Case 11-2005 — A 32-Year-Old Pregnant Woman with an Abnormal Fetal Karyotype
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《新英格兰医药杂志》
Presentation of Case
A 32-year-old woman in the 17th week of her first pregnancy was referred for genetic counseling after her fetus had been found to have an abnormal karyotype. She had had an uneventful first trimester. In the 14th week, transabdominal and transvaginal ultrasonography, performed to evaluate an ovarian cyst, revealed a single intrauterine gestational sac containing a normal fetus with a normal heart rate. The area of nuchal translucency was 3.7 mm in diameter (normal, 2.5 mm) (Figure 1 shows an ultrasound study of a similar fetus). There were two cysts within the left ovary; a clear cyst, 4.1 cm in diameter, and a second cyst, 4.5 cm in diameter, that was consistent with an endometrioma. It was recommended that amniocentesis be performed at 15 weeks of gestation. In a meeting with a genetic counselor at that time, the patient declined to provide a detailed family history.
Figure 1. Midsagittal Ultrasound Study of the 13-Week Fetus of Another Patient, Showing Increased Nuchal Translucency.
The area of nuchal translucency (indicated by the cursors) is enlarged, indicating fetal edema. This fetus had trisomy 21, and the nasal bone was absent.
Two weeks later, a follow-up ultrasound study demonstrated a regular fetal heartbeat and fluid within the fetal stomach. The cranium and spine appeared to be well formed for gestational age. There were bilateral nuchal hygromas. An amniocentesis was performed. The analysis of amniotic fluid revealed a normal alpha-fetoprotein level for gestational age. The amniotic fluid karyotype was 46,XY, add(18)(p11.2), indicating the presence of additional material on the short arm of chromosome 18 at band 11.2 (Figure 2).
Figure 2. Balanced and Unbalanced Translocations Involving Chromosomes 1 and 18.
In Panel A, the karyotype of the fetus, determined from the amniotic fluid, shows additional material on the long arm of one copy of chromosome 18 (arrow). In Panel B, the karyotype of the mother shows a reciprocal translocation between the long arm of one copy of chromosome 1 and the short arm of one copy of chromosome 18 (arrows). A color-coded diagram (Panel C) shows the t(1;18) translocation. In the derived chromosome 1, the distal portion of the long arm (q42.1ter), has been replaced by the distal portion of the short arm of chromosome 18 (p11.2ter). The derived chromosome 18 shows the translocated portion of chromosome 1.
Two weeks later, the parents returned to the Prenatal Diagnosis Unit. They were told that the fetus could be affected by an unbalanced chromosomal translocation. The mother reported that her paternal aunt had had two sons with birth defects. The father had no relatives with known birth defects or mental retardation. No other major risk factors were identified. The mother had smoked in the past but had stopped smoking before she conceived. She drank two to three alcoholic beverages per week but had stopped when she learned she was pregnant.
A diagnostic test was performed.
Differential Diagnosis
Dr. Lewis B. Holmes: Dr. Nadel, would you show us the ultrasound studies?
Dr. Allan S. Nadel: In the late first and early second trimester, fetal edema is usually most prominent in the subcutaneous tissue of the neck; this produces an area of translucency in the nuchal soft tissues. Mild nuchal edema is quantified by measuring the nuchal translucency, the sonolucent space in the skin of the posterior neck, as seen on a midsagittal image (Figure 1). Mild nuchal edema before 14 weeks is associated with an increased risk of chromosomal abnormalities, structural anomalies, and a number of genetic syndromes.1 Therefore, when excessive nuchal edema is detected, evaluation of the fetal karyotype (by chorionic-villus sampling or amniocentesis) should be offered to the parents and followed by a detailed structural survey with special attention to the heart and great vessels. If all the findings are normal, the fetus is probably completely healthy, although a slightly increased risk of a genetic syndrome remains. This fetus had mild nuchal edema in the 14th week, and the mother was counseled to undergo amniocentesis after 15 weeks, when the risk of procedure-related complications is thought to be minimal.2 At that time, the structural survey of the fetus was limited by its early gestational age. Amniocentesis was performed.
Dr. Holmes: After the first ultrasound study, the parents were asked to meet with a genetic counselor to discuss the amniocentesis procedure, including its potential risks and the benefits. As part of that session, the genetic counselor asked about the medical histories of their families, but the parents were distracted by the news of the fetal abnormalities and preferred not to review the family history at that time.
A chromosomal abnormality was identified on amniocentesis, with additional chromosomal material on the short arm of chromosome 18, possibly reflecting an unbalanced translocation (Figure 2A). The parents returned for a discussion of this finding and its potential clinical significance and interpretation. I showed them the karyotype and pointed out the chromosomal abnormality identified on amniocentesis and told them that an unbalanced chromosomal translocation was one possible explanation of the finding. I explained that one of them could be a carrier of a balanced translocation, which, in turn, had led to their fetus's having an unbalanced chromosomal translocation. When we began to construct the family tree, the mother said that she must have the chromosomal translocation, because of her family's medical history. Both her parents, her brother and sister, and six nieces and nephews were healthy. However, she knew that one of her father's sisters had had two children with birth defects. The parents agreed to blood testing to determine whether one of them had a chromosomal translocation. We agreed to wait for the results of the chromosomal analysis before the woman asked her relatives for more information.
The couple returned two weeks later to review the findings. The genetic team described the process of chromosomal analysis and showed them images of normal (Giemsa-banded) karyotypes. We then showed them their karyotypes; the father's was normal 46,XY, and the mother's contained a balanced translocation (Figure 2B). A color-coded diagram (Figure 2C) was used to show the derived chromosome 1; the distal portion of the long arm (q42.1ter) was absent and had been replaced by the distal portion (p11.2ter) of one copy of the short arm of chromosome 18. The derived chromosome 18 had the translocated portion of chromosome 1. The mother's karyotype was thus 46,XX,t(1;18)(q42.1;p11.2), indicating that material from the long arm (q) of chromosome 1 at band 42.1 had switched positions with material from the short arm (p) of chromosome 18 at band p11.2, resulting in a balanced translocation — that is, no chromosomal material was missing. That of the fetus was 46,XY,der(18)t(1;18)(q42.1;p11.2)mat — indicating that the derivative (der) chromosome 18 was the result of the maternal (mat) balanced translocation. Since this fetus inherited the normal chromosome 1 from the mother, the translocation was unbalanced, and the fetus effectively had trisomy for the translocated portion of 1q and monosomy for the terminal portion of chromosome 18.
The parents chose to terminate this pregnancy because of the serious prognosis associated with the unbalanced translocation. The mother said that she would contact relatives for more information about the medical history of her cousins with birth defects. The pregnancy was terminated at 18 weeks. The parents returned to the genetics clinic to review the risk that future pregnancies would be abnormal. They were told that a future pregnancy could have one of three outcomes: a healthy child, a healthy child with the same balanced translocation as the mother's, or a child with either of two unbalanced translocations.
The mother had brought with her a copy of an article in a genetics journal, published in 1979, in which the same chromosomal translocation had been described in several of her father's relatives.3 She had not been told about the risk of this genetic abnormality because she was related to these people through her father; since only females had been identified as carriers, her relatives believed that her father was not at risk and that she was therefore not at risk either.
When we contacted the first author of the report, Ruth M. Liberfarb, M.D., Ph.D., she recalled that the first contact with the family occurred as a result of the evaluation of a child with multiple congenital anomalies and developmental delay. Both the mother of the child (Figure 3; II-2), a maternal aunt of the child (II-5), and the maternal grandmother (I-2) had had multiple spontaneous abortions and stillbirths, as well as live-born children who died in infancy with congenital anomalies. Chromosomal analysis identified the unbalanced translocation in the child; studies of his parents showed that his mother had a balanced translocation. Screening of the parents' phenotypically normal son and daughter, the maternal grandparents, and all the mother's siblings was recommended to determine whether they were carriers of this translocation. The mother of the child with birth defects invited her relatives to come to her home for a discussion with Dr. Liberfarb about the risk of carrier status and the option of undergoing chromosomal analysis. Her brother (II-6), the father of the woman whose pregnancy is discussed here, did not attend this meeting or undergo genetic testing. The pedigree of the family described by Liberfarb et al.,3 with the addition of information about the patient's generation (the patient is III-15) and offspring, is shown in Figure 3.
Figure 3. Pedigree of the Mother's Family.
The pedigree, reported in 1979 by Liberfarb et al.3 and updated to show the parents and fetus discussed here, shows that numerous family members of several generations had a balanced translocation, an unbalanced translocation, or phenotypic features suggesting an unbalanced translocation. Circles denote female family members, squares male family members, triangles aborted fetuses, solid symbols subjects with unbalanced translocations, half-solid symbols subjects with balanced translocations, shaded symbols subjects with malformations suggesting an unbalanced translocation, N within a symbol subjects with a normal karyotype, and slash marks deceased subjects. The arrow indicates the fetus in the case under discussion. Adapted from Liberfarb et al.,3 with the permission of the publisher.
During the discussion at our genetics center, the mother of the affected fetus asked about the new method of prenatal diagnosis called preimplantation genetic diagnosis. She was eager to know whether this approach would allow her to have a healthy infant and to avoid having to consider the difficult and traumatic option of termination of pregnancy if another fetus had an unbalanced chromosomal translocation. Since this procedure is not performed at this hospital, I referred the couple to our colleagues at Brigham and Women's Hospital, who are using preimplantation genetic diagnosis to monitor pregnancies at risk for an unbalanced translocation.
Discussion of Management
Dr. Antonio R. Gargiulo: The couple saw Dr. Louise Wilkins-Haug in the Maternal–Fetal Medicine Division. At that time, the patient was again pregnant. Dr. Wilkins-Haug provided genetic counseling during a second pregnancy and performed chorionic-villous sampling, which disclosed abnormal material on the short arm of chromosome 18. After the termination of their second consecutive pregnancy, the couple came to the Center for Reproductive Medicine at Brigham and Women's Hospital to explore options for preimplantation detection of fetal chromosomal aberrations.
The couple's reproductive history disclosed profound subfertility, with unprotected timed intercourse practiced for four years before their first conception. The results of the diagnostic workup for infertility included a normal semen analysis and elevation of the wife's serum estradiol level to 62 pg per milliliter on day 3 of her menstrual cycle, with a normal serum level of follicle-stimulating hormone (FSH) of 7.6 mIU per milliliter.
Carriers of reciprocal translocations, such as this woman, produce both balanced and unbalanced gametes. The chromosomal constitution of the gametes formed depends on the type of segregation after pairing of homologous chromosomes at meiosis in a quadriradial figure. Typically, a chromosomal segregation results in the formation of two chromosomally balanced gametes and four unbalanced gametes. When fertilization occurs with a normal haploid gamete, the balanced gametes will produce a phenotypically normal conceptus with either normal chromosomes or the same balanced translocation carried by a parent. Some translocations can affect implantation and thus fertility; however, the amount of chromosomal material involved in this case would be unlikely to explain this couple's infertility.
The woman had been followed by her gynecologist by means of serial pelvic ultrasonography for a persistent complex left ovarian cyst. In preparation for assisted reproduction, we performed a combined laparoscopy and hysteroscopy to evaluate the ovarian cyst, as well as to survey the anatomical features of the endometrial cavity, looking for abnormalities that might explain her subfertility. We found a left ovarian endometriotic cyst, which we excised, and otherwise normal pelvic anatomy, with patent fallopian tubes and a normal endometrial cavity. This degree of pelvic endometriosis probably caused her infertility.
The couple was counseled about the technical aspects of in vitro fertilization and preimplantation genetic diagnosis for the identification of embryos with unbalanced chromosomes.
Dr. Catherine Racowsky: Preimplantation genetic diagnosis entails the screening of embryos produced by in vitro fertilization for a variety of genetic abnormalities. Genetic analysis of the embryos involves delicate micromanipulation techniques to remove cellular material. One or two blastomeres are removed from the embryo at the 6-to-8-cell cleavage stage. Removal of two blastomeres instead of one has been shown to improve the accuracy of preimplantation genetic diagnosis, especially in couples with balanced chromosomal translocations, in which a high incidence of blastomere mosaicism is encountered.4,5
Once the cellular material is obtained, it can be analyzed by different techniques: fluorescence in situ hybridization, polymerase chain reaction, or comparative genomic hybridization. Fluorescence in situ hybridization is used to determine numerical or structural abnormalities of specific chromosomes, as well as for determination of sex chromosomes. It is the technique best suited to screening embryos before implantation to identify carriers of balanced translocations. Its application has been shown to reduce drastically the chance of chromosomally abnormal pregnancies in couples with such translocations.6
Dr. Gargiulo: Fluorescence in situ hybridization studies with use of DNA probes for the subtelomeric regions of the short arm of chromosome 18 (18ptel) and the long arm (18qtel) were performed initially on chromosomes in metaphase and on nuclei in interphase from peripheral-blood lymphocytes obtained from the woman and her husband; the results were compared with those for matched controls. Two hybridization signals specific for the 18ptel probe and two hybridization signals specific for the 18qtel probe were observed in 90 percent of nuclei obtained from the wife's peripheral-blood sample, in 94 percent of nuclei obtained from the husband's peripheral-blood sample, and in 92 percent of nuclei that were examined from a control specimen.
Once this preliminary test was completed, the couple began a cycle of in vitro fertilization. Controlled ovarian hyperstimulation was induced with high doses of recombinant human FSH (300 IU given subcutaneously twice a day), and pituitary down-regulation was achieved by administration of the gonadotropin-releasing–hormone (GnRH) agonist leuprolide acetate at a daily dose of 0.5 mg, given subcutaneously, starting in the luteal phase preceding the stimulation cycle. In spite of this aggressive stimulation protocol, only six measurable follicles were present after 11 days of gonadotropin administration, when dimension criteria (two leading follicles with a mean diameter of 18 mm) for final oocyte maturation were reached. A single dose of human chorionic gonadotropin (hCG) (10,000 IU) was administered intramuscularly, and follicular aspiration guided by transvaginal ultrasonography was performed after 36 hours. All clinical embryology procedures were carried out by the Brigham and Women's In Vitro Fertilization Laboratory.
Seven cumulus–oocyte complexes were identified. Four of these contained metaphase II oocytes (mature oocytes that have extruded the first polar body and can be fertilized normally). Intracytoplasmic sperm injection was performed on the four mature oocytes, all of which were fertilized. Given the low number of mature oocytes and subsequently of embryos obtained, as well as the expected 1:3 ratio of embryos with the balanced translocation to those with the unbalanced translocation, the couple was counseled to have all embryos frozen at the pronuclear stage of development (i.e., about 18 hours after intracytoplasmic sperm injection) and to undergo another cycle of in vitro fertilization to obtain more embryos. Reports of successful preimplantation genetic diagnosis with cryopreserved embryos had recently appeared, proving that this genetic analysis was still possible after embryos had been thawed.7
A second cycle of controlled ovarian hyperstimulation was started after a rest cycle. This time, an even more aggressive stimulation protocol was adopted, with daily doses with recombinant human FSH (300 IU given subcutaneously) and purified urinary luteinizing hormone (LH) with FSH (300 IU given subcutaneously). Moreover, leuprolide acetate was administered together with gonadotropins at doses of 0.05 mg given subcutaneously twice a day. This protocol allows a mild "flare" of endogenous secretion at the beginning of the cycle, while still providing sufficient pituitary suppression to prevent the LH surge later in the cycle. In spite of this adjustment in the protocol, the ovarian response was again low. Six cumulus–oocyte complexes were identified, all of which contained mature oocytes; however, the fertilization rate with intracytoplasmic sperm injection was lower than expected, and only two embryos were obtained. For the reasons described above, these embryos were also cryopreserved for future analysis.
A third cycle of controlled ovarian hyperstimulation was begun after a rest cycle. In this case, we used the same gonadotropin regimen as in the second cycle but substituted the GnRH antagonist cetrorelix for the GnRH agonist leuprolide acetate. Cetrorelix (250 μg given subcutaneously per day) was started after six days of gonadotropin treatment to achieve rapid pituitary suppression of the LH surge with no compromise of FSH levels during the early part of the stimulation cycle. Once again, six cumulus–oocyte complexes were identified, all of which contained mature oocytes and on which intracytoplasmic sperm injection was performed; four embryos were obtained and again frozen at the pronuclear stage.
A fourth and final cycle of controlled ovarian hyperstimulation was started after two rest cycles. In this cycle the same stimulation protocol was used as in the preceding cycle, and the same number of mature oocytes resulted. Three of the four oocytes were successfully fertilized with intracytoplasmic sperm injection.
Dr. Racowsky: At this point, the decision was made to proceed with preimplantation genetic diagnosis, and the 10 previously cryopreserved embryos were thawed in order to synchronize their development with that of the freshly produced embryos. All 10 cryopreserved embryos survived the thawing process. Blastomere biopsy was performed on day 3 of embryo development. Blastomeres were obtained from 9 embryos that had developed to the stage of having six or more blastomeres — that is, from the 3 fresh embryos and from 6 of the 10 thawed embryos. In six of the nine biopsied embryos, it was technically feasible to obtain two blastomeres. Blastomeres were then processed for chromosome spreading and fixation by a modification of the Tarkowski method.8 They were then submitted to the cytogenetics laboratory for analysis by fluorescence in situ hybridization. The results are shown in Table 1.
Table 1. Characteristics of All Embryos Produced by the Couple.
In two of the nine embryos, the nuclei of the fixated blastomeres could not be located by fluorescence in situ hybridization. Results were therefore available for seven embryos on the day of embryo transfer (day 4 of embryo development). Three chromosomally balanced embryos (which were either chromosomally normal or had a balanced chromosomal translocation) and four chromosomally unbalanced embryos (two with partial monosomy 18 and two with partial trisomy 18) were identified (Figure 4A and Figure 4B and Table 1). One of the balanced embryos had developed appropriately to the morula stage, whereas in the other two balanced embryos growth had been arrested after the blastomere biopsy. One of these two appeared strikingly abnormal, with multiple large fragments and cytoplasmic vacuoles. After thorough consultation, the couple elected to transfer the two normal-appearing, chromosomally balanced embryos (Figure 5) and to discard all the other embryos.
Figure 4. Results of Fluorescence in Situ Hybridization with Blastomeres from Three Chromosomally Balanced Embryos (Panel A) and Four Chromosomally Unbalanced Embryos (Panel B).
A green chromogen is linked to the DNA probe for the telomere of the short arm of chromosome 18 (18ptel), and a magenta chromogen is linked to the DNA probe for the long arm (18qtel). The normal blastomeres contain two green and two magenta signals, signifying either a normal karyotype or a balanced translocation. The abnormal blastomeres contain either one or three signals of either or both colors, indicating monosomy or trisomy for that locus — the result of an unbalanced translocation. (Images courtesy of Dr. Aida Nureddin and Dr. Stanislava Weremowicz, Brigham and Women's Hospital.)
Figure 5. Phase-Contrast Photomicrograph of the Two Chromosomally Balanced Embryos Selected for Transfer.
On the left is a morula from a fresh embryo, and on the right is a cleavage-stage embryo frozen and thawed before preimplantation genetic diagnosis.
Dr. Gargiulo: Transcervical embryo transfer with use of a soft-tip catheter was performed without difficulty. Luteal-phase progesterone supplementation was provided by daily intramuscular injections of progesterone in oil (50 mg). The serum level of the subunit of hCG two weeks after embryo transfer (649 mIU per milliliter) indicated early implantation, with a normal increase after 48 hours (to 1818 mIU per milliliter). The first clinical evidence of pregnancy was obtained at 5.9 weeks of gestation, when ultrasonography showed a single intrauterine pregnancy with a fetal heart rate of 111 beats per minute. A follow-up ultrasound examination at eight weeks of gestation confirmed an intrauterine pregnancy with a live single fetus, with normal interval growth and a normal fetal heart rate of 153 beats per minute. The patient was referred to her obstetrician for routine prenatal care. Given an expected rate of accuracy of 90 to 95 percent for preimplantation genetic diagnosis, depending on the condition analyzed and the technique used, amniocentesis is strongly recommended for all women whose embryos have undergone this selection procedure before transfer. This patient declined both so-called triple screening (measurement of estriol, the subunit of hCG, and alpha-fetoprotein) and amniocentesis.
The woman had an uneventful pregnancy. Labor was induced at 41 weeks of gestation, and delivery was eventually accomplished by primary low transverse cesarean section because of failed induction and a nonreassuring fetal heart rate. A healthy baby boy, weighing 3420 g and with one-minute and five-minute Apgar scores of 9 and 9, was evaluated by the pediatrician and discharged home with his mother.
A Physician: What is the risk to the fetus associated with removing blastomeres?
Dr. Gargiulo: The largest study done to assess the developmental steps of children born after preimplantation genetic diagnosis followed some such children until two years of age and found no developmental abnormalities.9 However, unlike in vitro fertilization or intracytoplasmic sperm injection, for which we now have cohorts of children followed all the way into school age and young adulthood, we do not at present have any published developmental data on children born after preimplantation genetic diagnosis for use in counseling prospective parents considering the procedure.
Dr. Racowsky: There is an uneven distribution of regulatory proteins even as early as the one-cell stage in the human embryo, and this uneven distribution becomes more apparent as the embryo undergoes cleavage to the morula stage.10 During preimplantation genetic diagnosis, critical proteins that are currently unidentified might be removed with the biopsied blastomere and might affect development down the road. The European Society of Human Reproduction preimplantation genetic diagnosis consortium follows babies born after use of this procedure throughout the world, and to date there do not appear to be any differences in the developmental timelines of these babies and babies whose births did not follow preimplantation genetic diagnosis.11 However, these are still early days with regard to the application of this invasive technique, and we must counsel our patients appropriately. Larger studies are needed to assess the long-term outcomes of these children.
Dr. Holmes: At the last follow-up, the infant was nine months old and progressing normally.
Anatomical Diagnosis
Balanced maternal chromosomal translocation — 46,XX,t(1;18)(q42.1;p11.2) — resulting in an unbalanced fetal translocation — 46,XY,der(18)t(1;18)(q42.1;p11.2)mat.
Source Information
From the Genetics and Teratology Unit, Mass General Hospital for Children (L.B.H.), and the Department of Obstetrics and Gynecology (A.S.N.), Massachusetts General Hospital; the Departments of Pediatrics (L.B.H.) and Obstetrics, Gynecology, and Reproductive Biology (A.R.G., A.S.N., C.R.), Harvard Medical School; and the Center for Reproductive Medicine, Brigham and Women's Hospital (A.R.G., C.R.).
References
Nicolaides KH, Sebire NJ, Snijders JM, eds. The 11-14 week scan: the diagnosis of fetal abnormalities. New York: Parthenon, 1999.
The Canadian Early and Mid-trimester Amniocentesis Trial (CEMAT) Group. Randomised trial to assess safety and fetal outcome of early and midtrimester amniocentesis. Lancet 1998;351:242-247.
Liberfarb RM, Breg WR, Atkins L, Holmes LB. Multiple congenital anomalies/mental retardation (MCA/MR) syndrome due to partial 1q duplication and possible 18p deletion: a study of four individuals in two families. Am J Med Genet 1979;4:27-37.
Iwarsson E, Malmgren H, Inzunza J, et al. Highly abnormal cleavage divisions in preimplantation embryos from translocation carriers. Prenat Diagn 2000;20:1038-1047.
Van de Velde H, De Vos A, Sermon K, et al. Embryo implantation after biopsy of one or two cells from cleavage-stage embryos with a view to preimplantation genetic diagnosis. Prenat Diagn 2000;20:1030-1037.
Munne S, Sandalinas M, Escudero T, Fung J, Gianaroli L, Cohen J. Outcome of preimplantation genetic diagnosis of translocations. Fertil Steril 2000;73:1209-1218.
Ciotti PM, Lagalla C, Ricco AS, Fabbri R, Forabosco A, Porcu E. Micromanipulation of cryopreserved embryos and cryopreservation of micromanipulated embryos in preimplantation genetic diagnosis. Mol Cell Endocrinol 2000;169:63-67.
Tarkowski AK. An air-drying method for chromosome preparations from mouse eggs. Cytogenetics 1966;5:394-400.
Strom CM, Levin R, Strom S, Masciangelo C, Kuliev A, Verlinsky Y. Neonatal outcome of preimplantation genetic diagnosis by polar body removal: the first 109 infants. Pediatrics 2000;106:650-653.
Antczak M, Van Blerkom J. Temporal and spatial aspects of fragmentation in early human embryos: possible effects on developmental competence and association with the differential elimination of regulatory proteins from polarized domains. Hum Reprod 1999;14:429-447.
Sermon K, Moutou C, Harper J, et al. ESHRE preimplantation genetic diagnosis Consortium data collection IV: May-December 2001. Hum Reprod 2005;20:19-34.(Lewis B. Holmes, M.D., An)
A 32-year-old woman in the 17th week of her first pregnancy was referred for genetic counseling after her fetus had been found to have an abnormal karyotype. She had had an uneventful first trimester. In the 14th week, transabdominal and transvaginal ultrasonography, performed to evaluate an ovarian cyst, revealed a single intrauterine gestational sac containing a normal fetus with a normal heart rate. The area of nuchal translucency was 3.7 mm in diameter (normal, 2.5 mm) (Figure 1 shows an ultrasound study of a similar fetus). There were two cysts within the left ovary; a clear cyst, 4.1 cm in diameter, and a second cyst, 4.5 cm in diameter, that was consistent with an endometrioma. It was recommended that amniocentesis be performed at 15 weeks of gestation. In a meeting with a genetic counselor at that time, the patient declined to provide a detailed family history.
Figure 1. Midsagittal Ultrasound Study of the 13-Week Fetus of Another Patient, Showing Increased Nuchal Translucency.
The area of nuchal translucency (indicated by the cursors) is enlarged, indicating fetal edema. This fetus had trisomy 21, and the nasal bone was absent.
Two weeks later, a follow-up ultrasound study demonstrated a regular fetal heartbeat and fluid within the fetal stomach. The cranium and spine appeared to be well formed for gestational age. There were bilateral nuchal hygromas. An amniocentesis was performed. The analysis of amniotic fluid revealed a normal alpha-fetoprotein level for gestational age. The amniotic fluid karyotype was 46,XY, add(18)(p11.2), indicating the presence of additional material on the short arm of chromosome 18 at band 11.2 (Figure 2).
Figure 2. Balanced and Unbalanced Translocations Involving Chromosomes 1 and 18.
In Panel A, the karyotype of the fetus, determined from the amniotic fluid, shows additional material on the long arm of one copy of chromosome 18 (arrow). In Panel B, the karyotype of the mother shows a reciprocal translocation between the long arm of one copy of chromosome 1 and the short arm of one copy of chromosome 18 (arrows). A color-coded diagram (Panel C) shows the t(1;18) translocation. In the derived chromosome 1, the distal portion of the long arm (q42.1ter), has been replaced by the distal portion of the short arm of chromosome 18 (p11.2ter). The derived chromosome 18 shows the translocated portion of chromosome 1.
Two weeks later, the parents returned to the Prenatal Diagnosis Unit. They were told that the fetus could be affected by an unbalanced chromosomal translocation. The mother reported that her paternal aunt had had two sons with birth defects. The father had no relatives with known birth defects or mental retardation. No other major risk factors were identified. The mother had smoked in the past but had stopped smoking before she conceived. She drank two to three alcoholic beverages per week but had stopped when she learned she was pregnant.
A diagnostic test was performed.
Differential Diagnosis
Dr. Lewis B. Holmes: Dr. Nadel, would you show us the ultrasound studies?
Dr. Allan S. Nadel: In the late first and early second trimester, fetal edema is usually most prominent in the subcutaneous tissue of the neck; this produces an area of translucency in the nuchal soft tissues. Mild nuchal edema is quantified by measuring the nuchal translucency, the sonolucent space in the skin of the posterior neck, as seen on a midsagittal image (Figure 1). Mild nuchal edema before 14 weeks is associated with an increased risk of chromosomal abnormalities, structural anomalies, and a number of genetic syndromes.1 Therefore, when excessive nuchal edema is detected, evaluation of the fetal karyotype (by chorionic-villus sampling or amniocentesis) should be offered to the parents and followed by a detailed structural survey with special attention to the heart and great vessels. If all the findings are normal, the fetus is probably completely healthy, although a slightly increased risk of a genetic syndrome remains. This fetus had mild nuchal edema in the 14th week, and the mother was counseled to undergo amniocentesis after 15 weeks, when the risk of procedure-related complications is thought to be minimal.2 At that time, the structural survey of the fetus was limited by its early gestational age. Amniocentesis was performed.
Dr. Holmes: After the first ultrasound study, the parents were asked to meet with a genetic counselor to discuss the amniocentesis procedure, including its potential risks and the benefits. As part of that session, the genetic counselor asked about the medical histories of their families, but the parents were distracted by the news of the fetal abnormalities and preferred not to review the family history at that time.
A chromosomal abnormality was identified on amniocentesis, with additional chromosomal material on the short arm of chromosome 18, possibly reflecting an unbalanced translocation (Figure 2A). The parents returned for a discussion of this finding and its potential clinical significance and interpretation. I showed them the karyotype and pointed out the chromosomal abnormality identified on amniocentesis and told them that an unbalanced chromosomal translocation was one possible explanation of the finding. I explained that one of them could be a carrier of a balanced translocation, which, in turn, had led to their fetus's having an unbalanced chromosomal translocation. When we began to construct the family tree, the mother said that she must have the chromosomal translocation, because of her family's medical history. Both her parents, her brother and sister, and six nieces and nephews were healthy. However, she knew that one of her father's sisters had had two children with birth defects. The parents agreed to blood testing to determine whether one of them had a chromosomal translocation. We agreed to wait for the results of the chromosomal analysis before the woman asked her relatives for more information.
The couple returned two weeks later to review the findings. The genetic team described the process of chromosomal analysis and showed them images of normal (Giemsa-banded) karyotypes. We then showed them their karyotypes; the father's was normal 46,XY, and the mother's contained a balanced translocation (Figure 2B). A color-coded diagram (Figure 2C) was used to show the derived chromosome 1; the distal portion of the long arm (q42.1ter) was absent and had been replaced by the distal portion (p11.2ter) of one copy of the short arm of chromosome 18. The derived chromosome 18 had the translocated portion of chromosome 1. The mother's karyotype was thus 46,XX,t(1;18)(q42.1;p11.2), indicating that material from the long arm (q) of chromosome 1 at band 42.1 had switched positions with material from the short arm (p) of chromosome 18 at band p11.2, resulting in a balanced translocation — that is, no chromosomal material was missing. That of the fetus was 46,XY,der(18)t(1;18)(q42.1;p11.2)mat — indicating that the derivative (der) chromosome 18 was the result of the maternal (mat) balanced translocation. Since this fetus inherited the normal chromosome 1 from the mother, the translocation was unbalanced, and the fetus effectively had trisomy for the translocated portion of 1q and monosomy for the terminal portion of chromosome 18.
The parents chose to terminate this pregnancy because of the serious prognosis associated with the unbalanced translocation. The mother said that she would contact relatives for more information about the medical history of her cousins with birth defects. The pregnancy was terminated at 18 weeks. The parents returned to the genetics clinic to review the risk that future pregnancies would be abnormal. They were told that a future pregnancy could have one of three outcomes: a healthy child, a healthy child with the same balanced translocation as the mother's, or a child with either of two unbalanced translocations.
The mother had brought with her a copy of an article in a genetics journal, published in 1979, in which the same chromosomal translocation had been described in several of her father's relatives.3 She had not been told about the risk of this genetic abnormality because she was related to these people through her father; since only females had been identified as carriers, her relatives believed that her father was not at risk and that she was therefore not at risk either.
When we contacted the first author of the report, Ruth M. Liberfarb, M.D., Ph.D., she recalled that the first contact with the family occurred as a result of the evaluation of a child with multiple congenital anomalies and developmental delay. Both the mother of the child (Figure 3; II-2), a maternal aunt of the child (II-5), and the maternal grandmother (I-2) had had multiple spontaneous abortions and stillbirths, as well as live-born children who died in infancy with congenital anomalies. Chromosomal analysis identified the unbalanced translocation in the child; studies of his parents showed that his mother had a balanced translocation. Screening of the parents' phenotypically normal son and daughter, the maternal grandparents, and all the mother's siblings was recommended to determine whether they were carriers of this translocation. The mother of the child with birth defects invited her relatives to come to her home for a discussion with Dr. Liberfarb about the risk of carrier status and the option of undergoing chromosomal analysis. Her brother (II-6), the father of the woman whose pregnancy is discussed here, did not attend this meeting or undergo genetic testing. The pedigree of the family described by Liberfarb et al.,3 with the addition of information about the patient's generation (the patient is III-15) and offspring, is shown in Figure 3.
Figure 3. Pedigree of the Mother's Family.
The pedigree, reported in 1979 by Liberfarb et al.3 and updated to show the parents and fetus discussed here, shows that numerous family members of several generations had a balanced translocation, an unbalanced translocation, or phenotypic features suggesting an unbalanced translocation. Circles denote female family members, squares male family members, triangles aborted fetuses, solid symbols subjects with unbalanced translocations, half-solid symbols subjects with balanced translocations, shaded symbols subjects with malformations suggesting an unbalanced translocation, N within a symbol subjects with a normal karyotype, and slash marks deceased subjects. The arrow indicates the fetus in the case under discussion. Adapted from Liberfarb et al.,3 with the permission of the publisher.
During the discussion at our genetics center, the mother of the affected fetus asked about the new method of prenatal diagnosis called preimplantation genetic diagnosis. She was eager to know whether this approach would allow her to have a healthy infant and to avoid having to consider the difficult and traumatic option of termination of pregnancy if another fetus had an unbalanced chromosomal translocation. Since this procedure is not performed at this hospital, I referred the couple to our colleagues at Brigham and Women's Hospital, who are using preimplantation genetic diagnosis to monitor pregnancies at risk for an unbalanced translocation.
Discussion of Management
Dr. Antonio R. Gargiulo: The couple saw Dr. Louise Wilkins-Haug in the Maternal–Fetal Medicine Division. At that time, the patient was again pregnant. Dr. Wilkins-Haug provided genetic counseling during a second pregnancy and performed chorionic-villous sampling, which disclosed abnormal material on the short arm of chromosome 18. After the termination of their second consecutive pregnancy, the couple came to the Center for Reproductive Medicine at Brigham and Women's Hospital to explore options for preimplantation detection of fetal chromosomal aberrations.
The couple's reproductive history disclosed profound subfertility, with unprotected timed intercourse practiced for four years before their first conception. The results of the diagnostic workup for infertility included a normal semen analysis and elevation of the wife's serum estradiol level to 62 pg per milliliter on day 3 of her menstrual cycle, with a normal serum level of follicle-stimulating hormone (FSH) of 7.6 mIU per milliliter.
Carriers of reciprocal translocations, such as this woman, produce both balanced and unbalanced gametes. The chromosomal constitution of the gametes formed depends on the type of segregation after pairing of homologous chromosomes at meiosis in a quadriradial figure. Typically, a chromosomal segregation results in the formation of two chromosomally balanced gametes and four unbalanced gametes. When fertilization occurs with a normal haploid gamete, the balanced gametes will produce a phenotypically normal conceptus with either normal chromosomes or the same balanced translocation carried by a parent. Some translocations can affect implantation and thus fertility; however, the amount of chromosomal material involved in this case would be unlikely to explain this couple's infertility.
The woman had been followed by her gynecologist by means of serial pelvic ultrasonography for a persistent complex left ovarian cyst. In preparation for assisted reproduction, we performed a combined laparoscopy and hysteroscopy to evaluate the ovarian cyst, as well as to survey the anatomical features of the endometrial cavity, looking for abnormalities that might explain her subfertility. We found a left ovarian endometriotic cyst, which we excised, and otherwise normal pelvic anatomy, with patent fallopian tubes and a normal endometrial cavity. This degree of pelvic endometriosis probably caused her infertility.
The couple was counseled about the technical aspects of in vitro fertilization and preimplantation genetic diagnosis for the identification of embryos with unbalanced chromosomes.
Dr. Catherine Racowsky: Preimplantation genetic diagnosis entails the screening of embryos produced by in vitro fertilization for a variety of genetic abnormalities. Genetic analysis of the embryos involves delicate micromanipulation techniques to remove cellular material. One or two blastomeres are removed from the embryo at the 6-to-8-cell cleavage stage. Removal of two blastomeres instead of one has been shown to improve the accuracy of preimplantation genetic diagnosis, especially in couples with balanced chromosomal translocations, in which a high incidence of blastomere mosaicism is encountered.4,5
Once the cellular material is obtained, it can be analyzed by different techniques: fluorescence in situ hybridization, polymerase chain reaction, or comparative genomic hybridization. Fluorescence in situ hybridization is used to determine numerical or structural abnormalities of specific chromosomes, as well as for determination of sex chromosomes. It is the technique best suited to screening embryos before implantation to identify carriers of balanced translocations. Its application has been shown to reduce drastically the chance of chromosomally abnormal pregnancies in couples with such translocations.6
Dr. Gargiulo: Fluorescence in situ hybridization studies with use of DNA probes for the subtelomeric regions of the short arm of chromosome 18 (18ptel) and the long arm (18qtel) were performed initially on chromosomes in metaphase and on nuclei in interphase from peripheral-blood lymphocytes obtained from the woman and her husband; the results were compared with those for matched controls. Two hybridization signals specific for the 18ptel probe and two hybridization signals specific for the 18qtel probe were observed in 90 percent of nuclei obtained from the wife's peripheral-blood sample, in 94 percent of nuclei obtained from the husband's peripheral-blood sample, and in 92 percent of nuclei that were examined from a control specimen.
Once this preliminary test was completed, the couple began a cycle of in vitro fertilization. Controlled ovarian hyperstimulation was induced with high doses of recombinant human FSH (300 IU given subcutaneously twice a day), and pituitary down-regulation was achieved by administration of the gonadotropin-releasing–hormone (GnRH) agonist leuprolide acetate at a daily dose of 0.5 mg, given subcutaneously, starting in the luteal phase preceding the stimulation cycle. In spite of this aggressive stimulation protocol, only six measurable follicles were present after 11 days of gonadotropin administration, when dimension criteria (two leading follicles with a mean diameter of 18 mm) for final oocyte maturation were reached. A single dose of human chorionic gonadotropin (hCG) (10,000 IU) was administered intramuscularly, and follicular aspiration guided by transvaginal ultrasonography was performed after 36 hours. All clinical embryology procedures were carried out by the Brigham and Women's In Vitro Fertilization Laboratory.
Seven cumulus–oocyte complexes were identified. Four of these contained metaphase II oocytes (mature oocytes that have extruded the first polar body and can be fertilized normally). Intracytoplasmic sperm injection was performed on the four mature oocytes, all of which were fertilized. Given the low number of mature oocytes and subsequently of embryos obtained, as well as the expected 1:3 ratio of embryos with the balanced translocation to those with the unbalanced translocation, the couple was counseled to have all embryos frozen at the pronuclear stage of development (i.e., about 18 hours after intracytoplasmic sperm injection) and to undergo another cycle of in vitro fertilization to obtain more embryos. Reports of successful preimplantation genetic diagnosis with cryopreserved embryos had recently appeared, proving that this genetic analysis was still possible after embryos had been thawed.7
A second cycle of controlled ovarian hyperstimulation was started after a rest cycle. This time, an even more aggressive stimulation protocol was adopted, with daily doses with recombinant human FSH (300 IU given subcutaneously) and purified urinary luteinizing hormone (LH) with FSH (300 IU given subcutaneously). Moreover, leuprolide acetate was administered together with gonadotropins at doses of 0.05 mg given subcutaneously twice a day. This protocol allows a mild "flare" of endogenous secretion at the beginning of the cycle, while still providing sufficient pituitary suppression to prevent the LH surge later in the cycle. In spite of this adjustment in the protocol, the ovarian response was again low. Six cumulus–oocyte complexes were identified, all of which contained mature oocytes; however, the fertilization rate with intracytoplasmic sperm injection was lower than expected, and only two embryos were obtained. For the reasons described above, these embryos were also cryopreserved for future analysis.
A third cycle of controlled ovarian hyperstimulation was begun after a rest cycle. In this case, we used the same gonadotropin regimen as in the second cycle but substituted the GnRH antagonist cetrorelix for the GnRH agonist leuprolide acetate. Cetrorelix (250 μg given subcutaneously per day) was started after six days of gonadotropin treatment to achieve rapid pituitary suppression of the LH surge with no compromise of FSH levels during the early part of the stimulation cycle. Once again, six cumulus–oocyte complexes were identified, all of which contained mature oocytes and on which intracytoplasmic sperm injection was performed; four embryos were obtained and again frozen at the pronuclear stage.
A fourth and final cycle of controlled ovarian hyperstimulation was started after two rest cycles. In this cycle the same stimulation protocol was used as in the preceding cycle, and the same number of mature oocytes resulted. Three of the four oocytes were successfully fertilized with intracytoplasmic sperm injection.
Dr. Racowsky: At this point, the decision was made to proceed with preimplantation genetic diagnosis, and the 10 previously cryopreserved embryos were thawed in order to synchronize their development with that of the freshly produced embryos. All 10 cryopreserved embryos survived the thawing process. Blastomere biopsy was performed on day 3 of embryo development. Blastomeres were obtained from 9 embryos that had developed to the stage of having six or more blastomeres — that is, from the 3 fresh embryos and from 6 of the 10 thawed embryos. In six of the nine biopsied embryos, it was technically feasible to obtain two blastomeres. Blastomeres were then processed for chromosome spreading and fixation by a modification of the Tarkowski method.8 They were then submitted to the cytogenetics laboratory for analysis by fluorescence in situ hybridization. The results are shown in Table 1.
Table 1. Characteristics of All Embryos Produced by the Couple.
In two of the nine embryos, the nuclei of the fixated blastomeres could not be located by fluorescence in situ hybridization. Results were therefore available for seven embryos on the day of embryo transfer (day 4 of embryo development). Three chromosomally balanced embryos (which were either chromosomally normal or had a balanced chromosomal translocation) and four chromosomally unbalanced embryos (two with partial monosomy 18 and two with partial trisomy 18) were identified (Figure 4A and Figure 4B and Table 1). One of the balanced embryos had developed appropriately to the morula stage, whereas in the other two balanced embryos growth had been arrested after the blastomere biopsy. One of these two appeared strikingly abnormal, with multiple large fragments and cytoplasmic vacuoles. After thorough consultation, the couple elected to transfer the two normal-appearing, chromosomally balanced embryos (Figure 5) and to discard all the other embryos.
Figure 4. Results of Fluorescence in Situ Hybridization with Blastomeres from Three Chromosomally Balanced Embryos (Panel A) and Four Chromosomally Unbalanced Embryos (Panel B).
A green chromogen is linked to the DNA probe for the telomere of the short arm of chromosome 18 (18ptel), and a magenta chromogen is linked to the DNA probe for the long arm (18qtel). The normal blastomeres contain two green and two magenta signals, signifying either a normal karyotype or a balanced translocation. The abnormal blastomeres contain either one or three signals of either or both colors, indicating monosomy or trisomy for that locus — the result of an unbalanced translocation. (Images courtesy of Dr. Aida Nureddin and Dr. Stanislava Weremowicz, Brigham and Women's Hospital.)
Figure 5. Phase-Contrast Photomicrograph of the Two Chromosomally Balanced Embryos Selected for Transfer.
On the left is a morula from a fresh embryo, and on the right is a cleavage-stage embryo frozen and thawed before preimplantation genetic diagnosis.
Dr. Gargiulo: Transcervical embryo transfer with use of a soft-tip catheter was performed without difficulty. Luteal-phase progesterone supplementation was provided by daily intramuscular injections of progesterone in oil (50 mg). The serum level of the subunit of hCG two weeks after embryo transfer (649 mIU per milliliter) indicated early implantation, with a normal increase after 48 hours (to 1818 mIU per milliliter). The first clinical evidence of pregnancy was obtained at 5.9 weeks of gestation, when ultrasonography showed a single intrauterine pregnancy with a fetal heart rate of 111 beats per minute. A follow-up ultrasound examination at eight weeks of gestation confirmed an intrauterine pregnancy with a live single fetus, with normal interval growth and a normal fetal heart rate of 153 beats per minute. The patient was referred to her obstetrician for routine prenatal care. Given an expected rate of accuracy of 90 to 95 percent for preimplantation genetic diagnosis, depending on the condition analyzed and the technique used, amniocentesis is strongly recommended for all women whose embryos have undergone this selection procedure before transfer. This patient declined both so-called triple screening (measurement of estriol, the subunit of hCG, and alpha-fetoprotein) and amniocentesis.
The woman had an uneventful pregnancy. Labor was induced at 41 weeks of gestation, and delivery was eventually accomplished by primary low transverse cesarean section because of failed induction and a nonreassuring fetal heart rate. A healthy baby boy, weighing 3420 g and with one-minute and five-minute Apgar scores of 9 and 9, was evaluated by the pediatrician and discharged home with his mother.
A Physician: What is the risk to the fetus associated with removing blastomeres?
Dr. Gargiulo: The largest study done to assess the developmental steps of children born after preimplantation genetic diagnosis followed some such children until two years of age and found no developmental abnormalities.9 However, unlike in vitro fertilization or intracytoplasmic sperm injection, for which we now have cohorts of children followed all the way into school age and young adulthood, we do not at present have any published developmental data on children born after preimplantation genetic diagnosis for use in counseling prospective parents considering the procedure.
Dr. Racowsky: There is an uneven distribution of regulatory proteins even as early as the one-cell stage in the human embryo, and this uneven distribution becomes more apparent as the embryo undergoes cleavage to the morula stage.10 During preimplantation genetic diagnosis, critical proteins that are currently unidentified might be removed with the biopsied blastomere and might affect development down the road. The European Society of Human Reproduction preimplantation genetic diagnosis consortium follows babies born after use of this procedure throughout the world, and to date there do not appear to be any differences in the developmental timelines of these babies and babies whose births did not follow preimplantation genetic diagnosis.11 However, these are still early days with regard to the application of this invasive technique, and we must counsel our patients appropriately. Larger studies are needed to assess the long-term outcomes of these children.
Dr. Holmes: At the last follow-up, the infant was nine months old and progressing normally.
Anatomical Diagnosis
Balanced maternal chromosomal translocation — 46,XX,t(1;18)(q42.1;p11.2) — resulting in an unbalanced fetal translocation — 46,XY,der(18)t(1;18)(q42.1;p11.2)mat.
Source Information
From the Genetics and Teratology Unit, Mass General Hospital for Children (L.B.H.), and the Department of Obstetrics and Gynecology (A.S.N.), Massachusetts General Hospital; the Departments of Pediatrics (L.B.H.) and Obstetrics, Gynecology, and Reproductive Biology (A.R.G., A.S.N., C.R.), Harvard Medical School; and the Center for Reproductive Medicine, Brigham and Women's Hospital (A.R.G., C.R.).
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