mainly as a result of women delaying childbearing until they are of advanced maternal age and the expanded use of assisted reproductive techniques. The risk of chromosomal abnormalities increases with advancing maternal age; however, it has also been reported that there is an increased risk of structural and genetic abnormalities in twins compared with singleton gestations. This chapter focuses on procedures for invasive karyotype analysis: chorionic villus sampling (CVS) and amniocentesis ( Chapters 111 and 113 ).


Description, Technique, and Equipment

CVS and amniocentesis should be performed by a physician experienced with the procedures and ultrasound (US). Similar to singleton pregnancies, unsensitized Rh(D)-negative women should be given Rh 0 (D) immune globulin after CVS and amniocentesis to prevent Rh sensitization.

When performing invasive prenatal karyotype analysis on monochorionic twin gestations, the question remains whether one or both fetuses should be sampled. Although monochorionic twins theoretically should have identical karyotypes, case reports exist showing monochorionic twins discordant for chromosomal abnormalities. Although this is a rare event, because of this reported risk of discordant karyotypes, we recommend sampling both sacs if the patient is undergoing invasive prenatal genetic testing.

Chorionic Villus Sampling in Multiple Gestations

Before a CVS procedure, a transvaginal US scan should be done to evaluate the number of embryos, chorionicity, fetal viability, and any early US evidence of fetal anomalies. CVS in multiple gestations is performed in the same way as for singletons (see Chapter 113 ). The procedure can be done using the transabdominal or the transcervical method, or both. If there are two separate placentas, both methods may be used to avoid twin-twin contamination. If the placentas are fused or the chorionicity is unclear, the transcervical catheter or the transabdominal spinal needle should be directed to the placental margin farthest from the fusion site or the area closest to the umbilical cord insertion site. This approach helps avoid sampling the same fetus twice. To eliminate contamination of the samples, the aspiration device should not pass through one placenta to reach the second placenta. Although deoxyribonucleic acid (DNA) polymorphism and cytogenetic results can assist in ensuring accuracy of the results, an amniocentesis may be needed later in the pregnancy if the CVS results are unclear.

Amniocentesis in Multiple Gestations

Before an amniocentesis, US should be performed to evaluate the number of fetuses, chorionicity, fetal positions, fetal heart rates, placental location, and placental umbilical cord insertion sites.

Multiple-Needle Technique

First, an optimal needle insertion site is selected for each sac. The optimal insertion site avoids the maternal bowel and bladder and, if possible, the placenta. Using color Doppler may help to avoid the umbilical cord insertion site and large chorionic vessels.

Next, one should ensure that the required equipment is available (supplies are listed in Chapter 111 ). The maternal abdomen is prepared and draped. The sterile cover is placed over the US probe, and sterile US gel is used to relocate the optimal fluid pocket. The spinal needle is inserted under continuous US guidance. When the needle is ideally located in the fluid pocket, the needle stylet is removed, and the sterile syringe is attached to the needle. An appropriate amount of amniotic fluid is withdrawn, and the syringe is removed. When available, indigo carmine dye (2–3 mL) can be injected into the sac before withdrawing the needle.

After withdrawing the first needle, the operator turns to the second sac and performs amniocentesis with a fresh needle. The amniotic fluid in the subsequent sac should be clear, which confirms that the first sac has not been sampled twice. In higher-order multiple gestations, indigo carmine (if available) is injected into each subsequent sac to ensure a new sac is sampled each time. Methylene blue dye should not be used for this purpose because it has been associated with fetal skin staining, fetal small bowel atresia, and methemoglobinemia in newborns. Also, the mother should be alerted to potential greenish discoloration of her own urine over several hours or days as the dye is eliminated from the circulation.

In the absence of indigo carmine, care should be taken to ensure that each needle is inserted as far as possible from the other twin’s sac, and patients counseled about the potential for sampling the same sac twice.

Single-Needle Insertion Technique

The single-needle insertion technique has two benefits: (1) it reduces maternal discomfort because only one needle is inserted to withdraw amniotic fluid from both amniotic sacs, and (2) it does not require injecting indigo carmine dye to confirm separate sacs are sampled. The supplies needed are otherwise identical to the supplies required for the multiple-needle procedure.

First, the operator localizes an area where both amniotic sacs and the dividing membrane are clearly seen. The operator inserts the spinal needle into the more anterior sac and aspirates the appropriate amount of amniotic fluid. After attaching a new syringe to the spinal needle, the operator advances the needle through the dividing membrane into the second sac and aspirates the amniotic fluid. The disadvantages of this technique include the potential for contamination of the second sample by the first and the risk of membrane tearing.


The most common indication for CVS is to diagnose conditions in which DNA analysis or diagnostic cytogenetic analysis is possible early in pregnancy. The most common indications for amniocentesis include prenatal diagnosis of abnormal conditions and fetal lung maturity. Other indications include amnioreduction, diagnosing an intraamniotic infection, and confirming preterm premature rupture of membranes with an amnio dye test.


The contraindications for CVS are listed in Chapter 113 , and the contraindications for amniocentesis are listed in Chapter 111 .

Outcomes and Complications

CVS and amniocentesis appear to be safe for multiple gestations when performed by experienced clinicians. The complications are similar to CVS and amniocentesis in singleton gestations. However, the exact procedure-related loss rate after CVS or amniocentesis in multiple gestations, especially monochorionic twins, is unclear because of varying definitions of pregnancy loss, the natural loss rate secondary to maternal age, and the natural increased loss rate in twins compared with singletons.

Chorionic Villus Sampling

Although miscarriage rates of 4.5% have been reported, studies by Wapner et al. and Antsaklis et al. showed that CVS in multiple gestations has postprocedural loss rates comparable to amniocentesis performed in the second trimester. The advantage of CVS versus amniocentesis is that it can be performed earlier in pregnancy and allows patients to undergo an early selective termination if abnormal results are discovered.


Several studies evaluated pregnancy loss rates after amniocentesis in twin gestations. The range of loss rates reported included loss at less than 20 weeks (0%–6.3%), at less than 24 weeks (1.1% –10.6%), and at less than 28 weeks (1.1%–12.7%). A systematic review of articles published between 1970 and 2010 found that there was significant heterogeneity in the definition of “pregnancy loss” after amniocentesis in twins. The pooled procedure-related loss rate at less than 24 weeks was 3.5% (95% confidence interval [CI], 2.6–4.7). The pooled loss rates at less than 28 weeks and to term could not be calculated, as the studies exhibited unacceptable heterogeneity of data. Seven of the included studies had a control (no amniocentesis) group. The reported pooled odds ratio in these studies for total pregnancy loss among cases was 1.8 (95% CI, 1.2–2.7).

Monochorionic Twins

At the present time, studies evaluating CVS in twins are limited, and studies specifically evaluating the loss rates in monochorionic twins are lacking. Only two studies reported loss rates after amniocentesis in monochorionic twins. Millaire et al. reported no losses at less than 24 weeks’ gestation in 45 patients with monochorionic twin pregnancies who underwent amniocentesis. Cahill et al. reported a significant difference in loss rates before 24 weeks’ gestation in patients with monochorionic twins who had an amniocentesis compared with patients who did not have an amniocentesis (7.7% versus 1.4%; P = .02).

Jul 7, 2019 | Posted by in OBSTETRICS & GYNAECOLOGY IMAGING | Comments Off on Diagnostic Procedures in Multiples
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