Hum. Reprod. Advance Access published online on June 12, 2008
Human Reproduction, doi:10.1093/humrep/den226
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Twin pregnancies with diploid hydatidiform mole and co-existing normal fetus may originate from one oocyte
1 Department of Clinical Genetics, University Hospital of Aarhus, 8000 Aarhus C, Denmark 2 Institute of Human Genetics, University of Aarhus, 8000 Aarhus C, Denmark
3 Correspondence address. Bragesvej 38, 8230 Aabyhoej, Denmark. Tel: +45-41695588; E-mail: in{at}dadlnet.dk
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Abstract |
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BACKGROUND: In twin pregnancies comprising a hydatidiform mole and a normal co-fetus, the ploidy of the mole is almost exclusively reported as diploid and very rarely as triploid. We aimed at understanding this unbalanced distribution of diploid and triploid moles in twin pregnancies by investigating the number of gametes involved.
METHODS: Using polymorphic DNA markers, we compared the alleles of seven moles with those of the normal co-fetuses and deduced the number of oocytes and spermatozoa represented in each twin pregnancy.
RESULTS: The genomes of all seven moles were androgenetic diploid; six moles were homozygous in all loci analyzed and one mole was heterozygous in several loci. In one homozygous mole, the paternal alleles were identical to those of the normal co-fetus in 13 non-linked informative microsatellite loci, indicating the involvement of one spermatozoon only, and thus of one oocyte only. Duplications of the paternal genome followed by abnormal cell division can explain this observation. In six moles, the paternal alleles were different from those of the normal co-fetus suggesting involvement of two (or more) spermatozoa. Overfertilization of one oocyte followed by abnormal cell division is a possibility.
CONCLUSIONS: It is possible that twin pregnancies comprising a diploid mole and a normal co-fetus most often derive from one single oocyte fertilized with one or more spermatozoa. This can explain why diploid moles are far more frequent than triploid moles in twin pregnancies.
Key words: hydatidiform mole/twin pregnancy/parental origin/ploidy
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Introduction |
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Hydatidiform mole is diagnosed in 1:1000 pregnancies in the western part of the world (Steigrad, 2003
). Morphologically, a molar pregnancy is classified as either complete or partial (Vassilakos et al., 1977; Szulman and Surti, 1978). In the complete mole, fetal development is absent (Vassilakos et al., 1977). Complete moles most frequently have a diploid genome, and in most diploid moles, all 46 chromosomes are paternally derived (Kajii and Ohama, 1977; Lawler et al., 1991; Lage et al., 1992). The majority of androgenetic diploid moles have two identical sets of paternal chromosomes (homozygous). Most probably, a diploid homozygous mole arises after fertilization of one oocyte without nuclear chromosomes by one spermatozoon, followed by duplication of the paternal chromosomes (Kajii and Ohama, 1977; Wallace et al., 1982). A small fraction of diploid moles, however, have two different sets of paternal chromosomes (heterozygous) indicating fertilization by two spermatozoa (Surti et al., 1982).
Fetal differentiation can be observed in the partial mole, but the fetus is malformed and non-viable (Vassilakos et al., 1977). Most partial moles are triploid. Triploid moles have one maternal set plus two paternal sets of chromosomes, suggesting fertilization of one oocyte with two spermatozoa (Lawler et al., 1979).
Twin pregnancies consisting of a hydatidiform mole and a normal co-fetus are observed in 1 in 20 000–100 000 pregnancies (Steller et al., 1994b; Sebire et al., 2002; Niemann et al., 2007b). This entity has been assumed to derive from two separate conceptions, of which one develops into a normal fetus and placenta, and the other gives rise to a molar pregnancy (Vejerslev, 1991; Fishman et al., 1998; Ishii et al., 1998; Chu et al., 2004). In twin pregnancies, the mole is most often, by far, reported as diploid or—if of unknown ploidy—complete (Fisher et al., 1982; Steller et al., 1994b; Matsui et al., 1999; Sebire et al., 2002). Twin pregnancies with a normal fetus and a triploid mole are extremely rare. To our knowledge, only three cases have been reported (Steller et al., 1994a; Nugent et al., 1996; Chu et al., 2004).
To explain the low frequency of triploid moles in twin pregnancies, we tried to elucidate the mode of formation of twin pregnancies with diploid mole and normal fetus by comparing microsatellite markers of seven moles with those of the respective co-fetuses. From these analyses, we deduced the minimum number of spermatozoa and oocytes involved in each twin pregnancy, and we here present a hypothesis for the mechanisms leading to a twin pregnancy comprising a diploid hydatidiform mole and a normal fetus.
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Methods |
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In the Danish Mole Project, unfixed representative samples from pregnancies clinically suspected of hydatidiform mole have been collected since 1986. Only samples subsequently classified as hydatidiform mole at a central histopathologic revision were included. Among 270 hydatidiform moles, we identified seven twin pregnancies consisting of a hydatidiform mole and a normal co-fetus with normal placenta.
We determined the ploidy and the parental origin of these seven hydatidiform moles and seven normal pregnancies. To represent the normal pregnancies, unfixed tissue from six of the normal placentas was used, whereas in one case, formalin-fixed paraffin-embedded fetal lung tissue was analyzed. DNA isolated from maternal blood samples was analyzed in all seven cases, and in one case (Case 301) paternal DNA was also used for analysis.
Three of the seven patients had had ovulation induction treatment prior to their pregnancy; two conceived with donor semen (Cases 333 and 537), whereas one patient had two in vitro fertilized oocytes placed in her uterus (Case 536).
Ploidy of the moles and of the normal pregnancies was determined by karyotyping, flow cytometric measurement of DNA contents in unfixed interphase nuclei (Vindelov et al., 1983) and/or by fluorescence in situ hybridization (FISH) techniques (Cheville et al., 1995).
The parental origin of the genomes of the moles and of the normal pregnancies was assessed by microsatellite analyses using up to 24 primer pairs specific for highly polymorphic loci as described elsewhere (Niemann et al., 2007a). A mole was classified as androgenetic when, in at least three loci, none of the alleles was identical with a maternal allele. An androgenetic mole was classified as homozygous (P1P1) when all, and at least four, loci showed homozygosity. An androgenetic mole was classified as heterozygous (P1P2) when heterozygous in at least two loci, and when none of the alleles was identical with a maternal allele. Biparental origin (PM) was considered proven when one allele in all loci was identical with a maternal allele, and when in at least two loci, the other allele could not be maternal.
Linkage between two loci on one chromosome was defined when the distance between two loci was shorter than 50 cm. The genetic distance between loci was determined from the Marshfield linkage maps.
We compared the alleles of the molar genome with those of the normal pregnancy genome in each twin pregnancy. When the mole and the normal pregnancy had different paternally derived alleles in one or several loci, we concluded that (at least) two spermatozoa had been involved.
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Results |
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Table I lists the ploidy and parental origin of the genomes of the mole and of the normal pregnancy in the seven twin pregnancies. All seven moles were diploid and androgenetic; six moles were homozygous, whereas one mole was heterozygous. The genome in all normal pregnancies was diploid and biparental. In Case 333, a healthy female child with a normal placenta distinct from a 3.5 kg hydatidiform molar mass was delivered. The six other pregnancies were terminated early either due to intrauterine death, obstetrical complications or following the physician's advise (Niemann et al., 2007b
). No morphologic or histopathologic abnormalities were observed in the aborted fetuses.
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We compared the paternal alleles of the mole with those of the normal pregnancy and calculated the number of identical and different paternal alleles in each twin pregnancy (Table II). In one case (Case 301), the paternal alleles of the homozygous mole and of the normal pregnancy were identical in all of 24 analyzed loci. Eighteen markers were informative, and 13 of these were non-linked (Table III). In the remaining six moles, of which five were homozygous and one was heterozygous, we observed both different and identical paternal alleles between the mole and the normal pregnancy.
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Discussion |
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One twin pregnancy with mole and normal co-twin did likely evolve from a fertilization involving one oocyte and one haploid spermatozoon. The paternal alleles of the mole and of the normal pregnancy were identical in 13 non-linked informative microsatellite loci dispersed on nine chromosomes. The chance of two independent conceptuses having identical paternal markers in 13 non-linked informative loci is 0.513 = 0.00012. This observation thus strongly suggests the involvement of one spermatozoon only. If only one spermatozoon was involved, only one oocyte could have been fertilized. Possibly, the paternal chromosomes were duplicated before fusion of the female and male pronuclei, resulting in a triploid zygote with one maternal pronucleus and two identical paternal pronuclei, as suggested by Kaiser-Rogers et al. (2006)
. If this triploid zygote underwent diploidization (Golubovsky, 2003) by detaching one paternal genome, a normal biparental cell plus a cell with a haploid set of paternal chromosomes would result. In the latter cell, the haploid set of chromosomes could duplicate producing a diploid homozygous androgenetic cell line developing into a mole (Fig. 1:1).
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This ‘one-oocyte-model’ is also compatible with the genetic constitution of the six other twin pregnancies. If a single oocyte was fertilized with two spermatozoa, again a triploid zygote would result. By diploidization and duplication of one set of paternal chromosomes, two cell lines, a homozygous androgenetic cell line and a biparental cell line, would result. This mechanism can explain the five twin pregnancies with a homozygous mole and a normal pregnancy (Fig. 1:2). In the single case with a heterozygous mole, however, either two spermatozoa (with the chromosomes of one spermatozoon subsequently duplicating) or three spermatozoa could have fertilized the oocyte, both producing a tetraploid zygote, which could divide into two diploid cell lines (Fig. 1:3.A and B).
The vast majority of reports on twin pregnancies with hydatidiform mole and healthy co-fetus concerns cases with a diploid and/or complete molar component (Steller et al., 1994b; Bristow et al., 1996; Fishman et al., 1998; Matsui et al., 1999; Sebire et al., 2002). In concordance with these reports, we only observed hydatidiform moles with a diploid chromosome count in all of the seven twin pregnancies. Extremely few reports of a normal fetus co-twinning with a triploid (or even partial) mole have been published (Steller et al., 1994a; Nugent et al., 1996; Chu et al., 2004).
This overrepresentation of diploid moles in twin pregnancies is interesting, since in singleton molar pregnancies, triploidy is just as frequent as diploidy—and maybe even more frequent (Genest, 2001). If twin pregnancies comprising a hydatidiform mole and a normal pregnancy develop from two independent oocytes, the frequencies of diploid and triploid moles in these twin pregnancies should reflect the incidence ratio of singleton diploid/triploid molar pregnancies. The shortage of triploid moles in twin pregnancies might be caused by an unknown disadvantage for the triploids in competition with the normal pregnancy. It is also possible, however, that the underrepresentation of triploids is caused by the number of oocytes involved. Twin pregnancies comprising a triploid mole and normal pregnancy most probably originate from two independent oocytes, although studies of the maternal genomic contribution to these rare twin mole pregnancies have not yet been published. In contrast, our observations demonstrate that twin pregnancies with an androgenetic diploid mole and a normal pregnancy can originate from one oocyte. Since dizygotic pregnancies generally are far less frequent than monozygotic pregnancies, this could explain the underrepresentation of triploid moles in twin molar pregnancies.
Approximately 50% of the patients with twin pregnancies comprising a mole and a normal pregnancy have received fertility treatment with ovulation inducing drugs prior to their pregnancy (Petignat et al., 2002). In this study, three of seven patients had received fertility treatment. The moles in these three cases were all homozygous, and the moles and the fetuses had different paternal alleles. The high frequency of fertility treatment in these twin pregnancies may be an argument for multiple oocyte involvement. It is also possible, however, that the infertility is caused by a tendency to faulty fertilization, or that the fertility treatment itself increases the risk of abnormal fertilization, e.g. fertilization with multiple spermatozoa.
In conclusion, our observations make it very likely that one twin pregnancy with diploid mole and normal pregnancy arose from one oocyte fertilized with one spermatozoon. In six other twin pregnancies, our observations are compatible with fertilization of one oocyte with two or more spermatozoa. The involvement of one oocyte, only, can explain the overrepresentation of diploid moles in twin pregnancies with hydatidiform mole and normal pregnancy.
Future investigations should determine why a triploid diandric zygote remains triploid, giving rise to a triploid mole, or divides before fusion of pronuclei, resulting in a twin pregnancy with a diploid mole and a normal co-fetus.
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Author's Role |
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I.N. has contributed with: (i) substantial contributions to conception and design, acquisition of data, analysis and interpretation of data, (ii) drafting the article and (iii) final approval of the version to be published.
L.B. has contributed with: (i) analysis and interpretation of data, (ii) revising the article critically for important intellectual content and (iii) final approval of the version to be published.
L.S. has contributed with: (i) substantial contributions to conception and design, acquisition of data, analysis and interpretation of data, (ii) revising the article critically for important intellectual content and (iii) final approval of the version to be published.
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Acknowledgement |
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We wish to thank histopathologist E.S. Hansen, who skillfully revised all hydatidiform moles.
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References |
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Submitted on March 13, 2008; resubmitted on April 24, 2008; accepted on April 30, 2008.
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