Hum. Reprod. Advance Access published online on October 22, 2008
Human Reproduction, doi:10.1093/humrep/den364
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Zygosity and chorionicity in triplet pregnancies: new data
1 Department of Prenatal Diagnosis, Robert Debre Hospital, Paris, France 2 Department of Genetic Biochemistry, Robert Debre Hospital, Paris, France 3 Department of Developmental Biology, Robert Debre Hospital, Paris, France 4 Department of Obstetrics and Gynecology, Beaujon Hospital, Clichy, France
5 Correspondance address. Service de Gynécologie Obstétrique, Hôpital de la Pitié-Salpêtrière, 47 boulevard de l'Hôpital, 75013 Paris, France. Tel: +33-1-42-17-77-10; Fax: +33-1-42-17-79-67; E-mail: romain.guilherme{at}psl.aphp.fr
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Abstract |
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BACKGROUND: In studies of twin and triplet pregnancies, molecular genetic techniques have rarely been used to confirm zygosity yet this is the most accurate approach. The aim of this study was to present the cross-distribution of chorionicity and zygosity in triplet pregnancies as a function of their mode of conception using such techniques. Rates of monozygosity were studied simultaneously.
METHODS: Forty-nine consecutive sets of triplets were observed in the study, including 18 sets of spontaneously conceived (SC) triplet pregnancies and 31 sets resulting from assisted reproduction technologies (ARTs). Zygosity was determined through PCR-amplified microsatellite analysis. Chorionicity was determined by placental analysis in our department of fetopathology. Sets of triplets were considered as twin pairs in order to determine the rate of monozygosity.
RESULTS: For SC triplet pregnancies, the rate of monozygotic (MZ) twin pairs was 48%; 30% of dichorionic (DC) triplet pregnancies were MZ and 70% dizygotic (DZ); 20% of trichorionic (TC) triplet pregnancies were DZ and 80% trizygotic (TZ). For triplet pregnancies conceived using ART, the rate of MZ twin pairs was 6.5%; 100% of DC triplet pregnancies were DZ; 4% of TC triplet pregnancies were DZ and 96% TZ.
CONCLUSIONS: This study is the first report to present the cross-distribution of chorionicity and zygosity in triplet pregnancies as a function of their mode of conception. In triplet pregnancies conceived using ART, DC triplets are always DZ, and TC triplets are almost always TZ.
Key words: zygosity/chorionicity/triplets/microsatellites/assisted reproduction technologies
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Introduction |
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Although the chorionicity of triplet pregnancies has been studied extensively (Adegbite et al., 2005
; Geipel et al., 2005; Bajoria et al., 2006), we have little information on their zygosity. Chorionicity refers to the type of placentation, unlike zygosity which refers to the type of conception. Zygosity indicates if triplets arose from one fertilized oocyte [monozygotic (MZ)], or from two oocytes fertilized by two different sperm [dizygotic (DZ)] or from three oocytes fertilized by three different sperm [trizygotic (TZ)]. The type of placentation depends on when the zygote divides into two genetically identical twin embryos. If the split occurs during the first 3 days following fertilization, the pregnancy will be dichorionic (DC) with unconnected or fused placentas. If the division occurs 4–8 days after fertilization, the two fetuses share the same placenta but each fetus has its own amniotic fluid cavity. When the division occurs between Days 8 and 13 post-fecundation, the result is monochorionic (MC) monoamniotic twins with fetuses sharing the same placenta and amniotic fluid cavity. In triplet pregnancies, diagnosis of chorionicity and amnionicity is established by comparing each triplet with one another by sequential analysis: fetus 1 with fetus 2, 1 with 3 and 2 with 3. One zygote can therefore produce two or three embryos through successive division. The number of placentas and shared amniotic fluid cavities depends on the time of the division.
We know that MC triplets are always MZ. DC triplets may be either MZ or DZ. Trichorionic (TC) triplets may be either MZ, DZ or TZ. On the basis of only two reports concerning spontaneous triplet pregnancies (Machin and Bamforth, 1996; Blickstein and Keith, 2005), it has been calculated that 19% of DC triplets are MZ and 81% DZ; and that 6% of TC triplets are MZ, 39% DZ and 55% TZ.
The true incidence of MZ triplets is of biological, clinical and epidemiological relevance. Zygosity can be of great significance in the field of prenatal diagnosis and genetic counseling. The knowledge of zygosity is helpful in instances where chorionicity remains indeterminate and is likely to influence clinical management, e.g. in prenatal diagnosis of genetic disease, management of fetal death and fetal pregnancy reduction. In the field of biology, the study of twinning in triplets following spontaneous conceptions, ovulation induction and IVF may help us to understand the mechanism of monozygosity. At the present time, assisted reproduction technology (ART) is the only biological mechanism capable of influencing MZ twinning (Hall, 2003). Equally important, recent studies have illustrated that mono- or dichorionicity in triplet pregnancies is clearly implicated in adverse perinatal outcomes (Adegbite et al., 2005; Geipel et al., 2005; Bajoria et al., 2006).
Only two base studies have reported the rate of monozygosity in spontaneously conceived (SC) triplets with the help of DNA highly polymorphic markers analysis, considered as the ‘gold standard’ for assessing zygosity in multifetal pregnancies. The East Flanders Prospective Twin Study (EFPTS) (Blickstein and Keith, 2005) is the largest series of triplets on which this kind of complete zygosity assessment has been performed. This study has served as the accepted base value for monozygosity in relevant literature since 1987 (Derom et al., 1987). Machin and Bamforth (1996) also reported a series of 15 consecutive sets of SC triplets, using highly polymorphic DNA marker analysis. Their approach of the zygotic splitting rate was particularly interesting because it permitted an analysis of the rate of monozygosity not in the general population but within the population of triplet pregnancies itself.
The purpose of the present study was to evaluate the rate of monozygosity in a series of 53 consecutive sets of triplets, with a complete assessment of zygosity by analyzing highly polymorphic DNA microsatellite markers of each triplet. Distribution of zygosity and chorionicity was studied simultaneously as a function of their mode of conception. Our results were discussed and compared with data from the literature.
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Materials and Methods |
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Case eligibility
Between March 1998 and February 2006, every set of triplets in which at least one of the children, live- or stillborn, weighed 500 g or more, and with a gestation period of at least 22 weeks, was used in the study. These 53 consecutive triplet pregnancies included SC triplets, triplets resulting from ovulation induction only (OIO), IVF and IVF–ICSI. All of the pregnancies had their prenatal care and delivery in our tertiary referral centre (Robert Debré Hospital, Paris, France). Triplet pregnancies resulting from embryo reductions of high-order multiple pregnancies (HOMP) were excluded from the study. Triplet pregnancies presenting discordance in final anatomopathological chorionicity and molecular zygosity were also excluded. If the procedure of DNA extraction failed for one triplet, the entire set of triplets was removed from the study.
DNA preparation
Samples (2 ml each) of cord blood were obtained from the live newborn triplets after birth. For the stillborn triplets, a piece of pulmonary tissue was collected in our department of anatomopathology with a signed parental agreement. DNA was extracted using a commercial DNA isolation kit (Wizard® Genomic DNA Purification Kit, Promega Corporation, Madison, WI, USA) and processed according to the manufacturer's instructions.
Genomic DNA amplification, sample preparation and electrophoresis
The PowerPlex® 16 System (Promega Corporation) allowed the co-amplification and three-color detection of 16 loci, 15 STR (short tandem repeat) loci and amelogenin, the gender determining marker. STR loci, or microsatellites, consist of short, repetitive sequence elements, 3–7 base pairs in length. These repeats are evenly distributed throughout the human genome and are a rich source of highly polymorphic markers, which may be detected using the PCR. Alleles of microsatellites are differentiated by the number of copies of the repeat sequence contained within the amplified region and are distinguished from one another using radioactive, silver stain or fluorescence detection following electrophoretic separation. The 16 microsatellite loci studied with PowerPlex® 16 System include Penta E, D18S51, D21S11, TH01, D3S1358, FGA, TPOX, D8S1179, vWA, amelogenin, Penta D, CSF1PO, D16S539, D7S820, D13S317 and D5S818. Methods for PCR amplification, sample preparation and electrophoresis for the PowerPlex® 16 System were performed according to the manufacturer's instructions. The raw data were collected and analyzed using an ABI PRISM® 3100 Genetic Analyser (Applied Biosystems, Foster City, CA, USA) and GeneScan® (GeneScan, Suite 200 Metairie, LA, USA) analysis software, respectively (Fig. 1).
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Interpretation of the analyses
The matching probability of the PowerPlex® 16 System ranges from 1 in 1.83 x 1017 for Caucasian-Americans to 1 in 1.41 x 1018 for African-Americans. Whenever all of the 15 unlinked loci as well as the gender-determining marker, amelogenin, were identical in the PCR amplified microsatellite analysis, the compared newborns or stillborns were determined as MZ. Otherwise, they were DZ.
Anatomopathological placental analysis
All placentas were immediately identified in relation to their triplets in the delivery room and transferred to our referral center of fetopathology for determination of their chorionicity. Placental examinations were carried out according to the practical methods of Benirschke (1961).
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Results |
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Within the 8 year period, 53 triplet pregnancies had their prenatal care and deliveries in our hospital. One set was excluded from the final analysis, because they were the result of an embryo reduction in a quadruplet pregnancy. We excluded another set of triplets due to discordance in placental analysis and molecular zygosity. Two other pregnancies did not enter into the analysis because of failure in DNA extraction. Results of the chorionicity and zygosity analysis are shown in Table I. Of the 49 sets of triplets, 6 were MZ, 14 DZ and 29 TZ.
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Of these 49 sets of triplets, 18 were SC, while 16 were conceived through OIO, 9 by IVF and 6 by IVF–ICSI. Zygotic characteristics of the 49 sets of triplets are presented in Table II as a function of their mode of conception. As in Machin's study (Machin and Bamforth, 1996
), we considered each triplet pregnancy as an association of three twin pairs: fetus 1 with fetus 2, 1 with 3 and 2 with 3. Using this method, an MZ triplet pregnancy is always composed of three MZ twin pairs, a DZ triplet pregnancy is always composed of one MZ twin pair and two DZ twin pairs, and a TZ triplet pregnancy is always composed of three DZ twin pairs. For each mode of conception, we calculated the MZ twin pair rate, which was equal to the sum of all MZ twin pairs divided by the sum of all twin pairs.
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The 18 SC triplet pregnancies contained 54 twin pairs of which 26 pairs (48%) were MZ. Among these MZ twin pairs, 11 were male and 15 were female. The 16 sets of triplets conceived by OIO contained 48 twin pairs of which 3 twin pairs (6.3%) were MZ. Among these MZ twin pairs, two were male and one was female. The 9 triplet pregnancies conceived by IVF contained 27 twin pairs of which 1 twin pair (3.7%) was MZ. This MZ twin pair was male. The 6 sets of triplets conceived by IVF–ICSI contained 18 twin pairs of which 2 twin pairs (11.1%) were MZ. One of these MZ twin pairs was male and one was female.
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Discussion |
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Assisted reproduction has changed the incidence of HOMP in the last 30 years. Very few stringent studies have reported the incidence of monozygosity in the case of triplet pregnancies. Even today, the true distribution of zygosity and chorionicity in spontaneous and iatrogenic triplets remains unknown.
In our report, we have decided to include only pregnancies in which at least one of the children weighed over 500 g for two reasons. First, we wanted to copy the inclusion criteria of the East Flanders Prospective Twin Study in the case of triplet pregnancy (Derom et al., 1987). We could thus compare our results with the Belgian population-based register of multiple births. Second, as we were a tertiary referral centre for a delimited geographic district, we only followed and organized the prenatal care of triplet pregnancies which correspond to our inclusion criteria. However, we must underline that this exclusion of pregnancies with all children <500 g might lightly distort the relative proportion of mono-/di-/trichorionic pregnancies assessed.
In fact if the determination of chorionicity has become a common anatomopathological post-delivery practice since Benirschke's plea in 1961 (Benirschke, 1961), the accuracy of zygosity determination remains a puzzle in HOMP cases and especially where ART is concerned (Blickstein, 2005). Until 1985, all zygosity assessments were determined by proxy estimations in live- and stillbirths through sequential analysis based on Weinberg's rule (James, 1992), fetal sex, chorionicity, interamniotic membranes, umbilical cord blood groups and placental alkaline phosphatase. Allen (1960) reported estimates and ratios of MZ, DZ and TZ triplets in a spontaneous triplet population, mainly considering numbers of same-sex and opposite-sex triplets. Nevertheless, current techniques of zygosity assessment have shown that these estimations, based on chorionicity and fetal sex, are poor indicators of the correct rate of monozygosity, with an underestimate of at least one-third of the MZ twin pairs in spontaneous multiple pregnancies (Blickstein and Keith, 2005). In the field of multiple pregnancies resulting from ART, this previous proxy vision is also refuted by numerous data (Derom et al., 1987; Saito et al., 2000; Sills et al., 2000; Alikani et al., 2003; Jain et al., 2004). A recent report on the Danish national cohort of twins conceived by ART demonstrated a different proportion of same-sex twin pairs than in SC twins (Pinborg et al., 2004). Studies that use indirect methods to assess MZ splitting in pregnancies resulting from ART should therefore be interpreted with caution. In the same manner, the estimation of zygosity resulting from the number of fetuses in excess of the transferred embryos is not exact. This theory does not take into account the possibilities of superfecundation (Hall, 2003) and the early loss of one fetus with a following twinning process.
Because MZ twins and triplets come from the same fertilized ovum, their genomes should be identical. Thus, the most accurate zygosity assessment uses various genetic markers, with the ‘gold standard’ being DNA fingerprinting (Hill and Jeffreys, 1985). In the present study, we used a commercial PCR kit comprising 15 autosomal codominant unlinked loci and a gender-determining marker, amelogenin. These 16 STR markers presented a power of exclusion of at least 0.9999994 between triplets in our population. Determination of zygosity in triplet pregnancies by analyzing 16 STRs in a multiplex PCR is more cost-effective and faster than previous indirect methods. It also yields greater sensitivity and precision (Yang et al., 2006).
As in our report, neither Derom et al. (1987) nor Machin and Bamforth (1996) have ever shown MC triplet pregnancies in which zygosity was DZ or TZ, or DC triplet pregnancies in which zygosity was TZ. However, two cases of triplet pregnancies including a DZ twin pair with MC placentation have been reported in medical literature (Souter et al., 2003; Yamaguchi et al., 2003). These two cases were the products of pregnancy by ART. In the case of spontaneous triplet pregnancies, our report has shown a cross-distribution of zygosity and chorionicity, closely related to those exposed by Derom (EFPTS) (Blickstein and Keith, 2005) and Machin and Bamforth (1996) (Table III). We observed almost the same high incidence of monozygosity (34%, 6/18) with a high level of monochorionicity (17%, 3/18) and dichorionicity (17%, 3/18) in our cohort of triplet pregnancies. Indeed, in these three reports, the mean rate of monozygosity in spontaneous triplet pregnancies was 29% (18/64), clearly above the rate estimated by Allen's mathematic formula (Allen, 1960). Unlike Derom (Blickstein and Keith, 2005) but in line with Machin's findings (Machin and Bamforth, 1996), we noticed an equal distribution of MZ triplets with an MC (3/18) and DC (3/18) placentation in our series. Moreover, we showed that almost all (7/8) of our DZ triplets had a DC placentation. This distribution corresponds with the results of Derom who noticed 77% (13/17) of DZ sets of triplets with a DC placentation. As far as chorionicity is concerned, our results of zygosity in DC triplet pregnancies revealed differences with results of Derom's study. With 3.2% (1/31) of MZ DC and 42% (13/31) of DZ DC sets of triplets, Derom's data fall well below our rate of MZ DC sets of triplets (17%, 3/18), and above our rate of DZ DC sets of triplets (39%, 7/18). These differences can be partially explained by the method of zygosity assessment used in the EFPTS, where same-sex fetuses of a triplet pregnancy with an identical blood group and the same DNA markers (six DNA variants studied) have a monozygosity calculation probability based on a lod-score method, which can artificially reduce the rate of monozygosity.
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Our report is the first to evaluate the cross-distribution of chorionicity and zygosity in triplet pregnancies conceived by ART (Table I). We noticed that in triplet pregnancies conceived using ART, DC triplets were always DZ (n = 5) and TC triplets were almost always TZ (96%, 25/26). Thus, chorionicity seems to be a strong marker for the zygosity assessment in the field of triplet pregnancies conceived using ART. These results are particularly interesting in clinical practice because they allow us to calculate the likelihood of monozygosity in triplet pregnancies conceived by ART thanks to an antepartum chorionicity diagnosis through ultrasonography, or post-partum with an anatomopathological analysis.
To evaluate the effects of ART on the twinning process, we calculated the rate of MZ twin pairs in triplet pregnancies resulting from OIO (6.3%, 3/48), IVF (3.7%, 1/27) and IVF–ICSI procedures (11.1%, 2/18) (Table II). Although recent studies play down the role of ICSI and micromanipulation on the zona pellucida in the mechanisms of monozygosity (Saito et al., 2000; Sills et al., 2000), our results showed an increased monozygosity in triplet pregnancies resulting from IVF–ICSI, as has been reported by Alikani et al. (2003). Otherwise, our results on MZ twin pair rates in triplet pregnancies resulting from OIO corroborate the historical findings of Derom et al. (1987) and Edwards et al. (1986) that identified an increase in monozygosity in the case of OIO pregnancies (Table II). Indeed, in comparing our data with those of Derom from the EFPTS (Blickstein and Keith, 2005) (Table IV), we observed a higher rate of monozygosity in our results. This tendency may be explained by the fact that a large proportion of our patients were of East African origin. HOMP and monozygosity are more frequently observed in women from this region than in women from Caucasian countries (Nylander and Corney, 1971).
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This study is the first to report the cross-distribution of chorionicity and zygosity in triplet pregnancies as a function of their mode of conception. We have used the most stringent technique for zygosity assessment, which is currently PCR-amplified microsatellite analysis. Chorionicity and zygosity are pivotal to evaluating the outcomes of HOMP for many reasons. First, monochorionicity or dichorionicity increases the risk of complications in triplet pregnancies (Adegbite et al., 2005
; Geipel et al., 2005; Bajoria et al., 2006). Second, prenatal zygosity assessment may be helpful in instances where chorionicity remains indeterminate and its knowledge is likely to influence clinical management, e.g. in the case of triplet pregnancies complicated by the intrauterine demise of one of the triplets, in early onset discordant fetal growth between triplets or prior to selective feticide. Moreover, prenatal diagnosis of zygosity is important for prenatal diagnosis of genetic diseases, or to evaluate the risk of structural anomalies (Bajoria and Kingdom, 1997). As in twin pregnancies, we showed notable differences in the rates of MZ triplet pregnancies as a function of their mode of conception. Globally, these rates seem to be higher than those known for twin pregnancies, whatever the mode of conception. These differences and their explanations are currently being studied in our department. We hope that this work will provide a more precise understanding of the biological mechanisms of monozygosity. |
Authors' Role |
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R.G.: Conception, design, acquisition of data, analysis and interpretation of data.
S.D.: Acquisition of data, interpretation and analysis in the field of Molecular Biology.
A-L.D.: Acquisition of data, interpretation and analysis in the field of Developmental Biology.
J-F.O.: Final approval of the published version.
D.L.: Critical revision of the article for intellectual content and final approval of the published version.
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Submitted on December 13, 2007; resubmitted on August 1, 2008; accepted on September 8, 2008.
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