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

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1 PRENATAL DIAGNOSIS Prenat Diagn 2008; 28: Published online in Wiley InterScience ( Successful pregnancy after preimplantation genetic diagnosis for carrier of t(2;7)(p11.2;q22) with high rates of unbalanced sperm and embryos: a case report Ewa Wiland 1, Calvin J. Hobel 2, David Hill 3 and Maciej Kurpisz 1 * 1 Institute of Human Genetics, PAN, Poznań, ul. Strzeszyńska 32, Poland 2 Department of OB/GYN, Cedars-Sinai Medical Center, Los Angeles, California, USA 3 ART Reproductive Center, 450 North Roxbury Drive, Beverly Hills, California, USA Background Regarding the literature on the results of preimplantation genetic diagnosis (PGD) in reciprocal chromosomal translocation carriers seems to prevail a view that this method reduces the frequency of miscarriages, and the pregnancy rate is directly proportional to the number of normal spermatozoa. Therefore, we compared the results of sperm karyotype analysis of a carrier of familial t(2;7)(p11.2;q22) with PGD results. The carrier was ascertained as his wife had had two miscarriages. Methods Empirical data from a pedigree of t(2;7)(p11.2;q22) carrier was collected. A tri-color fluorescence in situ hybridization method (FISH) was used to show the meiotic segregation pattern in sperm of the proband. PGD of blastomeres from a single ICSI cycle and standard prenatal diagnosis procedures to confirm the PGD results was performed. Results Meiotic segregation pattern showed only 34.2% of normal/balanced spermatozoa. The high rate (42%) of miscarriages was observed in this family, which could be explained by chromosomal unbalanced karyotypes as a product of fertilization by unbalanced spermatozoa found with a frequency of approximately 66%. The lack of unbalanced progeny at birth suggests a natural selection of unbalanced fetuses. The 37.5% of normal/balanced embryos received after a single ICSI cycle and PGD was similar to the percentage of normal/balanced spermatozoa (34.2%). After 38 weeks a healthy girl with normal karyotype was born. Conclusion The presented study is an optimistic message for translocation carriers showing that even in case with more than 60% of genetically unbalanced sperm there is a reasonable chance for reproductive success. Copyright 2008 John Wiley & Sons, Ltd. KEY WORDS: chromosomal reciprocal translocation; meiotic segregation pattern; PGD; human embryos INTRODUCTION Reciprocal chromosomal translocations (RCT) are the most common structural rearrangements in humans. The incidence of RCT in a population of newborns was estimated as 1 : 712 (Nielsen and Wohlert, 1991) and the frequency at the time of prenatal diagnosis by amniocentesis was about 1 : 250 (Van Dyke et al., 1983). However, among couples with recurrent abortions and/or abnormal offspring, and in infertile males with various degrees of oligoasthenoteratozoospermia this frequency is about ten times higher (Munne et al., 2000; Morel et al., 2004). During meiosis, the chromosomes involved in balanced reciprocal translocation pair their homologous segments forming a quadrivalent figure, which can segregate in five different ways: alternate (producing normal or balanced gametes), adjacent I, adjacent II, 3 : 1, and 4 : 0 (all producing only unbalanced gametes). In a particular carrier of RCT, the proportions between sperm *Correspondence to: Maciej Kurpisz, Institute of Human Genetics, PAN, Poznań, ul. Strzeszyńska 32, Poland. kurpimac@rose.man.poznan.pl karyotypes that come from specific types of meiotic segregation compose the so-called segregation pattern. It seems that the segregation pattern for translocated chromosomes depends greatly on the morphology and genetic content of the chromosomes involved in the individual chromosomal translocation, on the localization of the breakpoints on particular chromosomes, on the length of the interstitial and translocated segments, and on the number and the localization of chiasmata. Gametes carrying the unbalanced reciprocal translocations may cause unfavorable pregnancy outcomes (spontaneous miscarriage, stillbirth, early death of newborn, and congenital malformations in live born progeny). The frequency of reproductive failures is strictly related to the production of a high proportion of gametes with an unbalanced genetic complement and can reflect the different survival rate of different types of unbalanced embryo/fetuses (Jalbert et al., 1980). Meiotic segregation patterns were analyzed so far in nearly 100 RCT carriers (for review see Benet et al., 2005). Translocations are usually unique with regard to the type of chromosomes involved and the breakpoint position and some authors suggest that in most cases of RCT no general rules can be drawn, and each one must be considered as a particular case (Geneix et al., 2002). Copyright 2008 John Wiley & Sons, Ltd. Received: 5 January 2007 Revised: 23 March 2007 Accepted: 2 October 2007

2 PREIMPLANTATION GENETIC DIAGNOSIS 37 Preimplantation genetic diagnosis (PGD) has been offered to RCT carriers as an alternative to prenatal diagnosis and pregnancy termination of unbalanced fetuses. It also aims to decrease the number of spontaneous miscarriages (Munne, 2005). Results of PGD indicate that in the case of RCT carriers this method reduces the frequency of miscarriages, but the pregnancy rate is directly proportional to the number of normal spermatozoa (Munne et al., 1998, 2000). From most of nearly 20 cases published so far where meiotic segregation patterns were compared with PGD results, it can be implied that sperm sample with more than 60% abnormal forms indicates a poor prognosis for a normal term birth (Van Assche et al., 1999; Mackie Ogilvie and Scriven, 2002; Munne, 2005; Yakut et al., 2006). We collected empirical data from a pedigree with a high (42%) incidence of miscarriages among 31 pregnancies of four carriers of the familial translocation t(2;7)(p11.2;q22.1) and performed the analysis of meiotic segregation pattern in the spermatozoa of one carrier. PGD of blastomeres from a single intracytoplasmic sperm injection (ICSI) cycle with the spermatozoa of this carrier was also performed. We compared the contribution of unbalanced sperm with the proportion of abnormal embryos resulting after the first cycle of ICSI and PGD. After 9 months a healthy, chromosomally normal female child was born. Patients MATERIALS AND METHODS The pedigree of the family is shown in Figure 1. The proband, a 43-year-old male who was a carrier of balanced translocation t(2;7)(p11.2;q22.1) (II 10 )was ascertained as his 29-year-old wife (II 11 ) had two miscarriages both at 2 to 3 months (samples from miscarriages were not karyotyped). The origin of the chromosomal translocation of the proband was a paternal one. The father of the (II 10 ), was an 86-year-old male with progeny of five phenotypically normal children (II 2,II 4,II 6,II 8,andII 10 ) and five miscarriages (II 1 ). The older sister (II 2 ) of the proband inherited the same familial reciprocal translocation from the father. She had two miscarriages, one at 8 weeks and one at 4 months. The oldest brother (II 4 ) died in an accident, but his wife (II 5 ) had no history of miscarriages. The older brother (II 6 ) was also a carrier of paternal translocation. His wife (II 7 ) had two miscarriages, one at 5 months and one at 7 weeks. The other pedigree members were inaccessible for chromosome evaluation; however, the wife of proband s older brother (II 8 )had two miscarriages both at 7 to 8 weeks. Examinations of karyotypes of proband and family members were performed by classic GTG banding Figure 1 The pedigree of the carrier t(2;7)(p11.2;q22.1)

3 38 E. WILAND ET AL. Figure 2 Schematic representation of breakpoint positions according to ISCN with marked position of tricolor FISH probes with quadrivalent figure at meiotic prophase explaining the disjunctional possibilities and derivative combinations of signals in spermatozoa methods on metaphase chromosomes from cultured lymphocytes according to standard methods. A resolution of 550 bands per haploid set was obtained. Breakpoint position identification was performed according to ISCN (1996). FISH analysis of spermatozoa Seminological analyses on proband s sperm were performed according to standard WHO criteria (World Health Organization, 1992) and showed normal seminal parameters (normozoospermia). The meiotic segregation pattern in spermatozoa of proband (II 10 ) was examined by tricolor fluorescence in situ hybridization (FISH) as described earlier (Midro et al., 2006). Shortly: sperm nuclear decondensation was obtained by immersing the slides with fixed spermatozoa into a solution of 25 mm dithiothreitol (DTT; Sigma) in a0.1m Tris-HCL(pH8.5)for5to10minin43 C. Four commercially available directly labeled probes (CYTOCELL, England) were used for the analysis of the meiotic segregation pattern: two centromeric probes of chromosome 7 (locus D7Z1), namely, 7c Green and 7c Red, and two telomeric probes, namely, 2p Red (Tel 2p) and 7q Green (Tel 7q). A tricolor FISH was performed in a combination of mixed 7c Red and 7c Green probes (= yellow signal), 2p Red probe, and 7q Green probe. The used probes did not distinguish between normal and balanced spermatozoa. The FISH procedure was performed using the standard protocol provided by the manufacturer. Hybridization signals were observed using an Olympus BX 41 microscope fitted with a triple-band-pass filter. Subsequent image acquisition was performed using a CCD camera with Isis (in situ imaging system) (MetaSystems, Germany). Two thousand and five hundred sperm nuclei per carrier were scored with efficiency of FISH hybridization 98% (in lymphocytes 99.9%). The expected quadrivalent configuration of the translocation at meiosis I with the marked position of FISH probes is shown in Figure 2. ICSI-PGD cycle The 29-year-old wife (II 11 ) of proband (II 10 ) underwent her first ICSI cycle due to two miscarriages and the high rate of miscarriages observed in the family of her husband (Figure 1). Ovarian stimulation was performed with recombinant FSH and human menopausal gonadotrophin (HMG) in a down-regulated cycle according to the short protocol. Twelve oocytes were aspirated from follicular puncture, and ten of them were in metaphase II. Nine oocytes were fertilized and further cultured for 72 h. One blastomere per embryo at the eight-cell stage was biopsied, washed in culture medium, lysed in 2 µl drop of 0.01 N HCl with 0.1% Tween-20 on a Superfrost plus microscope slide (LaboNord), air dried, and fixed. Slides were washed in 1 phosphate-buffered saline (PBS) and dehydrated in ethanol dilutions from 70 to 100%. FISH analysis of blastomeres FISH on eight blastomeres, one from each embryo biopsied, was performed with two sequential hybridizations. The first hybridization assessed chromosomes 2 and 7 involved in translocation and was performed with commercially available directly labeled probes: 2p-Spectrum Orange, 7p-Spectrum Green, and 7q-Spectrum Green (Vysis Inc., Downers Grove, Il.). The used probes did not distinguish between a normal and a balanced translocation. Because patients were also interested in identifying the common aneuploidies of chromosomes 21, X and Y, the hybridization was performed also with the following probes: X alfa-satellite-spectrum Aqua, Yq12 alfa satellite III-Spectrum Green, and 21q- Spectrum Orange-labeled (21q only on normal/balanced blastomeres) (Vysis Inc., Downers Grove, Il.). The FISH procedure was performed according to the manufacturer s instructions. Sperm analysis RESULTS The frequencies of different types of sperm segregation are listed in Table 1. About 34% of the sperm resulted from an alternate segregation and this was the most common segregation type. Adjacent I segregants had a

4 PREIMPLANTATION GENETIC DIAGNOSIS 39 frequency of about 33%. The true frequencies of alternate and adjacent I genotypes will be affected by the presence of crossing-over in interstitial segments. One crossing-over prior to alternate segregation will give the same signal pattern as adjacent I segregation and vice versa. In this study, we assumed that these conversions compensate one another. Adjacent II segregants (without recombination in the interstitial segments) were less common (about 15%). About 12.7% of spermatozoa were generated by a 3 : 1 segregation and all possible phenotypes were observed (5.7% of the tertiary segregation and 7% of the interchange segregation). About 5% of spermatozoa had ambiguous signals: about 3% were probably the result of recombination within the interstitial segments or, alternatively, of nondisjunction at anaphases II (the observed FISH signals were identical in both events) and in about 2% of spermatozoa no signals were present. The lack of signals could probably be explained by hybridization failure. Table 1 Frequencies (%) of different types of sperm segregation and scheme of derivative chromosome combinations in the case of t(2;7)(p11.2;q22.1) carrier PGD results The results of FISH analysis on blastomeres are shown in Table 2. Among nine embryos received in one ICSI cycle, eight were biopsied and only one was uninformative because of nuclear degeneration (Table 2). Following the first hybridization and re-hybridization three embryos were typed as normal/balanced and transferred. Two embryos were implanted. A triplet pregnancy with monozygotic (monochorionic, diamniotic) twins and singleton was ultrasonographically confirmed. Because performing PGD did not reduce the risk of a genetically unbalanced pregnancy to zero, prenatal diagnosis on week 13 via chorionic villous sampling (CVS) was recommended and performed. The twins were boys, carriers of paternal t(2;7)((p11.2;q22.1). The singleton was a girl with normal karyotype. After counseling, because of cervical incompetence and high risk of miscarriage, the patient underwent embryo reduction of the twins, continuing her gestation carrying the single pregnancy. On the 38th week a healthy girl was born. At the time of writing this happy girl is 1 year old. Comparison between PGD and sperm analysis results The percentage of normal/abnormal spermatozoa and blastomeres is presented in Table 3. Both the percentage of normal/abnormal spermatozoa and normal/abnormal blastomeres, and also the percentage of products of unbalanced 2 : 2 segregation are similar. DISCUSSION Though the meiotic segregation pattern in spermatozoa from nearly 100 different reciprocal translocation carriers is known (Benet et al., 2005), only in less than 20 cases the comparison with PGD results was performed (Van Assche et al., 1999; Mackie Ogilvie and Scriven, 2002; Munne, 2005; Yakut et al., 2006). Compared to the percentage of unbalanced spermatozoa, the embryo data seemed to produce higher rates of unbalanced embryos than expected. It is very probable that population selection may be biased by the fact that patients choosing PGD for translocations usually have a severe reproductive history (Munne, 2005). Escudero et al. (2003) found a correlation between the percentage of genetically unbalanced spermatozoa and the percentage of abnormal blastomeres. It was observed that more than 60% of spermatozoa with abnormal genotypes indicated a poor prognosis for pregnancy. A predictive equation was proposed for a correlation between the percentage of abnormal spermatozoa and the percentage of

5 40 E. WILAND ET AL. Table 2 Preimplantation genetic diagnosis (PGD) in blastomere(s) and karyotype of spermatozoa of t(2;7)(p11.2;q22.1) carrier as the result of FISH signal analysis Table 3 Comparison of FISH results on spermatozoa and embryos at PGD Genotype Sperm (%) Blastomere(s) (%) Normal/balanced Unbalanced 2 : 2 Adjacent I 16.2 } 25.0 } Adjacent I % % Unbalanced 3 : Haploid? 12.5 Unexplained/no result abnormal embryos: A = 55 + (1.9 B), whereais the percentage of abnormal embryos and B the percentage of abnormal sperm (Munne, 2005). According to this equation, in the case of the carrier of t(2;7)(p11.2;q22.1) in whom 65.8% of abnormal sperm was found, the predictive percentage of abnormal embryos would be 70%. In the first ICSI cycle, result of PGD of 62.5% of abnormal embryos was lower and more similar to the percentage of unbalanced spermatozoa (Table 3). It appears in this light that the obtained results were better than theoretically expected, and the number of three normal/genetically balanced embryos occurred to be sufficient for a successful pregnancy. This result is in agreement with earlier observations indicating that a good

6 PREIMPLANTATION GENETIC DIAGNOSIS 41 chance for conceiving and carrying on the pregnancy to term is dependent on the fact whether enough chromosomally normal/balanced embryos are available for transfer (Munne et al., 2000). We would like to emphasize, however, that in our view the described reproductive success was also influenced by the age of the embryo recipient (less than 30 years) as well as apparently very good implantation conditions, so the relatively low number of the obtained embryos was not an obstacle to this assisted reproduction intervention. Therefore our case seems to be an optimistic message for translocation carriers who have to decide whether to proceed with PGD or to opt for sperm donation. ACKNOWLEDGEMENTS Special thanks are due to the wife of the proband for her efficient collaboration. REFERENCES 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 111: Escudero S, 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 79: Geneix A, Schubert B, Force A, Rodet K, Briancon G, Boucher D Sperm analysis by FISH in a case of t(17;22)(q11;q12) balanced translocation. Hum Reprod 17: ISCN An international system for human cytogenetic nomenclature. In Recommendation of the International Standing Committee on Human Cytogenetic Nomenclature, Mitelman F (eds). S. Karger: Basel. Jalbert P, Sele B, Jalbert H Reciprocal translocations: a way to predict the mode of imbalanced segregation by pachytene-diagram drawing. Hum Genet 55: Mackie Ogilvie C, Scriven PN Meiotic outcomes in reciprocal translocation carriers ascertained in 3-day human embryos. Eur J Hum Genet 10: Midro AT, Wiland E, Panasiuk B, Leoniewicz R, Kurpisz M Risk evaluation of carriers with chromosome reciprocal translocation t(7;13)(q34;q13) and concomitant meiotic segregation analyzed by FISH on ejaculated spermatozoa. Am J Med Genet 140A: Morel F, Douet-Guilbert N, Le Bris M-J, et al Meiotic segregation of translocations during male gametogenesis. Int J Androl 27: Munne S Analysis of chromosome segregation during preimplantation genetic diagnosis in both male and female translocation heterozygotes. Cytogenet Genome Res 111: Munne S, Sandalinas M, Escudeo T, Fung J, Gianaroli L, Cohen J Outcome of preimplantation genetic diagnosis of translocations. Fertil Steril 73: Munne S, Morrison L, Fung J, et al Spontaneous abortions are reduced after pre-conception diagnosis of translocations. J Assist Reprod Genet 15: Nielsen J, Wohlert M Chromosome abnormalities found among 34,910 newborn children: results from a 13-year incidence study in Arhus, Denmark. Hum Genet 87: Van Assche E, Staessen C, Vegetti W, et al Preimplantation genetic diagnosis and sperm analysis by fluorescence in situ hybridization for the most common reciprocal translocation t(11;22). Mol Hum Reprod 5: Van Dyke DL, Weiss L, Roberson JR, Babu VR The frequency and mutation rate of balanced autosomal rearrangements in man estimated from prenatal genetics study for advanced maternal study. Am J Hum Genet 35: World Health Organization WHO Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus Interaction (3rd end). Berlin: Springer Verlag. Yakut T, Ercelen N, Acar H, Kimya Y, Egeli U Meiotic segregation analysis of reciprocal translocations both in sperms and blastomeres. Am J Med Genet 140A: