CASE REPORT. MATERIALS AND METHODS Couple

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1 CASE REPORT Preimplantation genetic diagnosis (PGD) for extremes successful birth after PGD for a consanguineous couple carrying an identical balanced reciprocal translocation Cagri Beyazyurek, M.S., a Cumhur Gokhan Ekmekci, M.D., Ph.D., a Yaman Saglam, M.D., Ph.D., a,c Cigdem Cinar, M.S., a and Semra Kahraman, M.D., Ph.D. b a Reproductive Genetics Laboratory, Istanbul Memorial Hospital, Sisli, Istanbul; b In Vitro Fertilisation Unit, Istanbul Memorial Hospital, Sisli, Istanbul; c Genetic Diagnosis Center, Goztepe Medical Park Hospital, Goztepe, Istanbul, Turkey Objective: To report a healthy birth after preimplantation genetic diagnosis (PGD) performed for a consanguineous couple carrying an identical familial reciprocal translocation in both partners. Design: Case report. Setting: In vitro fertilization (IVF) clinic and genetic laboratory in a private hospital. Patient(s): Consanguineous couple carrying the same balanced reciprocal translocation: 46,XX,t(1;16)(q12;q11.2) and 46,XY,t(1;16)(q12;q11.2). Intervention(s): 25 oocyte cumulus complexes were retrieved 36 hours after human chorionic gonadotropin injection; metaphase II oocytes were fertilized by intracytoplasmic sperm injection; single blastomere biopsy was performed on 15 embryos on day 3; one embryo was found to be normal or balanced according to fluorescent in situ hybridization studies, embryo transfer was performed on day 4. Main Outcome Measure(s): Healthy birth of homozygous double translocation carrier twins with 46,XY,t(1;16)(q12;q11.2)mat,t(1;16)(q12;q11.2)pat karyotype. Result(s): Healthy monozygotic male twins were born at 36 weeks of gestation. Karyotype studies of the babies revealed that they are double translocation homozygotes: 46,XY,t(1;16)(q12;q11.2)mat,t(1;16)(q12;q11.2)pat. They are healthy and more than 4 years old later show no physical or mental abnormalities. Conclusion(s): To our knowledge, this is the first PGD study performed for a couple who carry the same reciprocal translocation. The twins born after this study are rare examples in the literature of healthy balanced reciprocal translocation homozygotes. (Fertil Steril Ò 2010;93:2413.e1 e5. Ó2010 by American Society for Reproductive Medicine.) Key Words: Consanguinity, double translocation homozygosity, FISH, monozygotic dichorionic diamniotic twin, PGD, reciprocal translocation Chromosomal rearrangements are seen in 0.2% of the newborn population, but they are much more prevalent among infertile men (1) and couples experiencing repeated implantation failures (2.5%) and recurrent miscarriages (9.2%) (2). Reciprocal translocations are formed by the exchange of two terminal segments between nonhomologous chromosomes. Carriers have a poor chance of producing normal or balanced gametes, although the proportion of unbalanced forms depends on the characteristics of the reorganization and may vary from Received September 17, 2009; revised December 7, 2009; accepted December 10, 2009; published online February 1, C.B. has nothing to disclose. C.G..E. has nothing to disclose. Y.S. has nothing to disclose. C.C. has nothing to disclose. S.K. has nothing to disclose. Presented as a poster at the Sixth International Symposium on. Preimplantation Genetics, May 19 21, 2005, London, United Kingdom; and at the 7th National Prenatal Diagnosis and Medical Genetics Congress, March 17 20, 2006, Kayseri, Turkey. Reprint requests: Cagri Beyazyurek, M.S., Reproductive Genetics Laboratory, Istanbul Memorial Hospital, 34385, Sisli, Istanbul, Turkey (FAX: þ ; cagribeyazyurek@gmail.com). 23% to 81% (3, 4). However, preimplantation genetic diagnosis (PGD), which selects chromosomally normal or balanced embryos fertilized by in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), could offer an alternative for translocation carrier couples. With the aid of florescent in situ hybridization (FISH), individual blastomeres biopsied from embryos can be analyzed for the chromosomes and the parts of those chromosomes in question (5). Our study reports a healthy birth after a PGD study performed for a consanguineous couple who carried an identical familial reciprocal translocation: 46,XX,t(1;16)(q12;q11.2) and 46,XY,t(1;16)(q12;q11.2). To the best of our knowledge, ours is the first report of a PGD cycle performed for a couple in whom both partners carry the same reciprocal translocation. MATERIALS AND METHODS Couple A consanguineous couple (26-year-old woman and 35-year-old man at the time of intake) were referred to Istanbul Memorial Hospital IVF and /10/$36.00 Fertility and Sterility â Vol. 93, No. 7, May 1, e1 doi: /j.fertnstert Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.

2 FIGURE 1 Pedigree of the family. The numbers inside the circles and squares indicate the number of children born with the same gender. I.1: 46,XY,t(1;16)(q12;q11.2)mat,t(1;16)(q12;q11.2)pat. I.2: 46,XY,t(1;16)(q12;q11.2)mat,t(1;16)(q12;q11.2)pat. II.1: 46,XX,t(1;16)(q12;q11.2). II.2: 46,XY,t(1;16)(q12;q11.2). Reproductive Genetics Center. The couple were first-degree cousins. They had an 8-year history of secondary infertility, during this period, they had had three spontaneous abortions at (in chronologic order) 4, 8, and 20 gestational weeks. Karyotype analysis of the couple had been performed in another laboratory. Reports indicated a translocation in both the female and the male partner between chromosomes 1 and 16; however, the breakpoints of the translocations were different: 46,XX,t(1;16)(q24;q24) and 46,XY, t(1;16)(p22;p13) (6). Only the last fetus had been examined, revealing anencephaly, spina bifida, and split hand and foot deformities. The cytogenetic result of this fetus was even more confusing: 46,XY,t(1;16)(p22;p13). After we had noted the family history and pedigree (Fig. 1), we provided appropriate genetic counseling to the couple, indicating the need to replicate a karyotype study before beginning IVF treatments. The gynecologic examination revealed that the woman was normal. According to spermiogram testing, the man was found to have asthenoteratozoospermia ( /ml; 2% motility and 1% morphology). He had undergone a left varicocelectomy 6 years before this IVF treatment. Set-up Study: Cytogenetic and FISH Studies Karyotype analysis from peripheral blood and subsequent FISH analysis to confirm the breakpoints were performed as previously described elsewhere (1). Theoretical Estimation of Different Segregation Products Each partner could potentially produce 14 different gametes arising from alternate, adjacent I and II, and 3:1 segregation modes when crossing over between the rearranged points is excluded (3, 4, 7). From these 14, only two would give rise to normal or balanced embryos. In this case, because both parents were carrying the same reciprocal translocation, the difficulty of the study was doubled, and the chance of finding a normal or balanced embryo was further lowered. The types of all combinations of different forms of gametes, the resulting embryos, and the expected signal patterns in the PGD study were all documented before initiation of IVF and PGD studies. It was estimated that the couple would produce 1 normal and 15 balanced products out of approximately 196 (14 14) different combinations of gametes. Out of 15 balanced products, 12 of them would be uniparental disomy (UPD), and the remaining 3 would be either balanced (two) or balanced double translocation homozygote (one). IVF and PGD Study The couple underwent IVF treatment at the IVF and Reproductive Genetics Center of Memorial Hospital in Istanbul, Turkey. For ovarian stimulation, pituitary down-regulation was performed with a gonadotropin-releasing hormone agonist (GnRH-a, Decapeptyl; Ferring A.B., Lausanne, Switzerland), and controlled ovarian hyperstimulation was done with follicle-stimulating hormone (FSH, Gonal-F; Serono, Geneva, Switzerland). Twenty-five oocytecumulus complexes were retrieved by use of transvaginal ultrasound guidance 36 hours after human chorionic gonadotropin (hcg) injection. Written informed consent was obtained from the couple before they underwent IVF treatment, PGD procedures, and embryo transfer procedures. The metaphase 2 (MII) oocytes were fertilized with ICSI. Biopsy was performed for embryos that had at least six blastomeres on day 3. One cell was taken from each embryo using a calcium magnesium free biopsy medium (EB-10; Vitrolife, Gothenburg, Sweden) and a noncontact diode laser system (Research Instruments, Cornwall, United Kingdom). Individual blastomeres were fixed with Carnoy s fixative, as described previously elsewhere (8). In the first round, every individual nucleus was hybridized with Tel 16p (TelVysion 16p, Vysis) and Tel 16q (TelVysion 16q, Vysis). In the second round, Tel 1p (TelVysion 1p, Vysis) and Tel 1q (TelVysion 1q, Vysis) were used for those that had given a normal or balanced signal pattern. Finally, for the third round hybridization, centromeric probes for chromosomes 1 and 16 were used for the blastomeres that had given a normal or balanced signal pattern after both first and second round hybridizations. Because two very close or fused centromeric signals would indicate a break and fusion between centromeres, this third hybridization was to help to discriminate between normal and balanced forms, as if a breakpoint spanning approach (9) were used. Simultaneous denaturations of DNA and probe mix were carried out as described previously by Findikli et al. (10), and the criteria for signal scoring were adapted from Munne et al. (11) were used. RESULTS Cytogenetic analyses revealed that both the woman and man were carrying the same reciprocal translocation: 46,XX,t(1;16)(q12;q11.2) and 46,XY,t(1;16)(q12;q11.2), respectively (Fig. 2A, B). Detailed FISH analysis (see Fig. 2C, D) also revealed that the breakpoint positions had been incorrectly designated by the previous genetic study (6) e2 Beyazyurek et al. PGD for extremes Vol. 93, No. 7, May 1, 2010

FIGURE 2 Results of cytogenetic studies. Karyotype of (A) the mother 46,XX,t(1;16)(q12;q11.2) and (B) the father 46,XY,t(1;16)(q12;q11.2). FISH analysis on peripheral blood metaphase plaques of (C) mother and (D) father.

3 FIGURE 2 Results of cytogenetic studies. Karyotype of (A) the mother 46,XX,t(1;16)(q12;q11.2) and (B) the father 46,XY,t(1;16)(q12;q11.2). FISH analysis on peripheral blood metaphase plaques of (C) mother and (D) father. (E) Karyotype of monozygotic twin babies: 46,XY,t(1;16)(q12;q11.2)mat,t(1;16)(q12;q11.2)pat. (F) FISH analysis on peripheral blood metaphase plaques of the twins. Probes: Telomeric 16p spectrum green (SG) (TelVysion 16p, D16S3400, GDB: ; Vysis, Abbott Inc.), Telomeric 16q spectrum orange (SO) (TelVysion 16q, GenBank Z23646; Vysis, Abbott Inc.), Centromeric probe (CEP) 1 (SO) (Satellite II/III, 1q12; Vysis, Abbott Inc.), and CEP 16 Spectrum aqua (SA) (D16Z3; Vysis, Abbott Inc.). On day 3 of embryonic development, 15 embryos were biopsied, and successful hybridization was achieved in all analyzed blastomeres. According to first-round and second-round studies, only one embryo had normal or balanced signals (Fig. 3A, B). To differentiate between balanced and normal, centromeric probes were applied to the nucleus of this blastomere, but the results were inconclusive (see Fig. 3C). The embryo was at early blastocyst stage at the time of transfer, approximately 100 hours after fertilization on day 4. The beta human chorionic gonadotropin (b-hcg level) was positive, and the ultrasound examination revealed a monozygotic dichorionic diamniotic pregnancy. Although it had been strongly recommended to them before, during, and after the treatment and although the family was informed of the risk of uniparental disomy UPD and balanced double translocation homozygosity, the couple refused Fertility and Sterility â 2413.e3

FIGURE 3 FISH images of three rounds of hybridization of the transferred embryo. (A) In the first round, two green (Telomeric 16p) and two orange signals (Telomeric 16q) were observed.

4 FIGURE 3 FISH images of three rounds of hybridization of the transferred embryo. (A) In the first round, two green (Telomeric 16p) and two orange signals (Telomeric 16q) were observed. (B) In the second round, two green (Telomeric 1p) and two orange (Telomeric 1q) signals were observed. (C) Third round was inconclusive: apart from two fused green and orange signals (Centromeric 1 and Centromeric 16), in the middle of the nucleus there is a signal overlap which made the interpretation difficult. Probes: Telomeric 16p spectrum green (SG) (TelVysion 16p, D16S3400, GDB: ; Vysis, Abbott Inc.), Telomeric 16q spectrum orange (SO) (TelVysion 16q, GenBank Z23646; Vysis, Abbott Inc.), Centromeric probe (CEP) 1 (SO) (Satellite II/III, 1q12; Vysis, Abbott Inc.), and CEP 16 Spectrum aqua (SA) (D16Z3; Vysis, Abbott Inc.). amniocentesis. Healthy male twins were born at 36 gestational weeks by caesarian section. Cytogenetic studies and FISH analysis of the monozygotic twin babies were done to confirm the diagnosis when they were 2.5 years old. It revealed that they were double translocation homozygotes: 46,XY,t(1;16)(q12;q11.2)mat,t(1;16)(q12;q11.2)- pat (see Fig. 2E, F). They were healthy, and at the time this report was written, they were more than 4 years old and showed no malformations and had no medical complaints. DISCUSSION For translocation carrier couples, PGD is a useful tool that can increase healthy birth rates and decrease recurrent abortions (12 15). The most limiting factor in a PGD study for reciprocal translocations is the low probability of finding a normal or balanced embryo to transfer (4, 16). In this special case, because both partners were carrying the same reciprocal translocation, the difficulty of the study was doubled, and the chances of finding a suitable embryo for transfer were even lower. Despite the difficulties, there were several parameters that increased the chance of conceiving for this particular couple: the familial nature of the translocation, the sperm quality and concentration, the age of the female partner, the good ovarian reserve she had at that time, and the breakpoint positions which were very close to the centromeres increasing the proportion of normal or balanced gametes (7). Although Tsuji et al. (17) had reported similar reproductive risks in couples in whom both partners were translocation carriers and in couples in whom one individual was a translocation carrier, the couple in our study had apparently more reproductive risks than other couples in the family who were said to be carriers of the same translocation by only one partner (information from Ozkul et al. [6]), which is evident from the absence of infertility or any history of repeated spontaneous abortions in other families (see Fig. 1). In this case, the twins born after PGD were double reciprocal translocation homozygotes. Balanced double translocation is a very rare condition, and most reported cases are double translocation heterozygotes (18). According to our search in PubMed, only limited information is available regarding the status of double reciprocal translocation carriers. Martinet et al. (19) reported a fetus having two identical balanced reciprocal translocations of biparental origin: 46,XX,t(17;20)(q21.1;p11.21)mat,t(17;20)(q21.1;p11.21)- pat. Unfortunately, although the fetus had an apparently balanced karyotype, the pregnancy was terminated because of multiple organ anomalies. Several other studies reported other abnormalities such as infantile seizures (20) and, more recently, hearing loss (21). In these conditions, it is possible that a gene (or genes) might have been disrupted by the breaks and that the offspring were homozygous for a recessive gene mutation, masked by the parents who were heterozygous carriers. In contrast to those previous reports, the twins in our study are healthy, with no physical or cognitive abnormalities. Although it is too early to draw firm conclusions, there may not have been a disturbance caused by the homozygous breakage of the chromosomes involved in the translocation. The breakpoints being in the centromeric heterochromatin region of both chromosomes 1 and 16 might have been an advantage. Besides the uniqueness of the case reported here, this study also has highlighted the importance of confirming the breakpoint positions by FISH analysis before initiation of any PGD cycle for translocation carriers. Both testing probes for hybridization efficiency and confirming breakpoint positions by FISH are key contributions to the quality of a PGD program designed for chromosomal rearrangements (22). Furthermore, if this prerequisite set-up study had not been performed, the first and incorrect report, which indicated 2413.e4 Beyazyurek et al. PGD for extremes Vol. 93, No. 7, May 1, 2010

5 different breakpoint positions, might have led us toward incorrect interpretations and misdiagnosis. To confirm the familial nature of this translocation, cytogenetic analysis of peripheral blood from siblings of the father and mother were requested, but none was sent for genetic analysis. Fortunately, Ozkul et al. (6) had reported the presence of identical translocations in relatives of this family in their study. The father has been informed of the persistent risk of unbalanced fetuses, even though his second marriage, after the mother of the twins died in a traffic accident, was to a nonrelative. The health of the twins is being monitored every 6 months by telephone inquiries. CONCLUSION Our study describes one of the most difficult examples of PGD for translocations. This was the first PGD study performed for a couple in whom both partners had an identical reciprocal translocation; the twins born in this case are a rare example in the literature of healthy reciprocal translocation homozygotes. REFERENCES 1. Kumtepe Y, Beyazyurek C, Cinar C, Ozbey I, Ozkan S, Cetinkaya K, et al. A genetic survey of 1935 Turkish men with severe male factor infertility. Reprod Biomed Online 2009;18: Stern C, Pertile M, Norris H, Hale L, Baker HW. Chromosome translocations in couples with in-vitro fertilization implantation failure. Hum Reprod 1999;14: Benet J, Oliver-Bonet M, Cifuentes P, Templado C, Navarro J. Segregation of chromosomes in sperm of reciprocal translocation carriers: a review. Cytogenet Genome Res 2005;111: Scriven PN, Handyside AH, Ogilvie CM. Chromosome translocations: segregation modes and strategies for preimplantation genetic diagnosis. Prenat Diagn 1998;18: Munne S. Preimplantation genetic diagnosis of numerical and structural chromosome abnormalities. Reprod Biomed Online 2002;4: Ozkul Y, Dundar M. A family with two different chromosomal translocations. Ann Genet 2002;45: Escudero T, Abdelhadi I, Sandalinas M, Munne S. Predictive value of sperm fluorescence in situ hybridization analysis on the outcome of preimplantation genetic diagnosis for translocations. Fertil Steril 2003;79: Munne S, Dailey T, Finkelstein M, Weier HU. Reduction in signal overlap results in increased FISH efficiency: implications for preimplantation genetic diagnosis. J Assist Reprod Genet 1996;13: Lersch RA, Fung J, Munne S, Pedersen RA, Weier HU. Case-specific, breakpoint-spanning DNA probes for analysis of single interphase cells. Genet Test 2000;4: Findikli N, Kahraman S, Kumtepe Y, Donmez E, Biricik A, Sertyel S, et al. Embryo development characteristics in Robertsonian and reciprocal translocations: a comparison of results with non-translocation cases. Reprod Biomed Online 2003;5: Munne S, Marquez C, Magli C, Morton P, Morrison L. Scoring criteria for preimplantation genetic diagnosis of numerical abnormalities for chromosomes X, Y, 13, 16, 18 and 21. Mol Hum Reprod 1998;4: Kyu Lim C, Hyun Jun J, Mi Min D, Lee HS, Young Kim J, Koong MK, et al. Efficacy and clinical outcome of preimplantation genetic diagnosis using FISH for couples of reciprocal and robertsonian translocations: the Korean experience. Prenat Diagn 2004;24: Verlinsky Y, Tur-Kaspa I, Cieslak J, Bernal A, Morris R, Taranissi M, et al. Preimplantation testing for chromosomal disorders improves reproductive outcome of poor-prognosis patients. Reprod Biomed Online 2005;11: Otani T, Roche M, Mizuike M, Colls P, Escudero T, Munne S. Preimplantation genetic diagnosis significantly improves the pregnancy outcome of translocation carriers with a history of recurrent miscarriage and unsuccessful pregnancies. Reprod Biomed Online 2006;13: Beyazyurek C, Cinar C, Onal B, Ekmekci CG, Aslan C, Unal S, et al. Outcomes of over 200 preimplantation genetic diagnosis cycles for translocation carriers [abstract]. Reprod Biomed Online 2009; Munne S, Sandalinas M, Escudero T, Fung J, Gianaroli L, Cohen J. Outcome of preimplantation genetic diagnosis of translocations. Fertil Steril 2000;73: Tsuji K, Narahara K, Yokoyama Y, Ninomiya S, Yonesawa S, Hiramatsu Y, et al. Reproductive risk in mating between two translocation carriers: case report and review of the literature. Am J Med Genet 1993;15(46): Bijlsma JB, de France HF, Bleeker-Wagemakers LM, Dijkstra PF. Double translocation t(7;12), t(2;6) heterozygosity in one family. A contribution to the trisomy 12p syndrome. Hum Genet 1978;40: Martinet D, Vial Y, Thonney F, Beckmann JS, Meagher-Villemure K, Unger S. A fetus with two identical reciprocal translocations: description of a rare complication of consanguinity. Am J Med Genet 2006;140: Wilmot PL, Shapiro LR, Casamassima AC. Disomic balanced reciprocal translocation. Clin Genet 1990;38: Schneider E, M arker T, Daser A, Frey-Mahn G, Beyer V, Farcas R, et al. Homozygous disruption of PDZD7 by reciprocal translocation in a consanguineous family: a new member of the Usher syndrome protein interactome causing congenital hearing impairment. Hum Mol Genet 2009;18: Thornhill AR, dedie-smulders CE, Geraedts JP, Harper JC, Harton GL, Lavery SA, et al. ESHRE PGD Consortium. Best practice guidelines for clinical preimplantation genetic diagnosis (PGD) and preimplantation genetic screening (PGS). Hum Reprod 2005;20: Fertility and Sterility â 2413.e5

Ewa Wiland 1, Calvin J. Hobel 2, David Hill 3 and Maciej Kurpisz 1 * INTRODUCTION

Ewa Wiland 1, Calvin J. Hobel 2, David Hill 3 and Maciej Kurpisz 1 * INTRODUCTION PRENATAL DIAGNOSIS Prenat Diagn 2008; 28: 36 41. Published online in Wiley InterScience (www.interscience.wiley.com).1899 Successful pregnancy after preimplantation genetic diagnosis for carrier of t(2;7)(p11.2;q22)