Hum. Reprod. Advance Access originally published online on June 13, 2006
Human Reproduction 2006 21(8):2175-2179; doi:10.1093/humrep/del133
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Determination of twin zygosity using a commercially available STR analysis of 15 unlinked loci and the gender-determining marker amelogenin – a preliminary report
1 Department of Obstetrics and Gynecology and 2 Section of Transfusion Medicine, Department of Medicine, Taipei Veterans General Hospital, National Yang-Ming University School of Medicine, Taipei, Taiwan, R. O. C
3 To whom correspondence should be addressed at: Department of Obstetrics and Gynecology, Taipei Veterans General Hospital, 201, Shih-Pai Rd., Sec. 2, Taipei 112, Taiwan, R. O. C. E-mail: mjyang{at}vghtpe.gov.tw
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Abstract |
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BACKGROUND: The aim of this preliminary study was to estimate the accuracy of the zygosity determination in twin pregnancies. METHODS: Seventy-three sets of twin pregnancies were enrolled in this study, including 48 sets of twins resulting from assisted reproductive techniques (ART) and 25 sets of spontaneously conceived twins. Determination of zygosity was made by PCR-amplified short tandem repeat (STR) analysis with a commercially available panel, comprising 15 autosomal, codominant, unlinked loci and the gender-determining marker, amelogenin. Monozygotic (MZ) twins were determined when all these unlinked loci and the gender-determining marker were identical. Chorionicity and placenta were examined after delivery of the newborns to check their relationships to the twin zygosities. RESULTS: Three of 48 (6.25%) sets of twins produced by ART and 18 of 25 (72%) sets of spontaneously conceived twins were MZ. Monozygosity could be evaluated based on ‘like sex’ in spontaneously conceived twins, because they had a greater incidence of MZs than those produced by artificial reproductive techniques. The MZ prediction rate was 78.6%, and the overestimated rate was 21.4% if the monochorionic like-sexed twins (LST) had a grossly single placenta. The underestimated rate of MZs was 26.7% when the dichorionic LST had apparently separate placentas. CONCLUSION: With the DNA-based 15 STR analysis amplified in a multiplex PCR, the determination of the zygosity in multifetal pregnancies is not only cost and time saving but also yields greater sensitivity and precision than conventional methods.
Key words: chorionicity/placenta/short tandem repeat/twins/zygosity
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Introduction |
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A significant proportion of monozygotic (MZ) twin sets are not identical, which may result from the following possibilities: postzygotic genetic changes, antenatal environmental effects, post-natal environmental experiences and unequal allocation of numbers of cells to MZ twins (Machin, 1996
). Every so often, confusion of twin zygosity comes into existence on the parents and the twins themselves. Additionally, for medical purposes, such as organ transplantation between twins, though not common, exclusion of the use of immunosuppressive agents is crucially based on the zygosity (St. Clair et al., 1998). Good health care or giving warning for one twin is important when the other twin suffers from cancer or some other hereditary diseases if they are MZ. Consequently, acknowledging to the parents, the zygosity of the twins has been advocated (Keith and Machin, 1997). Assisted reproductive techniques (ART) including artificial inseminations by husband (AIH), IVF and ICSI have long been used to treat couples with fertility problems. Induction of ovulation with clomiphene or gonadotrophins was given either for anovulatory patients or for timing of ovulation during ART programs. Unfortunately, this act produced a higher incidence of multifetal pregnancies. To understand the etiology of MZ twinning following ART, we need to understand whether the rate is actually higher than that which occurs in nature.
Blood types and genders of newborns, HLA typing, chorionicity evaluated by prenatal ultrasonography and the examinations of the placentas after delivery have been used to determine twin zygosity (Scardo et al., 1995). However, these tests are not completely accurate because misinterpretations may occur; for example, like blood type and/or like gender newborns are not necessarily MZ twins, dichorionicity and separate placentas are not necessarily dizygotic (DZ) twins (Scardo et al., 1995). DNA fingerprinting analysis is currently used in various kinship studies (Tzeng et al., 2000a) and population studies (Pu et al., 1998; Tzeng et al., 2000b; Kim et al., 2003). Because the MZ twins come from the same fertilized ovum, their genomes should be identical. On this basis, PCR-amplified short tandem repeat (STR) analysis with multiple unlinked loci had been used for the determination of MZ twins for various purposes (Becker et al., 1997; Somkuti et al., 2000; Wachtel et al., 2000; Steinkampf et al., 2003; Fang et al., 2004; Wang et al., 2004). Results tend to be more accurate when more unlinked loci are used. In this present study, a commercially available panel, AmpFLSTR® IdentifilerTM, composing of 15 autosomal, codominant, unlinked loci and the gender-determining marker amelogenin, was used for the determination of twin zygosity that would provide a more precise incidence of MZ twins. At the same time, chorionicity and placenta would be examined after delivery of the newborns to determine their relationships to the twin zygosities.
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Materials and methods |
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Case eligibility
Within the 2-year period, from January 2002 to December 2003, all the twin pregnancies, including the spontaneously conceived twins and the twins produced by ART, delivered in the same hospital were enrolled in this study. Samples (2 ml each) of cord blood were obtained from the newborn twins after birth in EDTA-coated VacutainerR tubes.
DNA preparation
DNA was extracted from the cord blood using a commercial DNA isolation kit (Puregene, Gentra Systems, Minneapolis, MN, USA) and processed according to the manufacture’s instruction.
Genomic DNA amplification, sample preparation and electrophoresis
The AmpFLSTR® IdentifilerTM PCR Amplification kit (PE Applied Biosystems, Foster City, CA, USA) used in this study is a STR multiplex assay that amplifies 15 tetranucleotide (tetrameric) repeat loci and amelogenin, the gender-determining marker, in a single PCR amplification. Methods for PCR amplification, sample preparation and electrophoresis for the AmpFLSTR® IdentifilerTM kit (comprising of 15 loci: D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, FGA, D5S818) were performed according to the manufacturer’s instructions. Briefly, 30 µl of the AmpFLSTR® IdentifilerTM Master Mix (mixing AmpFLSTR® PCR Reaction Mix, AmpliTaq GoldTM DNA polymerase and AmpFLSTR® IdentifilerTM Primer together) was dispensed, and template DNA (1–2.5 ng) was added to make the final volume to 50 µl for the PCR. Cycling conditions were 95°C, 11 min before incubation, which was followed by 28 cycles of 94°C for 1 min, 59°C for 1 min, 72°C for 1 min and a final extension at 60°C for 45 min in a thermal cycler (GeneAmp PCR System 9600, PE Applied Biosystems). One microlitre aliquot of PCR product dissolved in 24 µl of formamide and the LIZ-labelled size standard (GeneScan-500, PE Applied Biosystems) was then processed by using a genetic analyser (ABI PrismTM. 310, PE Applied Biosystems).
The sample was electrokinetically injected for 5 s at 15 kV, and then run at 15 kV for 28 min at a constant temperature of 60°C. The raw data were collected and analysed by the resident software (ABI PrismTM. 310 Collection Software, version 1.0.2 and GeneScan Analysis Software, version 2.1, respectively) using LIZ DNA size standards (range, 35–500 base pairs), then electrophoresis was run in parallel as an internal standard for the determination of fragment sizes. Genotypes were determined by comparing the size of the unknown fragments to the allelic ladders provided by the manufacturer.
Reading of analysis
Although postzygotic mutation might occur to the tested loci, whenever all the 15 unlinked loci as well as the gender-determining marker, amelogenin, were identical in the PCR-amplified STR analysis, the newborns were determined as MZ twins. Otherwise, they were DZ.
Statistical analysis was determined with t-test and Chi-square test. P value < 0.05 was considered significant.
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Results |
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Within the 2-year period, there were a total number of 3305 deliveries in the same hospital. Eighty-two cases were multifetal pregnancies. Four of the multifetal pregnancies were triplets, and the remaining 78 pregnancies were twins. Five twins were excluded due to incomplete data as well as the four triplets. Cord blood samples of the remaining 73 sets of twins were examined in this study and divided into two groups. Group A composed of 48 twin pregnancies produced by ART – including 11 AIH, 29 IVFs, two ICSI and six cases of taking clomid for ovulation induction. The remaining 25 cases, group B were spontaneously conceived twins. Excluding twins produced by ART, the incidence of spontaneously conceived twins in the local area was around one in 133 births.
The average age (±SD) of pregnant women in group A was 33.2 ± 2.7 years old; ranging from 27 to 44 years. There were 23 sets of like-sexed twins (LST); of the 23 sets, three of them were MZ (6.25%) – including two sets of male and one set of female. Two of the three MZ twin sets came from IVF, and the remaining one was the result from taking clomid for ovulation induction. All pregnant women who gave birth to these MZ twins had no family history of multifetal gestations. The remaining 45 sets of twins in this group were DZ (93.75%). (Table I)
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The age of pregnant women in group B ranged from 21 to 40 years with a mean (±SD) of 29.3 ± 5.3 years. The mean age of this group (spontaneously conceived women) was significantly lower than that of group A (t-test, P = 0.002). There were 21 sets of LST in this group. Eighteen of them were MZ (18/25, 72%) confirmed by the PCR-amplified STR analysis. Both sexes were in equal number. The rate of DZ twins in this group was 28% (7/25). (Table I)
Chorionicity and placentas were inspected after delivery in 68 and 64 of the 73 twin sets, respectively. Of the 16 monochorionic (MC) twins, 11 were MZ twins and the remaining five were DZ. Dichorionicity was found in the remaining 52 cases, of which nine were MZ and 43 were DZ twins. The sensitivity and the specificity were 68.8 and 82.7%, respectively, if the zygosity was evaluated based on the finding of chorionicity. The positive predictive rate (PPR) of MZ twins with monochorionicity was 55%, and the negative predictive rate (NPR) was 89.6%.
The placentation data in this study were retrospectively obtained to estimate the accuracy of zygosity determination based on that information versus information obtained by DNA fingerprinting. Basically, dichorionic (DC) twins have two separate placentas. But when they are implanted closely to each other, abutting placentas may have an appearance of a single placenta and cannot be easily separated. In addition, DZ twins can never have one placenta. The placenta in these twins can be fused or separate. MZ twins can have single, fused or separate placenta. As a result, there may be two types of placenta appearance – grossly single placenta or apparently separate placentas. Grossly single placenta was found in 32 sets of twins. Sixteen of them were MZ, and the remaining 16 were DZ twins. Thirty-two twin sets had apparently separate placentas with only four of them being MZ, and the remaining 28 being DZ. The sensitivity and the specificity were 50.0 and 87.5%, respectively, if zygosity was evaluated based on the number of placenta. The PPR was 80%, whereas the NPR was 63.6%. (Tables II and III)
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Totally, there were 44 sets of LST in this study. Twenty-one of them were MZ, and the remaining 23 were DZ. Over half of them would be misinterpreted as MZ twins when only ‘like sex’ was used for the evaluation of monozygosity. However, 18 sets of the 21 LST in group B were MZ. As a result, when they were LST, there was a higher probability of MZs in spontaneously conceived twins. (Table IV)
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Because no monoamnionic twins were found in this study, placentation was used in combination with chorionicity and LST for the evaluation of zygosity. Twenty-seven sets of LST were DC. Nine of them were MZ, and the remaining 18 were DZ. Eleven of the DC LST had a grossly single placenta. Five of them were MZ, and the remaining six were DZ. Fifteen of the remaining DC-LST had apparently separate placentas. Four of them were MZ, and the remaining 11 were DZ. Here, the underestimated rate of MZs was 26.7% (4/15). There were 14 MC LST with a grossly single placenta. Eleven of them were MZ, and the remaining three were DZ. As a result, the overestimated rate of MZ twins was 21.4% (3/14) (Table IV).
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Discussion |
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Currently, genetic analysis is widely used in forensic medicine and paternity testing for personal identification, population genetics and gene mapping (Pu et al., 1998
; Tzeng et al., 2000a,b; Kim et al., 2003). Identical genetic fingerprinting is expected in MZ twins because they are derived from the same fertilized ovum.
Twin zygosity was determined by the analysis of multiple restriction enzyme site polymorphisms with several different DNA probes in Derom’s study (Derom et al., 1985); however, a substantial percentage of false-positive diagnosis of MZ was produced. Analysis of twin zygosity with minisatellite probes [variable number of tandem repeats (VNTRs)] provides more precise allelic determinations (Jeffery et al., 1988; Boerwinkle et al., 1989; Jeffery et al., 1990), but the large size of the PCR products makes them less suitable for general application than STR, which have a repeated motifs of a range only 2–7 base pairs (bp) rather than 15–70 bp. Besides, there are some disadvantages in the application of VNTRs in gene mapping, population genetics and personal identification; these include: (i) the low frequency (Amour et al., 1990) and asymmetric distribution (Royle et al., 1988) of these repeats in the genome, (ii) the inability to determine alleles precisely with Southern hybridization-based detection schemes resulting in homozygote excess (Devlin et al., 1990) and (iii) time consuming and costliness.
The frequency of STRs in the human genome is estimated at 400 000 or one STR/10 kb (Edwards et al., 1991). Unlinked STR loci are used in genetic testing, and the greater the amount, the more precise the results. In addition to the gender-determining marker, amelogenin, the kit used in this study composed of 15 unlinked, tetrameric repeated loci that have a very high cumulative power of exclusion (0.9999997) for local population’s personal identification (Tzeng et al., 2000b), can be amplified faithfully and is therefore suitable for application to DNA typing and genetic mapping. This test amplifies all the tetrameric STRs of the kit simultaneously in a multiplex PCR from a single DNA sample with good internal controls that provide precise genotype data, is cost effective and less time consuming. Consequently, the high MZ rate in the spontaneously conceived twin pairs in this study is convincing.
The frequency of spontaneously conceived MZ twins (51%, 152/300) is nearly equal to that of DZ twins (49%, 148/300), in the study of Machin et al. (1995), of which zygosity was determined by VNTRs analysis. Although, the rate of MZ twin sets could be verified if STRs analysis was used. However, in agreement with Machin’s finding, our results on the rate of MZ twin sets by using PCR-STR analysis can overturn that of the MacGillivray’s report (1986), in which spontaneously conceived DZ twinning rate was twice that of MZ twin pairs calculated by less-reliable Weinberg’s rule (St. Clair et al. 1998).
A similar study in the Taipei area was done by Chen et al. (1999). In that study, based on the PCR amplification analysis with five genetic loci, the DZ-to-MZ ratio of the LST was 1 : 4.53 for the adolescent twins and 1 : 4.88 for the child twins. Most of both the twin groups could be rendered as spontaneously conceived twins, because they were born in the era with less prevalence of ART. In our study, the DZ-to-MZ ratio of the spontaneously conceived LST was 1 : 6 and was 1 : 0.91 if all the LST were enrolled including those produced by ART. In addition to the weakness by using a less-powerful kit (which comprised only five STR loci) for PCR amplification analysis, there was a bias in Chen’s study population, which mainly consisted of twins registered with the ‘Taipei city Twins Association’. On the contrary, our study subjects contain only newborn twins from a single institution. No matter which twin populations are taken into consideration, multifetal pregnancy in the Taipei area is characterized by a higher rate of monozygosity based on the findings of the two studies.
Despite the small sample size of this study, it is difficult to collect a large number of multifetal pregnancies within a short period of time (two years) in Asia where the incidence of spontaneously conceived multifetal pregnancies is the lowest in the world (MacGillivray, 1986). The result of this study, however, reveals a unique finding of the local Chinese population in Taiwan.
In our study, most of the fetal membranes (68 twin sets) and the placentas (64 sets) were examined after delivery for their relationships to twin zygosities. High specificity (82.7%) and NPR (89.6%) were obtained when chorionicity was examined for twin monozygosity, meaning that when the twins were not MC, there was high probability of dizygosity (high specificity), and there was high probability of DC twins when they were DZ (high NPR). Nevertheless, there were high specificity (87.5%) and PPR (80.0%) when placental number was used for the evaluation of twin zygosities. It indicated that the twins had a high probability of being DZ when they had apparently separate placentas (high specificity), and when the twins were MZ, there was a high probability of having a grossly single placenta (high PPR). The sensitivity was 68.8% when monochorionicity was used for the evaluation of monozygosity. Five sets of MC twins were not MZ. Quintero et al. (2003) reported the similar finding in a case of twin–twin transfusion syndrome found in a DZ-MC-diamnionic twin pregnancy. The number in this study was not big enough to make a conclusion, and the mechanism was difficult to conclude. However, it is obvious that monochorionicity was not the sole and reliable criterion for the determination of monozygosity other than DNA ‘fingerprinting’.
Monochorionicity, grossly single placenta and like sex of twins were regarded as the basic factors for the evaluation of monozygosity, especially when these factors were used to determine the zygosity by prenatal ultrasound examinations. Over half of LST in this study were DZ. Consequently, MZ cannot be determined solely by ‘like sex’ unless they were spontaneously conceived twins. When MZ was evaluated based on the combination of monochorionicity, a grossly single placenta and LST, the predictive rate was 78.6%, the overestimated rate was 21.4% and the underestimated rate was 26.7%.
Nearly two-thirds of twins enrolled in this study were produced by ART. Not only was the patients’ age significantly higher than that of women with spontaneously conceived twins but also the rate of MZ twins was significantly lower than that of group B. Medical induction was the most likely aetiology resulting in multiple ovulations during the cycle of fertilization. In addition, women of group B had a higher family history of giving births to multifetal pregnancies compared with those of group A.
In summary, with the DNA-based 15 STR analysis amplified in a multiplex PCR, the determination of the zygosity of multifetal pregnancies is not only cost and time saving but also shows greater sensitivity and precision than conventional methods. Our study results clearly indicate that the frequency of the MZ twin in spontaneously conceived multifetal pregnancies is not as low as previously mentioned in the literatures. On the contrary, it is nearly three times the incidence of DZ twins. Hence, regardless of the mode of conception, patients diagnosed with a multifetal gestation should have the offspring assessed for monozygosity especially when they are of the same sex in order to establish their relation for future reference.
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Acknowledgements |
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The authors thank Ms. Pei-Shan Chen and Ms. Hsueng-Mei Liu for their excellent technical assistance in performing PCR-STR. This work was supported by a research grant VGH 92-A-0327 from the Taipei Veterans General Hospital.
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Submitted on August 15, 2005; resubmitted on October 14, 2005; resubmitted on December 16, 2005; resubmitted on March 3, 2006; resubmitted on March 29, 2006; accepted on April 4, 2006.
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