Multiple pregnancies have gradually become common because of the expanded use of assisted reproductive techniques (ART). As a result, twin pregnancies are also related to the incidence of fetal structural anomalies and Down syndrome, mainly because of the overall higher maternal age and gene imbalance of parents using ART (Hansen et al., Reference Hansen, Kurinczuk, Milne, de Klerk and Bower2013; Odibo et al., Reference Odibo, Lawrence-Cleary and Macones2003). Conventional serum screening has been widely used for singleton pregnancies in recent decades. Although first-trimester and second-trimester aneuploidy screening is available for twin pregnancies, conventional serum screening is less accurate for twin than for singleton pregnancies.
Invasive prenatal diagnosis in twin pregnancies is associated with a risk of pregnancy loss that is higher than the baseline risk of loss among twin pregnancies (Cleary-Goldman & Berkowitz, Reference Cleary-Goldman and Berkowitz2005; Spencer, Reference Spencer2000; Vink et al., Reference Vink, Wapner and D'Alton2012). Thus, there is an urgent need to develop better screening methods for twin pregnancies with a high risk of fetal aneuploidy. MPS technology has become increasingly available and has been widely used to screen for trisomies 21, 18, and 13 in singleton pregnancies. Massively parallel sequencing (MPS) testing for fetal Down syndrome has also gradually become more commonly used for twin pregnancies and has shown high sensitivity and specificity (Canick et al., Reference Canick, Kloza, Lambert-Messerlian, Haddow, Ehrich, van den Boom and Palomaki2012; Gromminger et al., Reference Gromminger, Yagmur, Erkan, Nagy, Schöck, Bonnet and Stumm2014; Huang et al., Reference Huang, Zheng, Chen, Zhao, Zhang, Liu and Wang2014; Lau et al., Reference Lau, Jiang, Chan, Zhang, Lo and Wang2013; Leung et al., Reference Leung, Qu, Liao, Jiang, Cheng, Chan and Lo2013). Here, we report the screening of trisomies 21, 18, and 13 with MPS testing for twin pregnancies. All samples were prospectively collected and assayed immediately in the same manner.
Materials and Methods
Sample Collection
In this study, 92 women with twin pregnancies were recruited from January 1, 2013 to October 1, 2016. The included criteria were twin pregnancies that required invasive prenatal diagnosis using amniocentesis or clinical examination and follow-up of the neonates. An ultrasound examination was performed at 11+0 to 13+6 weeks to determine gestational age based on the fetal crown–rump length (CRL), diagnose any major fetal anomalies, and measure the fetal nuchal translucency (NT) thickness. In the twin pregnancies, an ultrasound examination was used to determine the CRL of the larger fetus, and chorionicity was identified by examining the junction of the inter-twin membrane with the placenta. The measured NT was reported as the difference from the expected normal mean for gestation (delta value; Wright et al., Reference Wright, Kagan, Molina, Gazzoni and Nicolaides2008). In our study, the measured free beta human chorionic gonadotrophin (β-hCG) and pregnancy-associated plasma protein A (PAPP-A) were not used in combination to estimate the patient-specific risk for trisomies 21, 18, and 13 because of their relatively low precision in this group of patients. The indications for invasive tests included positive MPS test results, acatastatic sonographic signs, family genetic history, maternal age, and history of acatastatic fetal pregnancy. Women with intrauterine fetal demise at the time of sampling or without fetal karyotype results or clinical examination and follow-up were excluded from this study. Informed written consent was obtained from participants who had undergone extensive individual counseling and an ultrasound scan. The cffDNA was isolated from maternal peripheral blood. The results of MPS testing were presented within two weeks after the sample was received. Positive results of MPS testing of cffDNA for fetal aneuploidies were identified via karyotype analysis. Fluorescence in-situ hybridization (FISH) was used to examine the placenta postnatally in cases of false-positive results.
Maternal Plasma DNA Sequencing
In this study, 10 mL of peripheral blood was obtained from each participant and stored in EDTA-containing tubes before invasive procedures were performed. The maternal blood samples were centrifuged twice within 8 hours after collection to extract cffDNA. MPS testing was satisfactory for the fetal fractions (above 10%, see Table 1). All subsequent procedures, including cffDNA extraction, DNA library construction, and sequencing were carried out in China according to a previously reported workflow (Dan et al., Reference Dan, Wang, Ren, Li, Hu, Xu and Zhang2012).
TABLE 1 Clinical Cases and the Sequencing Outcome
DC = dichorionic twin; MC = monochorionic twin; F = female; M = male; ART = assisted reproductive techniques; OI = ovulation induction; NP = natural pregnancy;NA = not available or not done; N = normal; No = not done; −ve = screened negative; PD = premature delivery; TD = term delivery; TP = terminate pregnancy.
Bioinformatics Analysis for the Detection of Trisomies 21, 18, and 13
The analyses of the sequencing data and the detection of fetal aneuploidies were consistent with the methodology previously reported for singleton pregnancies. A bioinformatic analysis was performed, with z scores ≥3 indicating the presence of a fetal trisomy 21, 18, and 13 (Chen et al., Reference Chen, Chiu, Sun, Akolekar, Chan, Leung and Lo2011; Chiu et al., Reference Chiu, Akolekar, Zheng, Leung, Sun, Chan and Lo2011; Gregg et al., Reference Gregg, Gross, Best, Monaghan, Baja, Skotko and Watson2013). In brief, the data were analyzed using Illumina software, and an effective assessment of the standard deviations beyond the central estimate (z score) identified chromosomes 21, 18, and 13. If the A-value was >3 and the T-value was >3, the sample was in the high-risk zone. If either the A-value was >3 or the T-value was >3, the sample was in the warning zones. If the A-value was ≤3 and T-value ≤3, the sample was in the low-risk zone. If the sample result was in the ‘low-risk zone’, it was considered normal. If the sample result was in the ‘high-risk zone’, it was affected. If the sample fell within Warning Zone 1, the sample was considered affected by mosaicism or partial trisomy. Such cases were reported as high risk but were accompanied by appropriate comments. Samples in Warning Zone 2 were likely affected by inadequate fetal DNA concentrations. If clinically permitted, blood sampling and sequencing were repeated. Otherwise, a high-risk report was issued. This classification of twin pregnancies was the same as that used for singleton pregnancies in the laboratory. Of course, mosaicism cases with a high percentage of the acatastatic cell line might be in the high-risk zone, but those with a very low percentage of mosaicism might be regarded as low risk.
Karyotype Analysis and FISH Detection
Invasive diagnostic procedures for positive cases involved traditional amniotic fluid cell culture karyotype analysis. A karyotype analysis was performed using the conventional Giemsa banding (G-binding) method (Lanza et al., Reference Lanza, Castoldi, Castagnari, Todd, Moretti, Spisani and Traniello1998). FISH was performed on postnatal placenta tissues. To prepare the placental tissues and probes, tissues (0.5 g) were collected from each of the four quadrants of the placenta. FISH was performed according to the previously established method (Klinger et al., Reference Klinger, Landes, Shook, Harvey, Lopez, Locke and Dackowski1992).
Results
Study Population
In the 92 patients, four positive reports were issued, and those patients underwent the prenatal diagnosis. The basic characteristics of the 92 parents are summarized in Table 2. Their average age was 30.54 years, ranging from 23 to 41 years. Twenty-one were 35 years old and older. The median gestational age at the time of MPS testing was 17.92 weeks. There were 53 dichorionic twin pregnancies and 39 monochorionic twin pregnancies. The method of conception included two types of pregnancy, of which 40 patients (including six who underwent ovulation induction) conceived naturally while 57.60% (53/92) conceived using ART. The four patients who received positive MPS results chose to undertake amniocentesis. The pregnancy outcomes included three terminations, 47 premature deliveries, and 42 term deliveries.
TABLE 2 Basic Characteristics of the 92 Parents
Karyotype Results and FISH
Fetal outcomes were ascertained either by prenatal karyotype or by clinical examination of the newborn after delivery. Four patients underwent karyotype analysis via amniocentesis. There were two cases of trisomy 21 and one case of trisomy 15 in the twin pregnancies. However, only one of the two fetuses was affected by trisomy 21 in all positive cases. A false-positive trisomy 13 result of MPS testing was correctly identified by karyotype analysis.
Identification of Fetal Trisomies 21, 18, and 13 with MPS Testing
Using the bioinformatics analysis approach, four cases were identified as positive. The cases of trisomy 21 (n = 2) and trisomy 15 (n = 1) identified with MPS testing were correctly identified with a full karyotype analysis of the amniocentesis sample. The trisomy 13 result identified with MPS testing was identified as a normal karyotype and correctly diagnosed as confined placental mosaicism. In this cohort, fetal trisomy 21, detected with MPS testing of cffDNA, was consistent with the karyotype analysis. The sensitivity and specificity of MPS testing for fetal trisomy 21 were 100%. Although the finding of fetal trisomy 13 with MPS testing of cffDNA was a false positive, it was correctly identified as confined placental mosaicism. However, no cases of trisomy 18 were detected (see Figure 1).
FIGURE 1 Identification of fetal trisomies 21, 18, and 13. The risk of fetal aneupoloidy is described by the T-value (x-axis) and A-value (y-axis). Red circles represent the positive results and open circles represent the negative results. The high-risk zone is defined by a T-value >3 and A-value >3.
Follow-Up Investigations
All cases were followed-up after birth. Table 1 summarizes the outcomes of these subjects, including the karyotype analyses of amniocentesis samples, sonographic anomalies, and the results of follow-up. However, it is regrettable that in one case, the parents decided to terminate both twins because one of the fetuses had trisomy 21. Although the MPS result of one fetus showed trisomy 13, the full karyotype analysis of the amniocentesis showed a normal karyotype. Placental tissue was collected and confined placental mosaicism was diagnosed. The remaining 87 cases were classified as negative for trisomies 21, 18, and 13. Among the 87 patients with reported pregnancy outcomes, 87 delivered normal fetuses (Table 2). The delivery of twin fetuses was reported for 87 participants, 47 had premature delivery, and 45.98% (40/87) were delivered at term (see Table 2).
Discussion
In twin pregnancies, invasive diagnostic methods are more complex than in singleton pregnancies because of the higher possible incidence of procedure-related miscarriage and stillbirth, the possibility of sampling errors, and the increase in technical difficulty (Lau et al., Reference Lau, Jiang, Chan, Zhang, Lo and Wang2013). Non-invasive prenatal diagnosis is as formidable as invasive prenatal diagnosis. Many studies have confirmed the accuracy of MPS in singleton pregnancies for the prenatal testing of fetal trisomies 21, 18, and 13. However, there are only five published studies with a small amount of data for twin pregnancies (Canick et al., Reference Canick, Kloza, Lambert-Messerlian, Haddow, Ehrich, van den Boom and Palomaki2012; Gromminger et al., Reference Gromminger, Yagmur, Erkan, Nagy, Schöck, Bonnet and Stumm2014; Huang et al., Reference Huang, Zheng, Chen, Zhao, Zhang, Liu and Wang2014; Lau et al., Reference Lau, Jiang, Chan, Zhang, Lo and Wang2013; Leung et al., Reference Leung, Qu, Liao, Jiang, Cheng, Chan and Lo2013). One study detected two pregnancies with trisomy 18, one of which was a false-positive result (Huang et al., Reference Huang, Zheng, Chen, Zhao, Zhang, Liu and Wang2014). Another previously published study correctly detected one fetus with trisomy 13 and seven twin pregnancies with trisomy 21 out of 25 twin pregnancies. The sensitivity and specificity of MPS for fetal trisomies 21 and 13 were 100% (Canick et al., Reference Canick, Kloza, Lambert-Messerlian, Haddow, Ehrich, van den Boom and Palomaki2012). Five studies used MPS testing of cffDNA to identify trisomy 21 in twin pregnancies, with a sensitivity of 1.000 (95% CI [0.846, 1.000]) and a specificity of 0.999 (95% CI [0.999, 0.999]). A meta-analysis of twin pregnancies tested with cffDNA has been published; the diagnostic rate for trisomy 21 was 93.7% (95% CI [83.6, 99.2]), and the FPR was 0.23% (95% CI [0.00, 0.92]; Gil et al., Reference Gil, Quezada and Revello2015). There were also nine trisomy 18 pregnancies and two trisomy 13 pregnancies that were classified correctly (Canick et al., Reference Canick, Kloza, Lambert-Messerlian, Haddow, Ehrich, van den Boom and Palomaki2012; del Mar Gil et al., Reference del Mar Gil, Quezada, Bregant, Syngelaki and Nicolaides2014; Huang et al., Reference Huang, Zheng, Chen, Zhao, Zhang, Liu and Wang2014). It is feasible to use MPS of cffDNA in twin pregnancies. The fraction of cffDNA in maternal plasma in twin gravidas is about 5% ~ 30% , and increases with increasing gestational age (del Mar Gil et al., Reference del Mar Gil, Quezada, Bregant, Syngelaki and Nicolaides2014). There is evidence that each fetus in dizygotic twin pairs can contribute different amounts of cffDNA to the maternal circulation, and the difference can vary by nearly twofold. Twin pregnancies are more complex than singleton pregnancies because monozygotic twins are genetically identical and dizygotic twins are genetically different, in which case it is possible that only one fetus may have aneuploidy (Leung et al., Reference Leung, Qu, Liao, Jiang, Cheng, Chan and Lo2013; Qu et al., Reference Qu, Leung, Jiang, Liao, Cheng, Sun and Lo2013). However, monozygotic twins discordant for chromosomes 13, 18, and 21 are reported (Choi et al., Reference Choi, Ko, Shin, Yang, Choi and Oh2013; Ramsey et al., Reference Ramsey, Slavin, Graham, Hirata, Balaraman and Seaver2012; Reuss et al., Reference Reuss, Gerlach, Bedow, Landt, Kuhn, Stein and Eiben2011). There are several potential mechanisms that may lead to discordant karyotypes in monozygotic twins. Monochorionic–diamniotic twins may be discordant for karyotypes, for which anaphase lagging, chromosomal non-disjunction, and trisomy rescue may be the underlying reasons (Machin, Reference Machin2009). In twin pregnancies undergoing first-trimester screening for trisomies using cffDNA testing, the fetal fraction is lower and the failure rate is higher than those of singletons (Sarno et al., Reference Sarno, Revello, Hanson, Akolekar and Nicolaides2016).Therefore, our study was carried out during the second trimester, screening for trisomies using cffDNA testing. It is feasible to use MPS testing of cffDNA in dizygotic twin pregnancies that are discordant for aneuploidy; however, doing so could obtain an erroneous result regarding the risk of aneuploidy because a high contribution from the disomic cotwin could lead to a satisfactory total fetal fraction. To avoid this potential risk, researchers have proposed that for non-invasive prenatal diagnosis in twin pregnancies, the lower fetal fraction of the two fetuses, rather than the total fetal fraction, be used to assess the risk of aneuploidies (del Mar Gil et al., Reference del Mar Gil, Quezada, Bregant, Syngelaki and Nicolaides2014). However, an inevitable result of such a policy is that no-result findings may be more common in twin pregnancies than in singleton pregnancies (Bevilacqua et al., Reference Bevilacqua, Gil, Nicolaides, Ordoñez, Cirigliano, Dierickx and Jani2015). Although there are five studies that have reported the successful use of MPS in twin pregnancies, direct evidence is very limited.
Our results suggest that three positive results of the 92 twin pregnancies (two cases with discordant fetal trisomy 21 and one case with discordant fetal trisomy 15) were correctly identified with high precision. One case that had been classified as having trisomy 13 by MPS testing was determined to be a false positive when the karyotype analysis of amniotic fluid showed a normal karyotype. The fetus was found to have a normal karyotype, but the placenta had an acatastatic karyotype because of confined placental mosaicism. Mosaicism is a condition in which an individual has two or more genetically distinct cell lines that develop from a single zygote. Mosaic trisomies do occur with varying degrees of trisomic cells present in different tissues. Affected individuals have both trisomic and euploid cell lines in the case of trisomy 21 mosaicism and Down syndrome. However, the differing percentages of trisomic and euploid cells in individuals with mosaicism for trisomy 21 is related to the varying proportion and/or tissue distribution of the trisomic cells (Papavassiliou et al., Reference Papavassiliou, York, Gursoy, Hill, Nicely, Sundaram and Jackson-Cook2009). Everyone may carry some cells with an extra mosaicism for trisomy 21 in some tissues (Hultén et al., Reference Hultén, Jonasson, Iwarsson, Uppal, Vorsanova, Yurov and Iourov2013). Discordance between MPS testing and karyotype analysis may reflect the limitations of the positive predictive value of MPS testing. Confined placental mosaicism is the presence of an acatastatic cell line in the placenta of a fetus with a normal karyotype. It is diagnosed when both normal and acatastatic cell lines are detected in placenta samples, while only the normal cell line is detected in fetal blood samples and by amniocentesis (Kalousek & Vekemans, Reference Kalousek and Vekemans2000). It occurs in approximately 1–3% of viable pregnancies, according to chorionic villus sampling (CVS). A large proportion of pregnancies with confined placental mosaicism proceed uneventfully, resulting in normal liveborn infants. Confined placental mosaicisms have been classified as two different types: mitotic and meiotic. In mitotic confined placental mosaicism, the conception is diploid, and the acatastatic division takes place in the progenitors of specific placental cell lineages. In contrast, in meiotic confined placental mosaicism, the zygote has an initial acatastatic chromosomal complement (most often a trisomy), and a subsequent mitotic ‘error’ causes the loss of the acatastatic chromosome in the true embryonic cell lineage, leaving the acatastatic cell line confined to the placenta, a process known as trisomic zygote rescue (Baffero et al., Reference Baffero, Somigliana, Crovetto, Paffoni, Persico, Guerneri and Fedele2012; Chan Wong et al., Reference Chan Wong, Hatakeyama, Minor and Ma2012; Kalousek, Reference Kalousek2000). Amniotic fluid-derived stem cells are derived from the amniotic fluid of developing fetus and give rise to three germ layers: the endoderm, mesoderm, and ectoderm. However, the origin of the cffDNA is uncertain. Evidence suggests that cffDNA mostly derives from placental trophoblast cells (Alberry et al., Reference Alberry, Maddocks, Jones, Abdel Hadi, Abdel-Fattah, Avent and Soothill2007; Bianchi, Reference Bianchi2004; Flori et al., Reference Flori, Doray, Gautier, Kohler, Ernault, Flori and Costa2004; Gahan & Swaminathan, Reference Gahan and Swaminathan2008; Rosner & Hengstschläger, Reference Rosner and Hengstschläger2013). Therefore, the karyotype of amniotic fluid is truer than cffDNA in identifying the fetus karyotype. MPS testing is very precise, but MPS testing of cffDNA might yield a low false-positive result (Chen et al., Reference Chen, Chiu, Sun, Akolekar, Chan, Leung and Lo2011; Mao et al., Reference Mao, Wang, Wang, Liu, Li, Zhang and Chen2014). Although MPS testing can detect an affected fetus in twin pregnancies, it cannot accurately note which of the two fetuses is the affected one. Nonetheless, the high precision of MPS testing can correctly detect the vast majority of pregnancies with normal fetuses, thereby reducing the need for invasive prenatal diagnosis. With the limited evidence, it is still premature to offer MPS testing of cffDNA for trisomies 21, 18, and 13 as a routine clinical service for twin pregnancies. However, all reported evidence suggests that MPS testing of cffDNA for trisomies 21, 18, and 13 in twin pregnancies is likely to be as effective as such testing for singleton pregnancies. Therefore, MPS testing of cffDNA for trisomies 21, 18, and 13 should be an option for women with twin pregnancies at increased risk, in lieu of amniocentesis, especially for those who have been shown to be at high risk by conventional testing, after extensive counseling from specialists with experience in this specialized field. Our study suggested that MPS testing of cffDNA for trisomies 21, 18, and 13 in twin pregnancies is available. Moreover, other aneuploidies can correctly be detected. If the result of MPS testing of cffDNA is positive, genetic counseling may facilitate patient-focused decision making. Although MPS testing of cffDNA could enhance the diagnostic spectrum of non-invasive prenatal testing and decrease the invasive prenatal diagnosis of higher precision, the parents should consider the questions about pregnancy loss following first-trimester CVS and mid-trimester genetic amniocentesis in twins. In both amniocentesis and CVS, fetal loss rate does not affect significantly the outcomes evaluated (Agarwal & Alfirevic, Reference Agarwal and Alfirevic2012; Simonazzi et al., Reference Simonazzi, Curti, Farina, Pilu, Bovicelli and Rizzo2010). The evaluation of fetus, placenta, umbilical cord, and amniotic cavity by ultrasound scan is beneficial to amniocentesis.
If one fetus in a twin pregnancy is discordant for structural and chromosomal anomalies, genetic counseling may facilitate patient-focused decision making. Selective fetal reduction is performed for a variety of indications in multiple pregnancies. The reduction of multiple pregnancies using potassium chloride (KCl) is a usual method for reduction in cases of diamniotic twins, but this method is complicated in cases of monochorionic multiple pregnancies, in which KCl cannot be used. For monochorionic twins, vascular ablative techniques are used for selective reduction. Such techniques include bipolar cord occlusion (BCO), interstitial laser and, more recently, radiofrequency ablation (RFA; Nobili et al., Reference Nobili, Paramasivam and Kumar2013). In this scenario, the usual fetal reduction procedure of monochorionic twins could endanger both fetuses, which share a placenta; thus, normal saline was used to create a cardiac tamponade and realize cardiac asystole, which is a novel way of reducing the acatastatic fetus in monochorionic twins without an adverse effect on the co-twin and or a maternal spillage effect of the commonly used drug (Gupta et al., Reference Gupta, Vaid and Arora2016).
Conclusion
In summary, MPS testing of cffDNA for trisomies 21, 18, and 13 is precise and effective for successfully detecting discordant structural and chromosomal anomalies and reduces the need for invasive prenatal diagnosis. In the future, massive parallel sequencing tests will be gradually used for non-invasive screening for fetal trisomies 21, 18, and 13 in twin pregnancies. When twin fetuses are classed as discordant for structural and chromosomal anomalies, selective fetal reduction is performed to reduce the birth defects after genetic counseling by specialists.
Acknowledgments
This work was supported by Department of Obstetrics and Gynecology, Zhongnan Hospital of Wuhan University, Department of Obstetrics and Gynecology, Renmin Hospital, Hubei University of Medical, Shiyan, China. This work was also supported by National Natural Science Foundation of China (81370707) to W.-H. Z, Science and technology support program of Hubei Province (2015BCA310) to W.-H.Z. We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted
