Original Article
Brief Report
A True Hermaphrodite Chimera Resulting from Embryo Amalgamation after in Vitro Fertilization
N Engl J Med 1998; 338:166-169January 15, 1998
- Article
High rates of successful pregnancy after in vitro fertilization depend on placing more than one embryo into the mother, a practice resulting in a 30-to-35-fold increase in dizygotic-twin deliveries.1 Increased frequencies of twin-associated anomalies might also therefore be expected. Chimerism, the presence in a single person of cells derived from two or more zygotes, is one such rare anomaly. It is usually ascertained through anomalous blood-grouping results or (for XX/XY chimeras) sex reversal or intersex.
We used DNA polymorphisms to investigate a 46,XX/46,XY hermaphrodite conceived by in vitro fertilization. We found not only that the child is a chimera, but also that he must have resulted from amalgamation of two embryos, each derived from an independent, separately fertilized ovum.
Case Report
The mother was a 31-year-old woman with primary infertility. Hormonal and laparoscopic investigation indicated a normal pelvis and normal ovulation. Her partner, who was 41 years old, had had a child by another partner but was severely oligozoospermic. The woman was given buserelin and human menopausal gonadotropins, after which 18 oocytes were harvested, of which 15 were fertilized in vitro with anonymous donor sperm and maintained in separate dishes. Two days after insemination (the four-cell stage), three embryos were transferred to the woman. Ultrasonography 36 days after transfer showed a single fetus and sac. A 3.46-kg infant was delivered vaginally at term; he had a normal right testis and an undescended left testis, with otherwise normal male genitalia. At the age of six months, the left testis was palpable at the inguinal ring. Surgical exploration at the age of 15 months revealed a hernial sac containing an abnormal gonad and vas deferens. These structures were excised; they proved on histologic examination to be an ovary with a fallopian tube attached to a horn of uterus. Karyotyping of peripheral-blood lymphocytes then revealed two cell lines, one 46,XX and the other 46,XY.
At the age of 20 months, the infant's serum follicle-stimulating hormone and luteinizing hormone concentrations were normal for his age, both basally and in response to gonadotropin-releasing hormone. The basal serum testosterone concentration was normal (<20 ng per deciliter [<0.7 nmol per liter]) and rose normally to 180 ng per deciliter (6.3 nmol per liter) three days after a single intramuscular injection of 2000 IU of human chorionic gonadotropin. Ultrasonography at three years eight months revealed an apparently normal right testis in the scrotum and normal kidneys, bladder, and pelvic structures. At laparoscopy at four years four months, the right vas deferens and testicular vessels appeared to be normal; no female genital structures were seen. A skin biopsy was performed. Subsequently, the child has grown and developed normally, with height at the 90th percentile and weight at the 75th percentile. He has no neurodevelopmental abnormalities, and he attends a regular school.
Methods
Informed consent for all genetic investigations and publication of the information was obtained from the child's parents. Separate samples of DNA were prepared from his peripheral blood and each of three flasks of fibroblasts cultured from his skin-biopsy specimen. Short tandem-repeat polymorphisms corresponding to anonymous loci were analyzed after amplification by the polymerase chain reaction with fluorescein-labeled primers, as described previously.2 Primer sequences were obtained from the Genome Database or the Généthon linkage map.3
Results
Demonstration of Chimerism by Analysis of DNA Polymorphisms
We first examined X-chromosome markers, because the results were less likely to be uninformative due to allele sharing between the patient's mother and father (who was not available for testing). For DXS3 (chromosome Xq21.3) and DXS451 (chromosome Xp22.1), the patient had three alleles (Figure 1AFigure 1
Demonstration of Chimerism through the Use of DNA Polymorphisms. and Figure 1B). In each case, one was paternal, indicating (since the patient also has a Y chromosome) the involvement of two sperm. The presence of two other alleles indicates that two different maternal X chromosomes were present.Comparison of the results for the different samples of fibroblast DNA (Figure 1A and Figure 1B) revealed a consistent peak height (mass) ratio between the paternal allele and one of the maternal alleles. These alleles (paternal 2 and maternal 1 for DXS3; paternal 1 and maternal 3 for DXS451) must therefore be the two in the XX cell line. In contrast, because of the differing proportions of XY and XX cells in each fibroblast culture, the height of the other maternal allele (XY cell line) varied independently (peak 3 for DXS3; peak 2 for DXS451). Fibroblast culture 3 thus contained mostly XY cells, and culture 2 almost entirely XX cells. Fibroblast culture 1 and the patient's blood had intermediate proportions of XX and XY cells.
The lack of paternal DNA made the results of studies of many autosomal markers inconclusive, but for D17S1178 (chromosome 17q11.2–q12) we found four distinguishable parental alleles. Again, the different samples showed constant height ratios between pairs of peaks, indicating that alleles 4 (maternal) and 2 (paternal) were in the XX cell line, whereas 1 (maternal) and 3 (paternal) were in the XY cell line (Figure 1C; compare the reciprocal patterns indicated for fibroblast cultures 2 and 3). The uninformative marker D7S460 indicates that inadvertent mixing of patient and maternal DNA did not occur; the prominent maternal allele 2 is completely absent from all samples from the patient (Figure 1D). Therefore, the biallelic maternal contributions for other markers are genetic ones and not contamination artifacts.
Origin of Chimerism
The DNA results prove the involvement of two sperm but leave open several embryologic possibilities4: fertilization both of a mature ovum and its first polar body5; fertilization of an ovum and second polar body; or “complete” chimerism, the amalgamation of independent embryos, each derived from an independently ovulated and separately fertilized ovum. Discriminating among these mechanisms is important, since only the third could result from the transfer of multiple embryos after in vitro fertilization.
Because of recombination during the first meiotic division, distinguishing among these possibilities genetically requires analysis of polymorphisms near the chromosomal centromeres. Three patterns can be predicted. In the case of fertilization of the first polar body, the two cell lines in the chimera will inherit opposite maternal centromeres. For any informative marker near the centromere, therefore, both maternal alleles will be present in the chimera. After fertilization of the second polar body, in contrast, the two chimera cell lines will have the same maternal centromere. For any informative markers near the centromere, the chimera will inherit only one maternal allele. In the case of embryo amalgamation, the centromeres of each maternal chromosome pair will have segregated randomly and independently in each zygote. For markers close to some centromeres, the opposite maternal alleles will be transmitted to the two chimera cell lines, whereas for others, the same allele will be present in both cell lines.
We analyzed polymorphisms close to the centromeres of 15 different chromosomes (Table 1Table 1
Pericentromeric-Marker Segregation in DNA from the Patient and His Mother.). Markers on three chromosomes (D4S2996, D6S430, and D20S871) (Figure 2AFigure 2
Segregation of Pericentromeric Markers in DNA from the Patient and His Mother., 2B, and 2C) demonstrated transmission of only one maternal allele, ruling out the first mechanism. In contrast, one autosomal marker (D11S4109) (Figure 2D) and one X-linked marker (AR) (Figure 2E) clearly showed the transmission of both maternal alleles, ruling out the second mechanism.By comparing the peak heights of the samples with different proportions of XX and XY cells, we could also infer the transmission of both maternal alleles for two further markers — D9S1874 (Figure 2F) and D19S49 (Figure 2G). Thus, four pericentromeric markers reveal the transmission of both maternal alleles, and three the transmission of only one maternal allele (Table 1).
Though each marker has a small chance of error due to recombination with its centromere, the findings for at least three chromosomes are inconsistent with fertilization of either the first or the second polar body as a mechanism. They instead strongly suggest independent segregation of maternal centromeres in two separate meioses (the third mechanism). We conclude that two embryos, independently fertilized in vitro, fused, presumably after transfer into the mother, because ova or embryos were not cocultured during or after fertilization.
Discussion
It is standard practice to replace more than one embryo after in vitro fertilization (preferably two embryos, because of high rates of the delivery of triplets after the placement of three or more7,8). The resulting proportion of twin pregnancies in most in vitro fertilization programs is 20 to 25 percent.1,9 The perinatal or postnatal complications of these twin gestations are generally thought to be those of multiple conception and not specific for in vitro fertilization,9,10 although the discordance in birth weight between twins seems to be greater than in natural pregnancies.11 The increase in the frequency of dizygotic twins after in vitro fertilization by a factor of approximately 33 implies a similarly increased risk of rare twin-associated anomalies such as chimerism.
The natural incidence of chimerism is unknown. Phenotypes of XX/XY chimeras range from normal fertile males12,13 through males with hypospadias or ambiguous genitalia and hermaphroditism14-18 and fertile female hermaphrodites19 to phenotypically normal, fertile females.20 This sparse literature is undoubtedly biased toward cases with sexual ambiguity or other gonadal problems, and many XX/XY chimeras may go unnoticed. Same-sex chimeras should be almost invariably phenotypically normal.
The observation of chimerism after in vitro fertilization should therefore be taken seriously. Not only does the great rarity of XX/XY chimerism suggest a causal link to the in vitro fertilization, but also its incidence could be higher than suspected.
Source Information
From the Human Genetics Unit, University of Edinburgh, Western General Hospital, Edinburgh (L.S., D.T.B.); the Department of Medical Genetics, Aberdeen Royal Infirmary (J.C.S.D.), and the Assisted Reproduction Unit, University Department of Obstetrics and Gynaecology, Aberdeen Maternity Hospital (M.P.R.H.), Aberdeen — all in the United Kingdom.
Address reprint requests to Dr. Bonthron at the Human Genetics Unit, Molecular Medicine Centre, Western General Hospital, Edinburgh EH4 2XU, United Kingdom.
- References
References
1
Friedler S ,Mashiach S ,Laufer N . Births in Israel resulting from in-vitro fertilization/embryo transfer, 1982-1989: National Registry of the Israeli Association for Fertility Research. Hum Reprod 1992;7:1159-1163
Web of Science | Medline2
Strain L ,Warner JP ,Johnston T ,Bonthron DT . A human parthenogenetic chimaera. Nat Genet 1995;11:164-169
CrossRef | Web of Science | Medline3
Dib C ,Faure S ,Fizames C , et al. A comprehensive genetic map of the human genome based on 5,264 microsatellites. Nature 1996;380:152-154
CrossRef | Web of Science | Medline4
Ford CE . Mosaics and chimaeras. Br Med Bull 1969;25:104-109
Web of Science | Medline5
Bieber FR ,Nance WE ,Morton CC , et al. Genetic studies of an acardiac monster: evidence of polar body twinning in man. Science 1981;213:775-777
CrossRef | Web of Science | Medline6
Collins A ,Frezal J ,Teague J ,Morton NE . A metric map of humans: 23,500 loci in 850 bands. Proc Natl Acad Sci U S A 1996;93:14771-14775
CrossRef | Web of Science | Medline7
Roest J ,van Heusden AM ,Verhoeff A ,Mous HV ,Zeilmaker GH . A triplet pregnancy after in vitro fertilization is a procedure-related complication that should be prevented by replacement of two embryos only. Fertil Steril 1997;67:290-295
CrossRef | Web of Science | Medline8
Staessen C ,Janssenswillen C ,Van den Abbeel E ,Devroey P ,Van Steirteghem AC . Avoidance of triplet pregnancies by elective transfer of two good quality embryos. Hum Reprod 1993;8:1650-1653
Web of Science | Medline9
Olivennes F ,Kadhel P ,Rufat P ,Fanchin R ,Fernandez H ,Frydman R . Perinatal outcome of twin pregnancies obtained after in vitro fertilization: comparison with twin pregnancies obtained spontaneously or after ovarian stimulation. Fertil Steril 1996;66:105-109
Web of Science | Medline10
Saunders K ,Spensley J ,Munro J ,Halasz G . Growth and physical outcome of children conceived by in vitro fertilization. Pediatrics 1996;97:688-692
Web of Science | Medline11
Bernasko J ,Lynch L ,Lapinski R ,Berkowitz RL . Twin pregnancies conceived by assisted reproductive techniques: maternal and neonatal outcomes. Obstet Gynecol 1997;89:368-372
CrossRef | Web of Science | Medline12
Watkins WM ,Yates AD ,Greenwell P , et al. A human dispermic chimaera first suspected from analyses of the blood group gene-specified glycosyltransferases. J Immunogenet 1981;8:113-128
CrossRef | Medline13
Schoenle E ,Schmid W ,Schinzel A , et al. 46,XX/46,XY Chimerism in a phenotypically normal man. Hum Genet 1983;64:86-89
CrossRef | Web of Science | Medline14
Fitzgerald PH ,Donald RA ,Kirk RL . A true hermaphrodite dispermic chimera with 46,XX and 46,XY karyotypes. Clin Genet 1979;15:89-96
CrossRef | Web of Science | Medline15
Farag TI ,Al-Awadi SA ,Tippett P , et al. Unilateral true hermaphrodite with 46,XX/46,XY dispermic chimerism. J Med Genet 1987;24:784-786
CrossRef | Web of Science | Medline16
Poissonnier M ,Janvier D ,Cabrol S ,Reviron M ,Noel L ,Reviron J . Hermaphrodisme vrai chez un enfant porteur d'une chimère chromosomique XX/XY, d'une double population erythrocytaire et d'une phénotype Lewis (a+ b+) inhabituel. J Genet Hum 1987;35:119-129
Medline17
Green AJ ,Barton DE ,Jenks P ,Pearson J ,Yates JR . Chimaerism shown by cytogenetics and DNA polymorphism analysis. J Med Genet 1994;31:816-817
CrossRef | Web of Science | Medline18
Sawai T ,Yoshimoto M ,Kinoshita E , et al. Case of 46,XX/47,XY,+21 chimerism in a newborn infant with ambiguous genitalia. Am J Med Genet 1994;49:428-430
CrossRef | Web of Science | Medline19
Verp MS ,Harrison HH ,Ober C , et al. Chimerism as the etiology of a 46,XX/46,XY fertile true hermaphrodite. Fertil Steril 1992;57:346-349
Web of Science | Medline20
Bromilow IM ,Duguid J . The Liverpool chimaera. Vox Sang 1989;57:147-149
CrossRef | Web of Science | Medline
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Citing Articles
1
Ronald M. Green. 2011. Ethical Considerations
. , 1117-1130.
CrossRef2
Johanna Winberg, Peter Gustavsson, Kristina Lagerstedt-Robinson, Elisabeth Blennow, Johanna Lundin, Erik Iwarsson, Anna Nordenström, Britt-Marie Anderlid, Marie-Louise Bondeson, Agneta Nordenskjöld, Ann Nordgren. (2010) Chimerism resulting from parthenogenetic activation and dispermic fertilization. American Journal of Medical Genetics Part A 152A:9, 2277-2286
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CrossRef5
Daniel W. Rust, Diana W. Bianchi. (2009) Microchimerism in Endocrine Pathology. Endocrine Pathology 20:1, 11-16
CrossRef6
Ann-Leslie Berger-Zaslav, Lakshmi Mehta, Jessy Jacob, Theresa Mercado, Inder Gadi, James H. Tepperberg, Lane S. Palmer. (2009) Ovotesticular Disorder of Sexual Development (True Hermaphroditism). Urology 73:2, 293-296
CrossRef7
M. Ramsay, W. Pfaffenzeller, E. Kotze, L. Bhengu, F. Essop, T. De Ravel. (2009) Chimerism in Black Southern African Patients with True Hermaphroditism 46,XX/47XY,+21 and 46,XX/46,XY. Annals of the New York Academy of Sciences 1151:1, 68-76
CrossRef8
M. Binkhorst,, N. de Leeuw,, B.J. Otten,. (2009) A Healthy, Female Chimera with 46,XX/46,XY Karyotype. Journal of Pediatric Endocrinology and Metabolism 22:1, 97-102
CrossRef9
Valerie A. Arboleda and, Eric Vilain. 2009. Disorders of Sex Development. , 367-393.
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Edmond J. Yunis, Joaquin Zuniga, Viviana Romero, Emilio J. Yunis. (2007) Chimerism and tetragametic chimerism in humans: implications in autoimmunity, allorecognition and tolerance. Immunologic Research 38:1-3, 213-236
CrossRef11
Martin H. Bluth, Marion E. Reid, Noga Manny. (2007) Chimerism in the Immunohematology Laboratory in the Molecular Biology Era. Transfusion Medicine Reviews 21:2, 134-146
CrossRef12
Vivienne L. Souter, Melissa A. Parisi, Dale R. Nyholt, Raj P. Kapur, Anjali K. Henders, Kent E. Opheim, Daniel F. Gunther, Michael E. Mitchell, Ian A. Glass, Grant W. Montgomery. (2007) A case of true hermaphroditism reveals an unusual mechanism of twinning. Human Genetics 121:2, 179-185
CrossRef13
Guillermo Alonso, Titania Pasqualini, Julio Busaniche, Eduardo Ruiz, Héctor Chemes. (2007) True Hermaphroditism in a Phenotypic Male without Ambiguous Genitalia: An Unusual Presentation at Puberty. Hormone Research 68:5, 261-264
CrossRef14
V Malan, M Vekemans, C Turleau. (2006) Chimera and other fertilization errors. Clinical Genetics 70:5, 363-373
CrossRef15
Mira Mosebach, Andreas Parkner, Sybille Jakubiczka, Peter Wieacker, Marcell U. Heim. (2006) Dispermic chimerism identified during blood group determination and HLA typing. Transfusion 46:11, 1978-1981
CrossRef16
Camilla Drexler, Thomas Wagner. (2006) Blood group chimerism. Current Opinion in Hematology 13:6, 484-489
CrossRef17
Urvashi Surti, Lori Hoffner, Marta Kolthoff, Jeanette Dunn, Jennifer Hunt, Lynn Sniezek, Trevor Macpherson. (2006) Persistent Gestational Trophoblastic Disease After an Androgenetic/Biparental Fetal Chimera. International Journal of Gynecological Pathology 25:4, 366-372
CrossRef18
Chien-Feng Sun, Ding-Ping Chen, Ching-Ping Tseng, Wei-Ting Wang, Jui-Ping Liu. (2006) Identification of a novel A1v-O1v hybrid allele with G829A mutation in a chimeric individual of AelBel phenotype. Transfusion 46:5, 780-789
CrossRef19
A Ito, Y Honma, E Inamori, Y Yada, M Y Momoi, Y Nakamura. (2006) Developmental outcome of very low birth weight twins conceived by assisted reproduction techniques. Journal of Perinatology 26:2, 130-133
CrossRef20
Chih-Ping Chen, Schu-Rern Chern, Jin-Chern Sheu, Shuan-Pei Lin, Chin-Yuan Hsu, Tung-Yao Chang, Chen-Chi Lee, Wayseen Wang, Hsiao-En Cindy Chen. (2005) Prenatal diagnosis, sonographic findings and molecular genetic analysis of a 46,XX/46,XY true hermaphrodite chimera. Prenatal Diagnosis 25:6, 502-506
CrossRef21
Camilla Drexler, Barbara Glock, Maria Vadon, Erika Staudacher, Eva-Maria Dauber, Silvia Ulrich, Rosemarie B.K. Reisacher, Wolfgang R. Mayr, Gerhard Lanzer, Thomas Wagner. (2005) Tetragametic chimerism detected in a healthy woman with mixed-field agglutination reactions in ABO blood grouping. Transfusion 45:5, 698-703
CrossRef22
Kiyonori Miura, Norio Niikawa. (2005) Do monochorionic dizygotic twins increase after pregnancy by assisted reproductive technology?. Journal of Human Genetics 50:1, 1-6
CrossRef23
Denise I. Quigley, Jeffery Sailus, Kathleen Kaiser-Rogers, Kathleen W. Rao, Muge Calikoglu, Stuart Gold, Shawn E. McCandless. (2005) A clinical report of a patient with two abnormal cell lines: 46,XX,del(21)(q22.1) and 47,XX,+3. American Journal of Medical Genetics Part A 132A:1, 101-105
CrossRef24
Holzelova, Eliska, Vonarbourg, Cédric, Stolzenberg, Marie-Claude, Arkwright, Peter D., Selz, Françoise, Prieur, Anne-Marie, Blanche, Stéphane, Bartunkova, Jirina, Vilmer, Etienne, Fischer, Alain, Le Deist, Françoise, Rieux-Laucat, Frédéric, . (2004) Autoimmune Lymphoproliferative Syndrome with Somatic Fas Mutations. New England Journal of Medicine 351:14, 1409-1418
Full Text25
Regina Ensenauer, Syed Jalal, Reid Meyer, Dusica Babovic-Vuksanovic. (2004) Unbalanced cryptic 5p deletion/17p duplication identified by subtelomeric FISH in a family with a boy with chimerism and a balanced t(4;5). American Journal of Medical Genetics 125A:1, 86-91
CrossRef26
R Lisker. (2003) Ethical and legal issues in therapeutic cloning and the study of stem cells. Archives of Medical Research 34:6, 607-611
CrossRef27
Shemin Zeng, Shivanand R. Patil, Jerome Yankowitz. (2003) Prenatal detection of mosaic trisomy 1q due to an unbalanced translocation in one fetus of a twin pregnancy following in vitro fertilization: A postzygotic error. American Journal of Medical Genetics 120A:4, 464-469
CrossRef28
Souter, Vivienne L., Kapur, Raj P., Nyholt, Dale R., Skogerboe, Kristen, Myerson, David, Ton, Carl C., Opheim, Kent E., Easterling, Thomas R., Shields, Laurence E., Montgomery, Grant W., Glass, Ian A., . (2003) A Report of Dizygous Monochorionic Twins. New England Journal of Medicine 349:2, 154-158
Full Text29
Ronald Green. 2002. Human Subjects Research, Ethics, Research on Human Embryos. .
CrossRef30
Yu, Neng, Kruskall, Margot S., Yunis, Juan J., Knoll, Joan H.M., Uhl, Lynne, Alosco, Sharon, Ohashi, MarinaClavijo, OlgaHusain, Zaheed, Yunis, Emilio J., Yunis, Jorge J., Yunis, Edmond J., . (2002) Disputed Maternity Leading to Identification of Tetragametic Chimerism. New England Journal of Medicine 346:20, 1545-1552
Full Text31
Helen Pearson. (2002) Human genetics: Dual identities. Nature 417:6884, 10-11
CrossRef32
Elizabeth J. Parks. (2002) Changes in fat synthesis influenced by dietary macronutrient content. Proceedings of the Nutrition Society 61:02, 281-286
CrossRef33
Michael Golubovsky. (2002) Paternal Familial Twinning: Hypothesis and Genetic/Medical Implications. Twin Research and Human Genetics 5:2, 75-86
CrossRef34
J. Savulescu. (2000) The ethics of cloning and creating embryonic stem cells as a source of tissue for transplantation: time to change the law in Australia. Australian and New Zealand Journal of Medicine 30:4, 492-498
CrossRef35
M Montero, R Mendez, D Valverde, JL Fernández, M Gómez, C Ruíz. (1999) True hermaphroditism and normal male external genitalia: a rare presentation. Acta Paediatrica 88:8, 909-911
CrossRef36
Jose M. Ordovas, Ernst J. Schaefer. (1999) Treatment of dyslipidemia: Genetic interactions with diet and drug therapy. Current Atherosclerosis Reports 1:1, 16-23
CrossRef37
Jose M. Ordovas, Ernst J. Schaefer. (1999) Genes, variation of cholesterol and fat intake and serum lipids. Current Opinion in Lipidology 10:1, 15-22
CrossRef38
Roelof-Jan Oostra, Bob Baljet, Ben W.J.M. Verbeeten, Raoul C.M. Hennekam. (1998) Congenital anomalies in the teratological collection of museum Vrolik in Amsterdam, the Netherlands. v: conjoined and acardiac twins. American Journal of Medical Genetics 80:1, 74-89
CrossRef39
Van Steirteghem, André, . (1998) Outcome of Assisted Reproductive Technology. New England Journal of Medicine 338:3, 194-195
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