A True Hermaphrodite Chimera Resulting from Embryo Amalgamation after in Vitro Fertilization

Abstract

IGH 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 dizygot-ic-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 chime-ras) 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 gonad-otropins, 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. Ultra-sonography 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 histo-logic examination to be an ovary with a fallopian tube attached H 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 cho-rionic 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 polymer-ase 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 (chro-mosome Xp22.1), the patient had three alleles (Fig. 1A and 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 (Fig. 1A and 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 be