Rapid determination of zygosity and common aneuploidies from amniotic fluid cells using quantitative fluorescent polymerase chain reaction following genetic amniocentesis in multiple pregnancies

Chih-Ping Chen1,2,3, Schu-Rern Chern2 and Wayseen Wang2

1 Department of Obstetrics and Gynecology, Mackay Memorial Hospital and 2 Department of Medical Research, Mackay Memorial Hospital and National Yang-Ming University, Taipei, Taiwan, Republic of China


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Following second-trimester twin amniocentesis, we used quantitative fluorescent polymerase chain reaction (QF-PCR) assays and polymorphic small tandem repeats (STR) for rapid determination of zygosity and common aneuploidies from amniotic fluid (AF) cells in four pregnancies with like-sex twins, fused placentae and inconclusive chorionicity. The first and the second cases were suspected to have inadvertent sampling of the same amniotic cavity twice. The first case showed a dizygotic (DZ) pattern and repeat amniocentesis was thus avoided. The second case was monozygotic (MZ) and was complicated by discordant fetal growth and twin–twin transfusion syndrome. The third case was associated with a co-twin malformation, occipital encephalocele. DNA studies revealed MZ twinning with a discordant structural defect. The fourth case was associated with co-twin abnormalities of cystic hygroma and hydrops fetalis. DNA studies showed DZ twinning with discordant structural and chromosomal defects. The QF-PCR assay with STR has the advantages of rapid determination of zygosity and common aneuploidies in AF cells. This simple test appears to be useful in the instances of possible inadvertent puncture of the same amniotic cavity twice during amniocentesis and of discordant fetal structural and/or chromosomal abnormalities following genetic amniocentesis in multiple pregnancies with uncertain chorionicity.

Key words: amniocentesis/aneuploidy/fluorescent polymerase chain reaction/twins/zygosity


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Zygosity determination at birth on all like-sex twins has been recommended by geneticists and epidemiologists (Derom et al., 1991Go; Machin, 1996Go; Bajoria and Kingdom, 1997Go; Keith and Machin, 1997Go; St Clair et al., 1998Go). This information is useful in organ transplantation compatibility, in twin genetic research concerning the biology and pathology of monozygotic (MZ) twinning, and in the investigation of discordance and concordance for many genetic diseases and disorders with multifactorial inheritance (Hall and López-Rangel, 1997Go). Prenatal zygosity can be determined sonographically by chorionicity, i.e. monozygosity in monochorionic–diamniotic (MC-DA) or monochorionic–monoamniotic (MC-MA) placentation, or by fetal gender, i.e. dizygosity in multiple pregnancy with discordant fetal external genitalia. Prenatal zygosity can also be determined by analysis of DNA polymorphism from amniotic fluid (AF) and placental samples. Recently, quantitative fluorescent polymerase chain reaction (QF-PCR) assays using small tandem repeats (STR) have become a useful method for rapid detection of fetal aneuploidies (Mansfield, 1993Go; Adinolfi et al., 1997Go; Pertl et al., 1997Go, 1999Go; Findlay et al., 1998Go; Tóth et al., 1998Go; Verma et al., 1998Go). However, its use in twin amniocentesis has not previously been described. This study aimed to use QF-PCR assays and STR to determine zygosity and common aneuploidies from AF cells following genetic amniocentesis in multiple pregnancies when chorionicity is not known definitively.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Clinical subjects
Case 1, a 28-year-old woman, gravida 2 para 1, was referred for genetic amniocentesis at 16 gestational weeks because of familial history of congenital anomalies. She did not receive any assisted reproduction treatment. Sonographic examination before amniocentesis revealed two male fetuses, a dividing membrane and fused placentae. Sonographic evidence of dichorionicity was inconclusive. The AF around each fetus appeared normal. Twin amniocentesis was performed under sonographic guidance without the use of a marker dye but with some difficulty. From each gestational sac, 22 ml of AF was obtained, of which 16 ml was used for cytogenetic analysis, 3 ml for alpha-fetoprotein (AFP) analysis and 3 ml for measurement of optical density (OD) by spectrophotometry at a wavelength of 340 nm. In the two AF samples, the OD values measured 0.384 and 0.381, and AFP values measured 13 125 and 13 138 ng/ml respectively. The differences in OD and AFP between two samples were so small that the physicians suspected the same amniotic cavity might have been punctured twice. SRY sequence was detected in both AF samples. We therefore used QF-PCR and STR from the AF cells for rapid determination of zygosity and common aneuploidies (Table IGo). The result showed a DZ pattern without common aneuploidies (Table IIGo). Repeat amniocentesis was thus avoided. Cytogenetic analysis of each AF sample revealed a 46,XY karyotype. The woman had an elective Caesarean section at 38 gestational weeks and delivered normal twins. Co-twin A weighed 2364 g and had Apgar scores of 10 and 10 at 1 and 5 min. Co-twin B weighed 2258 g and had Apgar scores of 10 and 10 at 1 and 5 min. Molecular studies using cord blood samples confirmed dizygosity. Pathological examination revealed dichorionic (DC) DA fused twin placentae.


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Table I. Primer sequences used in QF-PCR assays
 

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Table II. Genotypic information of the twins and their parents at eight STR markers specific for chromosomes 13, 18, 21 and X obtained by separate QF-PCR assays*
 
Case 2, a 30-year-old primigravida woman was referred for genetic amniocentesis at 22 gestational weeks. She did not receive any assisted reproduction treatment. Ultrasound revealed two female fetuses, a dividing membrane and fused placentae. Monochorionicity could not be excluded sonographically. Twin amniocentesis was performed under sonographic guidance without the use of a marker dye but with some difficulty. From each gestational sac, 26 ml of AF was obtained, of which 20 ml was used for cytogenetic analysis, 3 ml for AFP analysis and 3 ml for measurement of OD by spectrophotometry at a wavelength of 340 nm. In the two AF samples, the OD values measured 0.301 and 0.302, and AFP values measured 7181 and 6775 ng/ml respectively. The difference in OD between two samples was so small that the physicians suspected that the same amniotic cavity might have been punctured twice. No SRY sequence was detected in either AF sample. QF-PCR and STR were used for rapid determination of zygosity and common aneuploidies from the AF cells (Table IGo). The result showed a MZ pattern without common aneuploidy (Table IIGo). Repeat amniocentesis was suggested. During the repeat procedure, a marker dye indigo carmine was injected into the first amniotic sac after withdrawal of fluid to distinguish it from the second sac. Molecular studies of the AF cells showed a MZ pattern consistent with earlier studies. Cytogenetic analysis of each AF sample revealed a 46,XX karyotype. The pregnancy was later complicated by discordant fetal growth and twin–twin transfusion syndrome. The woman underwent serial amniocentesis procedures. She had a Caesarean section due to malpresentation at 38 gestational weeks and delivered twins with discordant growth. Co-twin A weighed 2518 g and had Apgar scores of 9 and 9 at 1 and 5 min. Co-twin B weighed 1102 g and had Apgar scores of 10 and 9 at 1 and 5 min. Molecular analysis using cord blood samples confirmed monozygosity. Pathological examination revealed MC DA fused twin placentae.

Case 3, a 20-year-old primigravida woman was referred for genetic amniocentesis at 20 gestational weeks due to a twin pregnancy discordant for fetal malformation, occipital encephalocele. She did not receive any assisted reproduction treatment. Ultrasound revealed a normal male fetus and an abnormal male fetus with occipital encephalocele, a dividing membrane and fused placentae. Sonographic determination of chorionicity was inconclusive. Twin amniocentesis was performed under sonographic guidance with the use of a marker dye. Indigo carmine was injected into the first amniotic sac containing the abnormal fetus after withdrawal of fluid to distinguish it from the second sac containing the normal fetus. From each gestational sac, 23 ml of AF was obtained, of which 20 ml was used for cytogenetic analysis and 3 ml for AFP analysis. The AFP values measured 55 416 and 30 183 ng/ml respectively. The SRY sequence was detected in both AF samples. QF-PCR and STR were used for rapid determination of zygosity and common aneuploidies from the AF cells (Table IGo). The result showed a MZ pattern without common aneuploidies (Table IIGo). Cytogenetic analysis of each AF sample revealed a 46,XY karyotype. Careful ultrasound follow-ups did not find the development of polyhydramnios. The parents refused selective fetocide and insisted on an expectant management. The woman had a Caesarean section due to malpresentation at 38 gestational weeks and delivered twins with a co-twin occipital encephalocele. Co-twin A, the normal neonate weighing 2300 g, had Apgar scores of 9 and 10 at 1 and 5 min. Co-twin B, the malformed neonate weighing 2100 g, expired after birth. The chorionicity could not be determined due to poor configuration of the placenta after difficult manual removal.

Case 4, a 32-year-old primigravida woman was referred for genetic amniocentesis at 16 gestational weeks due to a twin pregnancy with a structurally abnormal co-twin. She did not receive any assisted reproduction treatment. Ultrasound revealed an abnormal female co-twin A with cystic hygroma and hydrops fetalis, a normal female co-twin B, a dividing membrane with uncertain chorionicity, and fused placentae. Twin amniocentesis was performed under sonographic guidance with the use of a marker dye. Indigo carmine was injected into the first amniotic sac containing the abnormal fetus after withdrawal of fetal ascitic fluid and amniotic fluid to distinguish it from the second sac containing the normal fetus. From each gestational sac, 20 ml of AF was obtained, of which 17 ml was used for cytogenetic analysis and 3 ml for AFP analysis. A 10 ml sample of fetal ascitic fluid was obtained for cytogenetic analysis. In the two AF samples, the AFP values measured 11 086 and 10 003 ng/ml respectively. No SRY sequence was detected in either AF sample. QF-PCR and STR were used for rapid determination of zygosity and common aneuploidies from the AF cells (Table IGo). The result showed a DZ pattern discordant for monosomy X (Table IIGo). Cytogenetic analysis revealed a 46,XX karyotype in the AF (46,XX = 20/20) of the normal co-twin B and a 45,X karyotype in the AF (45,X = 43/43) of the abnormal co-twin A. The ascitic fluid of co-twin B had 45,X/46,XX mosaicism (45,X/46,XX = 14/86). The pregnancy was later complicated by preterm labour and pre-eclampsia. The woman opted to terminate the pregnancy at 20 gestational weeks. A hydropic abortus with cystic hygroma was delivered with a weight of 952 g and a normal co-twin B with a weight of 338 g. Examination of the placentae revealed DC-DA fused twin placentae. Molecular studies using tissues from the abortuses confirmed DZ twinning. Cytogenetic analysis of the malformed abortus showed 45,X/46,XX mosaicism in the placental tissue (45,X/46,XX = 37/63) and the blood (45,X/46,XX = 23/77), and a 45,X karyotype in the liver (45,X = 100/100) and skin (45,X = 100/100). The blood lymphocytes of the normal abortus had a 46,XX karyotype (46,XX = 20/20).

DNA analysis
Fetal DNA was extracted from 3 ml AF and parental DNA from 5 ml peripheral blood using a DNA extraction kit (Qiagen, Hilden, Germany). Fetal sex was quickly determined by PCR with primers specific for the SRY gene (Berta et al., 1990Go). QF-PCR and STR were later used for rapid determination of zygosity as well as common aneuploidies, i.e. trisomies 13, 18, and 21, and sex chromosomal abnormalities. Eight pairs of polymorphic markers D13S217, D13S285, D18S70, D18S59, D21S1252, D21S1446, DXS987, and DXS1001 were separately used for PCR amplification (http://www.gdb.org. and http://lpg.nci.nih.gov/CHLC. 1999 Internet Web of STR). The primer sequences are listed in Table IGo. Each of the forward primers was labelled at the 5' end with one of the following fluorescent dyes: 6-FAM (6-carboxyfluorescein), HEX (4,7,2',4',5',7'-hexachloro-6-carboxyfluorescein), or TET (4,7,2',7'-tetrachloro-6-carboxyfluorescein) to enable the visualization and analysis of the PCR products. The 6-FAM fluorescent dye is visualized as blue, HEX as yellow, and TET as green using the filter C. The PCR analysis was carried out in a total volume of 15 µl containing 60 ng DNA, 0.33 µmol/l of each primer, 0.25 mmol/l dNTPs, 1x PCR buffer, 1.5 mmol/l MgCl2, and 0.6 U AmpliTaq (Perkin Elmer, Norwalk, CT, USA). The thermal profile was according to the manufacturer's protocol. After initial denaturation at 95°C for 5 min, 10 cycles of PCR amplification were done: 15 s denaturation at 94°C, 15 s annealing at 55°C, and 30 s extension at 72°C, after which 20 cycles of PCR amplification were performed: 15 s denaturation at 89°C, 15 s annealing at 55°C, and 30 s extension at 72°C. The final extension was at 72°C for 10 min. The DNA fragments were diluted 10 times (HEX-labelled products) or 20 times (6-FAM- or TET-labelled products) and were mixed together with formamide and Genescan-500 TAMRA size standard (Applied Biosystems, Foster City, CA, USA). The DNA fragments were resolved on a 6% denaturing polyacrylamide gel and analysed on an automated DNA sequencer (ABI PRISM 377, Applied Biosystems) running Genescan Analysis 2.1 software (Applied Biosystems). The fluorescence intensities were calculated based on the peaks on the electrophoretograms. The amplification products were sized accurately. The same procedures were applied to cord blood or aborted tissues for confirmatory studies.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Table IIGo shows allelic distribution of eight STR specific for chromosome 13 (D13S217, D13S285), chromosome 18 (D18S70, D18S59), chromosome 21 (D21S1252, D21S1446), and chromosome X (DXS987, DXS1001) in the four pairs of concordant-sex twins and their parents. The twin AF samples from case 1 revealed genetic diversity in five STR markers D13S217, D13S285, D18S59, D21S1252, and DXS987, disomy for chromosomes 13, 18, and 21 and monosomy for chromosome X specific markers. The result showed a DZ pattern without common aneuploidies. The twin AF samples from case 2 revealed genetic identity in all eight STR markers and disomy for chromosomes 13, 18, 21, and X specific markers. The result showed an MZ pattern without common aneuploidies. The samples from case 3 revealed genetic identity in all eight STR markers, disomy for chromosomes 13, 18, and 21 and monosomy for chromosome X specific markers. The result showed an MZ pattern without common aneuploidies. The samples from case 4 revealed genetic diversity in six STR markers D13S217, D13S285, D18S59, D21S1252, DXS987, and DXS1001 and disomy for chromosomes 13, 18 and 21. For chromosome X, co-twin A was monosomic, whereas co-twin B was disomic. The result showed a DZ pattern discordant for monosomy X. Figures 1 and 2GoGo illustrate electrophoretograms of an MZ pattern and a DZ pattern respectively.



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Figure 1. Partial electrophoretograms of case 3 revealed a monozygotic pattern. Informative microsatellite analysis showed two alleles, 245 bp and 249 bp for D13S217 and one allele 214 bp for DXS987 in both twins.

 


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Figure 2. Partial electrophoretograms of case 4 reveal a dizygotic pattern discordant for monosomy X. Informative microsatellite analysis showed two alleles, 245 bp and 249 bp in co-twin A and two alleles 247 bp and 249 bp in co-twin B for D13S217, and one allele 212 bp in co-twin A and two alleles 212 bp and 214 bp in co-twin B for DXS987.

 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
DZ twins are always DC-DA. However, 8–10% of DC-DA like-sex twins are MZ (Derom et al., 1991Go). Therefore, in the case of DC-DA like-sex twins, it is impossible to determine from chorionicity alone whether the twins are MZ or DZ. MC twins are at a higher risk of fetal loss, perinatal mortality, preterm birth and intrauterine growth retardation than DC twins due to fetoplacental vascular anastomoses (Bajoria and Kingdom, 1997Go; Sebire et al., 1997Go; Sepulveda, 1997Go). Routine determination of chorionicity during the first trimester has been recommended in multiple pregnancies and the optimal timing for determination of chorionicity through the lambda or twin-chorionic peak sign is at around 10–14 weeks' gestation (Sepulveda et al., 1996Go, 1997). As pregnancy advances from the first trimester to the second trimester, the lambda sign is less prominent and the determination of chorionicity becomes more difficult. On the other hand, the DNA polymorphism from AF cells obtained through genetic amniocentesis can be used to determine zygosity and indirectly provides clues to the chorionicity (Norton et al., 1997Go). If the like-sex twins are DZ, they must be DC; whereas if they are MZ, they may be either MC or DC. An MZ result should raise consideration of possible monochorionicity and prompt colour Doppler studies in identifying communicating superficial placental vessels and evaluating regional flow patterns in the twin fetuses.

Unlike chorionicity, routine prenatal zygosity determination is rarely indicated because it seldom influences the fetal outcome or obstetric management of multiple pregnancies (Bajoria and Kingdom, 1997Go). However, simultaneous prenatal zygosity determination following genetic amniocentesis can provide useful information for obstetricians in the case of a co-twin's demise or impending fetal death of one twin in a like-sex pair in the second or third trimester without exact knowledge of the chorionicity. If the twins are DZ, it may be safe to continue the pregnancy. If the twins are MZ, rapid delivery is recommended whenever the surviving co-twin is of sufficient maturity (Norton et al., 1997Go). Furthermore, MZ twins may be genetically and/or phenotypically discordant for structural defects, X-linked traits, autosomal traits and chromosomal abnormalities (Hall and López-Rangel, 1997Go). Structural or karyotypic discordance in MZ twins has important implications for prenatal diagnosis and management, particularly in considering selective termination of the affected co-twin. At least 70% of MC twins have inter-twin vascular anastomoses in the placentae that can possibly be visualized by colour Doppler ultrasonography (Hecher et al., 1995aGo,bGo). In order to minimize fetocide-related risk to the normal co-twin, selective termination in MC MZ twins discordant for structural and/or chromosomal abnormalities should preferably use umbilical vessel occlusion by either ligation of the umbilical cord or endoscopic coagulation of umbilical vessels rather than using intracardiac injection of lethal agents.

Fluorescent PCR has been shown to be highly efficient, accurate, and reliable (Van de Velde et al., 1999Go). In this study, we used a one-stage approach of eight pairs of highly polymorphic single-locus microsatellite probes on chromosomes 13, 18, 21 and X. An alternative two-stage approach can be more cost-effective by using two STR initially and only proceeding to more if the results are identical. DNA fingerprinting using multilocus probes to detect variable number of tandem repeats (VNTRs) has been applied for zygosity determination (Appelman et al., 1994Go; Bamforth, 1999Go). Multilocus VNTR probes such as 33.6 and 33.15 are more informative than single-locus probes. However, multilocus minisatellite probes have some disadvantages, i.e. (i) the sophisticated technical expertise needed to obtain good DNA patterns; (ii) the relatively large amount of DNA required for the Southern blotting studies; and (iii) the difficulty in interpretation of a large number of DNA fragments (Trent, 1997Go). On the other hand, the advantages of QF-PCR assays with STR for detection of zygosity and common aneuploidies in AF cells include: (i) the rapid testing and easy interpretation; (ii) the requirement of only a small number of AF cells for good results; (iii) the capability of detecting trisomies resulting from translocation and low levels of mosaicism; and (iv) the simultaneous determination of parental origin and of genetic occurrence at meiosis I or II (Adinolfi et al., 1997Go). Nevertheless, using this method may fail to detect some subtle structural chromosomal abnormalities such as inversions, cryptic translocations, minor duplications, deletions and insertions. In cases of monozygosity in which the patterns of single-locus markers are determined by PCR and more polymorphisms need to be studied, a Southern blot technique using multilocus VNTR probes may be required. The use of fluorescent in-situ hybridization (FISH) on nuclei in interphase on uncultured AF cells is limited to major numerical chromosomal abnormalities. Chromosome-specific probes (CSP) and FISH will miss many structural chromosomal abnormalities (Evans et al., 1999Go). QF-PCR with STR, like CSP-FISH, is a useful adjunct to karyotype for high risk pregnancies, but should not be seen as a replacement for karyotype. The QF-PCR assay with STR has the advantages of rapid determination of zygosity and common aneuploidies in AF cells. In this study, QF-PCR assay using STR appears to be useful in the instances of possible inadvertent puncture of the same amniotic cavity twice and of discordant structural and/or chromosomal abnormalities following genetic amniocentesis in twin pregnancies with uncertain chorionicity. Much more data and reflection are needed for better understanding of its utility in multiple pregnancies.


    Acknowledgments
 
The work was supported by a research grant (NSC-89–2314-B-195-011) from the National Science Council, Taiwan, R.O.C.


    Notes
 
3 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Mackay Memorial Hospital, 92, Section 2, Chung-Shan North Road, Taipei, Taiwan, Republic of China Back


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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on May 28, 1999; accepted on December 14, 1999.