Tail stump syndrome associated with chromosomal translocation in two brothers attempting intracytoplasmic sperm injection

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1 Tail stump syndrome associated with chromosomal translocation in two brothers attempting intracytoplasmic sperm injection Celia Ravel, M.D., a,b Sandra Chantot-Bastaraud, M.D., a,b Jean-Pierre Siffroi, M.D., a,b Denise Escalier, Ph.D., c Jean-Marie Antoine, M.D., d and Jacqueline Mandelbaum, M.D. a,b a AP-HP, Hôpital Tenon, Service d Histologie Biologie de la Reproduction Cytogénétique; b Université Pierre et Marie Curie-Paris6, EA1533; c Université Paris V, UFR Saints Pères; and d AP-HP, Hôpital Tenon, Service de Gynécologie et Médecine de la Reproduction, Paris, France Objective: To raise the possibility that a familial chromosomal translocation associated with teratozoospermia can disrupt a gene necessary for flagellum assembly. Design: Case report. Setting: University hospital. Patient(s): Two brothers with infertility related to anomalies of meiotic division and of the flagella assembly, presenting the same balanced 5-12 autosomal translocation. Intervention(s): Several intracytoplasmic sperm injection (ICSI) cycles in our IVF department for both couples. Main Outcome Measure(s): Sperm analysis, karyotypes, electron microscopy, and fluorescence in situ hybridization (FISH) analysis of spermatozoa performed using probes coding for chromosomes X, Y, 13, 18, and 21. Result(s): In both brothers, sperm analysis indicated a tail stump syndrome. Electron microscopy analysis displayed complex abnormalities, which were probably related to meiotic errors. The FISH analysis indicated an increase of diploid germ cells. Karyotypes of both patients revealed the same balanced chromosomal t(5;12) (p15.1; q21) translocation. Results of ICSI cycles were comparable for both couples. A twin pregnancy was achieved in one of these two couples, but a spontaneous miscarriage occurred at 10 weeks of gestation. Conclusion(s): The flagella anomalies raise the possibility that the translocation disrupts a gene necessary for the flagellum assembly, although a mutation in a gene unrelated to the chromosome breakpoints cannot be excluded. (Fertil Steril 2006;86:719e by American Society for Reproductive Medicine.) Key Words: Chromosomal translocation, flagella, ICSI, spermatozoa Idiopathic male factor infertility may have a genetic origin, leading to cytogenetic investigations in patients with severe oligozoospermia or nonobstructive azoospermia. Indeed, many studies have reported an increased frequency of balanced chromosomal abnormalities in men with infertility, particularly in those referred for intracytoplasmic sperm injection (ICSI) (1), although a higher frequency of chromosomal aberrations may also be observed in the female partners of infertile couples treated by ICSI (2, 3). After Klinefelter s syndrome, balanced reciprocal translocations are the most frequent structural chromosomal rearrangements found in men with infertility and may lead to a dramatic decrease in sperm count, which is usually associated with a polymorphic teratozoospermia. The existence of a monomorphic abnormality of sperm structure is a rare event, exceptionally well shown to be associated with a constitutional abnormal karyotype in patients. In such a case, Received October 14, 2005; revised and accepted December 27, Supported by funds from the Institute of Rare Diseases, Institut National de la Santé et de la Recherche Médicale, grant GIS 0334 (D.E.). Reprint requests: Dr. Jacqueline Mandelbaum, M.D., Service d Histologie, Biologie de la Reproduction et Cytogénétique. Hôpital Tenon, AP-HP, 4 rue de la Chine, Paris, France (FAX: (33) ; jacqueline.mandelbaum@tnn.ap-hop-paris.fr). autosomal balanced translocations may be of particular interest for determining candidate genes involved in spermatogenesis. We report the cases of two brothers who were referred for primary infertility. Semen analysis indicated a failure to assemble the flagella structures in all sperm cells, associated with a t(5;12) balanced chromosomal translocation. CASE REPORTS Both brothers were of Algerian origin, but belonged to a family without any evident consanguinity. Pedigree revealed that three brothers and one sister were infertile, whereas two other males and two other females had produced normal children, thus suggesting a classical story of familial infertility (Fig. 1). Both patients gave informed consent for a genetic analysis of their infertility according to French law. Case 1 The members of the first couple were both 31 years old and had been consulting for primary infertility for 3 years. The wife s clinical and biological analysis did not reveal anything particular except moderate hyperprolactinemia, which was treated. Results of the husband s physical examination were normal /06/$32.00 Fertility and Sterility Vol. 86, No. 3, September 2006 doi: /j.fertnstert Copyright 2006 American Society for Reproductive Medicine, Published by Elsevier Inc. 719.e1

2 FIGURE 1 Genealogy of the family. Semen Analysis. A semen analysis was performed in our laboratory, according to standard World Health Organization (WHO) criteria (4). Semen parameters in case 1 revealed a severe oligoasthenoteratozoospermia (sperm count: / ml) with 99% of spermatozoa that were immotile, while 80% were alive. Light microscopy analysis of sperm cells revealed a monomorphic teratozoospermia, with almost all spermatozoa showing a tail stump phenotype. Cytogenetic Evaluation and Familial Data. Chromosome analysis was performed on peripheral blood lymphocytes by conventional cytogenetic techniques using G-bands by trypsin using Giemsa (GTG) and R-bands by heating using Giemsa (RHG) banding. The patient s karyotype revealed an apparently balanced t(5;12) (p15.1;q21.2) translocation, which was confirmed by fluorescence in situ hybridization (FISH) analysis using 5p and 12p/12q telomere probes (Oncor, Gaithersburg, MD) (Fig. 2). Chromosome analysis results in the patient s wife were normal. The patient s mother had a normal karyotype, but cytogenetic investigations were impossible in the patient s father, who was dead. Transmission Electron Microscopy. Electron microscopy analysis of the sperm cells was performed as previously described (5) and revealed a lack of flagellum in almost all spermatozoa (Fig. 3a). Spermatozoa with a nucleus of normal size were rarely observed (Fig. 3a). The other sperm cells presented a giant nucleus (Fig. 3a) or were binucleated (not shown). Centriole pairs that can represent two to four proximal flagella regions were frequently observed close each other (Fig. 3b). Most frequently, the distal centrioles did not give rise to an axoneme (Fig. 3c). Flagellated spermatozoa exhibited a short flagellum with misassembly of the structures (Fig. 3d). The middle piece region was rarely observed and presented an incomplete and disorganized axoneme and an excess of dense fibers (Fig. 3e). Rare flagella able to assemble nine doublets of microtubules lacked the central pair of microtubules and displayed an incomplete microtubule B, absence of outer dynein arms and of some inner arms (Fig. 3f). These flagella also exhibited additional doublets and an accumulation of the fibrous sheath (Fig. 3f). Sperm Aneuploidy Study. Sperm aneuploidies for chromosomes 13, 21, 18, X, and Y were investigated in ejaculated sperm nuclei using three-color FISH analysis. Directly labeled centromeric DNA probes coding for chromosomes X, Y, 18 and locus-specific DNA probes coding for 13 and 21 (Aneuvysion Vysis, Naperville, IL) were used. After separation of sperm cells by gradient, spermatozoa were prepared for FISH as previously described (6). In situ hybridization was performed using standard methods, and analysis was made on an ultraviolet microscope equipped with an appropriate filter set and connected to a Geniken Imaging System (Alphelys, Plaisir, France). Only spermatozoa with well-defined boundaries were scored, and signals in a specific color were considered to be multiple when separated by at least one signal diameter. The 6-diamino-2-phenylindole stained spermatozoa with no FISH signals were eliminated. Slides were scored in a blind protocol by two independent investigators. A total of 1,054 spermatozoa were analyzed (Table 1). A fertile, normospermic male was used as a control subject, and a comparison was performed using a Fisher s exact test. An increased frequency of disomy for chromosomes 21 and 18 was observed (P.01), whereas the disomy rate for chromosome 13 and sex chromosomes was in the control range (Table 1). The diploidy rate was greater than the control range (P.001), confirming the meiotic division abnormalities observed by electron microscopy. In Vitro Fertilization. The couple underwent IVF with ICSI, according to the previously described procedure (7). Six ICSI cycles were performed (Table 2). The mean fertilization rate was 39%. The percentage of transferred and frozen embryos per microinjected oocyte was approximately 37%. The first ICSI cycle failed because of technical difficulties 719.e2 Ravel et al. Tail syndrome, translocation, and ICSI Vol. 86, No. 3, September 2006

3 FIGURE 2 (A) Partial karyotype displaying the two translocated chromosome pairs (chromosomal breakpoints are indicated by arrows). (B) FISH using 5p (green), 12p (green), and 12q (red) probes. The abnormal chromosome 5 carries only the 12q signal, whereas the translocated chromosome 12 carries two short arm signals. FIGURE 3 Electron microscopy of spermatozoa of case 1. (a) A general view of the sperm cells exhibiting an absence of elongated flagella. The sperm nuclei exhibit an irregular shape and are, most frequently, abnormally enlarged. (b) A section of the neck region of a spermatozoon displaying the formation of multiple connective pieces that can correspond to four centriole pairs. (c) A spermatozoon with an enlarged nucleus and two connective pieces. No other flagella structures are observed. (d) A spermatozoon displaying misassembly of the flagella structures in a cytoplasmic remnant. (e) A section of a flagella middle piece displaying missassembly and the absence of some axonemal components. The microtubule B wall is incomplete in four of the five doublets present. (f) A section at the level of the principal piece of the flagellum, which contains an axoneme lacking the central pair of microtubules and an absence of many dynein arms. As in e, the microtubule B wall is incomplete in four doublets. Additional doublets and dense fibers are located close to the membrane. The fibrous sheath presents several layers and is misassembled. Bars: a, c, d 0.5 m; b, e, f 0.2 m. for catching and injecting morphologically abnormal spermatozoa, but later cycles were more successful in obtaining fertilized oocytes and morphologically normal embryos. Recovery of very rare motile spermatozoa with apparently normal flagella or membrane destabilization by repeated aspiration through the micropipette probably helped to achieve fertilization success. At the fourth ICSI cycle, a twin pregnancy was obtained after the transfer of 3 embryos, but terminated spontaneously at 10 weeks of gestation. Both fetuses had a 46 XX normal karyotype. Two subsequent cycles failed to achieve a pregnancy. At present, two embryos are still cryopreserved for this couple. Case 2 The members of the second couple were 36 and 27 years old, respectively. The couple was referred to the laboratory because of primary infertility for 2 years. Cytogenetic investigations indicated that the male patient carried the same t(5;12) translocation as his brother. Semen analysis revealed a more severe oligoasthenoteratozoospermia (sperm count: /ml) with identical morphological abnormalities, particularly the absence of flagella. Fertility and Sterility 719.e3

4 TABLE 1 FISH analysis of spermatozoa of case 1. Chromosomal pattern Patient Control P-value Normal 23X 49.4% 51.6% NS 23Y 45.0% 46.5% NS Gonosomic disomy X-X % NS Y-Y NS X-Y % 0.5% NS Autosomic disomy % 0.7% NS X or Y 0.8% 0 P % 0 P.001 Diploidy X-X or Y-Y or X-Y-18-18, % 0.4% P.001 Note: FISH fluorescence in situ hybridization. NS not statistically significant. Transmission Electron Microscopy. The sperm cells of the patient in case 2 presented the same ultrastructural characteristics as in case 1, both at the head and flagellum levels. However, this patient was more affected by the meiotic division defects, as observed by the high proportion of binucleated sperm cells (Fig. 4a) and of spermatozoa with a large nucleus (Fig. 4b). The sperm cells appeared less differentiated than in case 1, as observed by the large cytoplasm TABLE 2 ICSI cycles of case 1 and case 2. Cycle Case Total Case 1 Sperm concentration (10 6 /ml) Motility (%) Some Some Some No. of oocytes No. of mature oocytes (M II) No. of 2PN oocytes No. of embryos transferred No. of embryos cryopreserved Pregnancy No No No Twin miscarriage No No Case 2 Sperm concentration (10 6 /ml) Motility (%) No. of oocytes No. of mature oocytes (M II) No. of 2 PN oocytes No. of embryos transferred No. of embryos cryopreserved Pregnancy No No No No Note: ICSI intracytoplasmic sperm injection. 719.e4 Ravel et al. Tail syndrome, translocation, and ICSI Vol. 86, No. 3, September 2006

5 FIGURE 4 Electron microscopy of spermatozoa of case 2. (a) A binucleated spermatozoon with a large cytoplasmic remnant devoid of axonemal and periaxonemal structures. (b) A spermatozoa with a large nucleus containing poorly condensed chromatin. Structures that can correspond to elements of the fibrous sheath are present in the cytoplasm, whereas elements of the axoneme and the dense fibers are not observed. Bars: a, b: 0.5 m. surrounding the nucleus (Fig. 4b). As in case 1, two to four centriole pairs were observed close to each other (not shown). Most frequently, the axoneme was absent, and few periaxonemal structures were observed (Fig. 4b). Few sperm cells were well conserved, whereas the other sperm cells displayed disruption of the plasma membrane and alteration of the nuclei. In Vitro Fertilization. Four ICSI cycles were attempted, with the same technical difficulties as in case 1 (Table 2). Nevertheless, the mean fertilization rate was 38%, and the percentage of embryos transferred or frozen per microinjected oocyte was 38%. However, although the patient s wife was 27 years old, the number of collected oocytes decreased by half from the first to the fourth attempt, and only 52% were mature. No pregnancy was achieved through the four transfers of fresh embryos. DISCUSSION Human male factor infertility related to oligoasthenoteratozoospermia may have a familial component (8), but, in most cases, familial investigations do not fit with an autosomal dominant or recessive segregation mode, suggesting that impaired spermatogenesis results from several different factors rather than a common genetic defect (9). However, in a few cases, specific and monomorphic alterations of sperm cell structure have been reported, which could be related to a particular genetic abnormality (6, 10 14) or to a chromosomal aberration. To our knowledge, very rare cases of balanced chromosomal rearrangements have been described as associated with an alteration of sperm cell structure, which leads to monomorphic teratozoospermia (15 19), whereas isolated and severe asthenoteratozoospermia is reportedly associated with an increased rate of sperm aneuploidy (20). Therefore, finding a familial translocation associated with specific teratozoospermia, as in our patients, is a rare event that raises the question of whether abnormal sperm structure may be the direct consequence of the chromosomal rearrangement. In patients carrying a chromosomal translocation, infertility appears to be the result of troubles in nuclear events at meiotic prophase I, such as the abnormal pairing behavior of translocated chromosomes and their frequent association with the XY body, rather than the consequence of gene disruption at chromosomal breakpoints (21, 22). In these cases, alteration of sperm parameters is usually restricted to a sperm count decrease that leads to oligozoospermia or azoospermia (3). The autosomal translocation in the present cases allowed spermatocytes to reach the meiotic divisions. However, the low number of spermatozoa suggested that high numbers of spermatocytes were arrested and eliminated at this step. The spermatocytes that escaped arrest gave rise to spermatozoa exhibiting multiple centriole pairs, giant nuclei, and diploidy, suggesting meiotic division anomalies but associated with abnormal flagella structures (either totally or partially). An association of the absence of meiotic divisions and the assembly of flagella structures has been previously described in humans (5, 23), but a gene mutation leading to a similar phenotype has never been identified, either in mouse models or in humans (24). In the family we studied, the t(5;12) translocation was observed in two infertile brothers, who carried the same abnormal sperm phenotype, as well as in another, fertile, brother and in two fertile sisters, thus bringing evidence that this translocation was inherited. Because the mother s karyotype was normal, the paternal origin of the translocation was obligatory, although analysis of the father s karyotype had not been feasible. Because of the father s normal fertility and the existence of a fertile brother carrying the translocation, a direct relationship between the familial translocation and the specific sperm abnormalities found in the two infertile brothers appears unlikely. However, assuming that these two cases of infertility could have a chromosomal origin, the association of the paternal translocation with a recessive maternal mutation, both affecting a gene involved in sperm structure and located at or nearby translocation breakpoints, has to be considered. The DNAH5 gene, coding for a heavy dynein chain, maps to the 5p15 region. Using FISH with a probe covering the near totality of the DNAH5 gene (RPCI A21: selected in accordance with the Genome Database May 2004, and received from the Welcome Trust Sanger Institute, Cambridge, UK), the translocation breakpoint on chromosome 5 was shown to be proximal to the probe, thus indicating that this gene was not involved in the chromosomal rearrangement (data not shown). Therefore, the genetic origin of the complex teratozoospermia in the present cases, as strongly suggested by the identical testicular phenotype in Fertility and Sterility 719.e5

6 the two brothers, is likely to be due to a recessive mutation inherited from both parents, although they were unrelated. Such hereditary transmission could explain the infertility of the third infertile brother, who does not carry the familial translocation. The only chance for the wives of our patients to achieve pregnancy was ICSI, but treating these couples in which the men carried both a chromosomal translocation and severe teratozoospermia raised major difficulties. In this particular case with very short flagella, we tried to perform the obligatory destabilization of sperm membrane by repeated sucking of the sperm head in and out the pipette. Despite the fact that this repeated aspiration was known to yield lower results (25), we obtained acceptable 2 PN fertilization and normal cleavage rates. Higher fertilization rates could perhaps be achieved by using a noncontact diode laser to destabilize the sperm membrane (26). The twin pregnancy achieved in couple 1, although terminated by a spontaneous miscarriage, confirms that tail stump spermatozoa can successfully fertilize oocytes by ICSI, giving rise to normal pregnancies and newborns (27, 28). Natural or medically assisted pregnancy by a translocation carrier can risk the birth of a child carrying a chromosomal imbalance, as the result of the abnormal segregation of the parental balanced karyotype rearrangement. Sperm karyotyping studies performed in 30 reciprocal translocation carriers have demonstrated that 19% 77%, with an average of approximately 50%, of spermatozoa were chromosomally unbalanced (29). Using the HC Forum databank ( the estimated risk of giving birth to an affected child, carrying multiple congenital abnormalities and developing later mental retardation, was estimated to be 10.9% for the t(5;12) translocation present in our cases. Although theoretical, this estimation indicates that this translocation presented a high risk for the offspring, complicated by the presence of twins, and that a prenatal diagnosis should have been proposed to the parents if the pregnancy had been carried to term. Moreover, in chromosomally normal infertile patients carrying severe teratozoospermia and/or asthenozoospermia, an increased rate of aneuploid spermatozoa has been described (30 32). In our case 1 patient, the frequency of disomic sperm cells for chromosome 21 was higher than in the normospermic control subject, suggesting either a direct relationship between spermatogenesis impairment and segregation errors of some autosomes at meiosis or a possible interchromosomal effect between the segregation of both translocated and nontranslocated chromosomes. Such an interchromosomal effect for chromosome 21 has already been described in 9 (41%) of 22 translocation carriers who presented a 1.4- to 6.6-fold increase of chromosome 21 disomic spermatozoa when compared with controls (33). In conclusion, this case report underlines the usefulness of karyotyping patients who carry a monomorphic structural abnormality of sperm cells and demonstrates that treatment of their infertility by ICSI may be attempted, although specific risks must be taken into account. Acknowledgments: The authors thank the IVF technical staff for participating in the ICSI procedures. REFERENCES 1. Van Assche E, Bonduelle M, Tournaye H, Joris H, Verheyen G, Devroey P, et al. Cytogenetics of infertile men. Hum Reprod 1996; 11(Suppl 4): Gekas J, Thepot F, Turleau C, Siffroi JP, Dadoune JP, Briault S, et al. Association des Cytogeneticiens de Langue Francaise. 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