Hum. Reprod. Advance Access originally published online on December 16, 2005
Human Reproduction 2006 21(4):976-979; doi:10.1093/humrep/dei427
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Meiotic segregation patterns and ICSI pregnancy outcome of a rare (13;21) Robertsonian translocation carrier: a case report
Department of Obstetrics and Gynaecology, University of British Columbia, Vancouver, British Columbia, Canada
1 To whom correspondence should be addressed at: Department of Obstetrics and Gynaecology, Room 313, Willow Pavilion, Vancouver Hospital and Health Sciences Centre, 855 West 12th Avenue, Vancouver, British Columbia, Canada V5Z 1M9. E-mail: sai{at}interchange.ubc.ca
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
t(13;21) is an uncommon Robertsonian translocation (RT) with limited information in the literature. Hereby, we assessed the meiotic segregation and interchromosomal effect (ICE) in sperm nuclei from a t(13;21) carrier. The pregnancy outcome following ICSI was also included as reference for physicians and patients. Dual-colour fluorescent in situ hybridization (FISH) was carried out to analyse the segregation pattern of chromosomes 13 and 21, while triple-colour FISH was used to investigate the possible concurrence of ICE. With respect to chromosomal constitutions of 13 and 21, 88.39% of the spermatozoa were normal or balanced due to alternative segregations, and 11.08% showed nullisomy or disomy as a result of adjacent segregations. However, for chromosome 18 and sex chromosomes, the proportion of normal haploids was 98.79%. The rate of disomy was not significantly higher than the controls for either chromosome 18 or X/Y. The rare t(13;21) case exhibited a similar pattern of meiotic segregation as in the common RTs. ICEs were not observed in the current case.
Key words: ICSI/interchromosomal effects/meiotic segregation/Robertsonian translocations/spermatozoa
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Robertsonian translocations (RTs), fusions between two acrocentric chromosomes, are the most common structural chromosomal rearrangements in humans and occur in approximately 1 in every 1000 newborns (Therman et al., 1989
). Among the possible combinations of the five acrocentric chromosomes (13, 14, 15, 21 and 22), translocations between chromosomes 13 and 14 and between chromosomes 14 and 21 are the most frequent, comprising about 75% and 10% of all RT cases, respectively. The other possible RT combinations exist to a much less extent. t(13;21) is one of the most rare RTs, estimated to be approximately 2% among all RTs (Therman et al., 1989).
Men carrying RTs often have some degree of infertility, for instance, oligozoospermia or azoospermia, but are otherwise phenotypically normal (Scriven et al., 2001). The aetiology of infertility is related to synaptic abnormalities during meiosis leading to meiotic arrest. The incidence of RTs in infertile men is approximately 2–3% compared to 0.12% in the general population. (Nielsen and Wohlert, 1991; Baschat et al., 1996; Testart et al., 1996). With current advances in assisted reproductive technologies (ART), such patients have increasingly been given the opportunity to conceive their own biological children. Thus, it is of concern that chromosomally unbalanced gametes from RT carriers may be incorporated into the conceptus.
In addition, RTs may adversely affect the meiotic segregations of uninvolved chromosome pairs. The presence of such interchromosomal effects (ICE) is still an open debate. Supporting data have been provided by several studies using multicolour fluorescent in situ hybridization (FISH) (Rousseaux et al., 1995; Morel et al., 2001; Baccetti et al., 2002; Anton et al., 2004). Gianaroli et al. (2002) examined 111 in vitro-generated embryos from RT carriers and reported a considerable amount of ICE such that 31% of the embryos had abnormalities on RT nonrelated chromosomes and 36% had abnormalities in both RT-related and nonrelated chromosomes. Conversely, other authors reported lack of ICE in RTs (Blanco et al., 2000; Pellestor et al., 2001), and some suggest that ICE is restricted to sperm with poor quality (Vegetti et al., 2000; Pellestor et al., 2001). As most studies on ICE were done on common RTs, the investigation on the rare t(13;21) case will not only add information to the debate on the presence of ICE but also provide insight into the association between the types of translocated chromosomes and ICE, if it exists.
Although meiotic segregation and ICE in spermatozoa have been repeatedly studied in the t(13;14) and t(14;21) cases, t(13;21) cases have yet to be well documented. The aim of the current study was to investigate meiotic segregation pattern of involved chromosomes and possible ICE in a man with a balanced t(13;21), which was also transmitted to his ICSI-facilitated conception.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Clinical information
A couple (female, 39 years old; male, 40 years old) presented with a 3-year history of primary infertility. The female partner displayed no evidence of tubal, ovulatory or pelvic infertility factors. The male partner (proband), however, was found to have oligoasthenoteratozoospermia from two consecutive semen analyses (sperm count, 7.4–8.4 x 106/ml; motility, 3–7%; and morphology, 2%). Subsequent karyotyping of the man’s peripheral blood revealed a balanced RT involving chromosomes 13 and 21. The breakpoint appears to be at the centromeres of both chromosomes. This translocation karyotype, 45,XY,t(13;21)(q10;q10), appeared in all cells examined. The proband’s brother was found to have the same translocation. However, the proband’s parents and the female sibling were not available for cytogenetic analysis. Both the proband’s brother and sister had no children: his brother underwent a vasectomy in his early thirties and his sister had a 5-year history of infertility. Because of the severity of the proband’s sperm parameters, IVF combined with ICSI was undertaken.
Fluorescent in situ hybridization
Detailed procedures for sperm preparation and FISH were described in our previous publication (Tang et al., 2004). Dual-colour FISH was carried out using LSI 13/21 probes (13q14 LSI13, SpectrumGreen/21q22.13-q22.2 LSI21, SpectrumOrange; Vysis, Downers Grove, IL, USA) for the detection of normal/balanced or unbalanced sperm. Triple-colour FISH was performed using 
-satellite (DNA) probes for chromosomes 18, X and Y (CEP 18, SpectrumAqua/CEP X, SpectrumGreen/CEP Y, SpectrumOrange; Vysis) to investigate the presence of ICE.
The analysis was carried out with an epifluorescent microscope (Nikon Elipse E600W) equipped with a triple bandpass filter (DAPI/FITC/Cy3), a dual bandpass filter (FITC/Cy3), and single bandpass filters for DAPI, Aqua, FITC and Cy3. Scoring was performed in areas with consistent hybridization. Only morphologically intact sperm nuclei with sperm tails were assessed according to the standard assessment criteria (Tang et al., 2004). At least 10 000 nuclei were scored for each probe set.
Data analysis
The control group used in the present study was derived from FISH results of sperm samples from fertile males with normal karyotypes previously studied in our laboratory. The experimental procedures for controls were followed exactly the same as for the patient. Chi-square test was used to compare the aneuploidy rates in the patient with those in the control group.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
ICSI outcome
A summary of results from three ICSI cycles is given in Table I. Of the 17 oocytes retrieved in three consecutive ICSI cycles (with 2-year interval), 12 metaphase II (MII) oocytes were used for ICSI. Of the 12 oocytes injected, 9 of them fertilized normally (75%). Four (4-, 6-, 7- and 8-cell stage, respectively), three (all 8-cell stage) and two (7- and 8-cell stage, respectively) embryos were transferred in three separate cycles. Among the nine transferred embryos, only two were of poor quality, while the others displayed normal development and good quality. In all three cycles, it was noted that sperm parameters had worsened at the time of ICSI procedure compared to previous semen analyses: sperm concentrations were all less than 3 x 106/ml, and very few motile sperm were present.
|
Pregnancy was achieved only in the first ICSI cycle, in which a total of four embryos were transferred. At the eighth week of gestation, the pregnancy spontaneously aborted. Cytogenetic analysis of cultured chorion revealed a male karyotype with a balanced RT of paternal origin [45,XY,t(13;21)].
FISH on sperm
The meiotic segregation analysis on chromosomes 13 and 21 was performed on a total number of 10 223 sperm nuclei (Table II). With respect to chromosomal constitutions of 13 and 21, the majority of spermatozoa (88.39%) were normal, 13q/21q, or balanced, der(13q;21q), results of alternate segregation in meiosis. Chromosomally unbalanced spermatozoa for chromosomes 13 and 21, derived from an adjacent segregation, account for 11.08%. This was significantly higher than in the fertile controls (0.6%) (P < 0.05). The rates of nullisomy for both chromosomes 13 and 21 were higher than the complementary disomy rates in the patient (P < 0.05); however, such discrepancy was not observed in the control group (P > 0.05). The rate for the 3:0 segregation and diploidy, indicated by two signals for each of 13 and 21, was 0.26%, whereas the control group had a frequency of 0.1%. In addition, there was 0.18% of 21q/21q and 0.09% of 13q/13q exclusively observed in the patient, which were categorized as ‘other’ modes of segregation in Table II.
|
The results of the ICE investigation are summarized in Table III. A total of 10 172 sperm nuclei were analysed with the 18, X, Y probe set, and 98.79% were normal haploid. The overall aneuploidy rate was not significantly higher in the patient compared to that of controls (P > 0.05). The rate of sex chromosomes disomy (0.15%) was about five times as high as disomy 18 (0.03%), which was also observed in the control group (0.39 versus 0.07%). The diploidy rate was 0.05% in the patient and 0.04% in the controls.
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Studies on meiotic segregation in RT carriers have been of great informative value in reproductive counselling. However, t(13;21) cases, along with other rare RTs, have few reports available in the literature, despite the involvement of the two chromosomes that account for the majority of trisomies among live births. To our knowledge, this is the first study that has investigated the meiotic segregation pattern and the possible ICE of t(13;21). The clinical information regarding the ICSI treatment and the pregnancy outcome were also reported thoroughly in order to provide further information for the physicians and patients considering ART.
The segregation analysis of chromosomes 13 and 21 in the current study showed that 88.39% of the spermatozoa were normal or balanced. This percentage is in agreement with previous FISH studies on sperm for other types of RTs (Martin et al., 1992; Mennicke et al., 1997; Honda et al., 2000; Ogawa et al., 2000; Morel et al., 2001). Normal or balanced sperm were present predominantly in all segregation studies, ranging from 60 to 96.6%, regardless of different methodologies used for analyses (heterospecific IVF, sperm injection into mouse oocytes or FISH). Among the most well-studied t(13;14) cases, the percentage of normal or balanced constitutions also ranges from 73.5 to 92.3% (reviewed by Anton et al., 2004). The high frequency of normal or balanced spermatozoa is presumably attributable to a selection in all RTs towards the cis-configuration of the trivalent during meiosis which leads to an alternate segregation (Sybenga, 1975).
The unbalanced chromosomal constitutions comprise of nullisomies and disomies derived from adjacent segregations, 3:0 or diploidy, and unexpected combinations (‘other’ in Table II). There was an obvious deviation from the expected 1 : 1 ratio of disomic to its complementary nullisomic spermatozoa. For both chromosomes 13 and 21, the nullisomy rates were significantly higher (P < 0.05). Similar observations were made previously by other researchers (Honda et al., 2000; Frydman et al., 2001; Morel et al., 2001; Anton et al., 2004). The discrepancy is possibly originated from two aspects. First, hybridization artefacts can be responsible for the higher frequency of nullisomy. However, it can probably be excluded because of the high efficiency of hybridization. Also, taking into the consideration that such inconsistency was not seen in the controls and that a hybridization failure would affect all combinations of signals rather than one category, it would be reasonable to state that in our case the discrepancy may not be due to experimental errors. The second factor that may contribute to the observation is meiotic checkpoints. As suggested by Honda et al. (2000), there may be a more stringent selection against disomic cells than nullisomic cells. Therefore, a higher frequency of nullisomy in the spermatozoa can be expected.
0.26% of spermatozoa displayed two signals for each of chromosomes 13 and 21, which is similar to that in studies on other RT types (0–0.8%; reviewed by Morel et al., 2001). This phenomenon is caused by either diploidy or 3:0 segregation. The distinction was aided by the comparison with the diploidy rate found from the ICE analysis. The diploidy rate was 0.05% according to the investigation on chromosomes 18, X and Y. Hence, the rate of 3:0 segregation is estimated to be approximately 0.2%. This confirms that the occurrence of 3:0 segregation is a much rarer event compared to alternate and adjacent patterns.
The unexpected abnormalities observed in this study (13q/13q and 21q/21q) were perhaps attributable to non-disjunction events in meiosis II following an adjacent segregation pattern. In other words, a non-disjunction event at MII may have prevented the formation of nullisomy 13 or 21. Instead, cells with disomy for one chromosome and nullisomy for the other chromosome (13q/13q and 21q/21q) can be formed. However, the theoretical ‘co-products’ of the non-disjunction, cells missing both 13 and 21, were not detected in the study. Those cells may have been arrested at cell-cycle checkpoints, as they are in genetically more intolerable forms. Investigating a larger number of nuclei may increase the chance to find such cells; however, in order to distinguish from those with hybridization failures, one would need to co-hybridize the slide with another chromosome marker as an internal control.
Although the existence of ICE remains controversial, a number of studies have supported its prevalence in patients with chromosomal arrangements including RTs. Gianaroli et al. (2002) suggested that ICE is more common in cases of RTs than other types of translocations. Several FISH studies on spermatozoa from RT patients have reported increased frequency of sex chromosome and certain autosomal disomies (reviewed by Shi and Martin, 2001). The current case did not provide evidence for the ICE on chromosome 18 and sex chromosomes. There was no significant difference in the aneuploidy rate of those chromosomes compared to the control group (P > 0.05). While Baccetti et al. (2002) observed ICE on sex chromosome disomy, disomy 18 and diploidy in a t(13;21) case, the disparity may depend on the region of chromosome involved, which may lead to certain meiotic configuration that enhances the formation of aneuploidies (Estop et al., 2000). Thus, the same type of RT may have variable ICE during spermatogenesis.
Although ICSI can possibly facilitate the transmission of the structural abnormality, in the current case, an unbalanced spermatozoon was not introduced to the conceptus. However, the pregnancy unfortunately ended as a spontaneous abortion. Although the fetus carried the same RT as in the father, the translocation is probably not responsible for the miscarriage because the breakpoint should be conserved; one may also exclude the female anatomical factors for the pregnancy loss, as the mother had no evidence of tubal, ovulatory or pelvic infertility factors. Thus, the cause for the loss remains unexplained with the available information at this point.
|
Acknowledgements |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
We gratefully thank the patient for donating the semen samples for this study. The authors also thank UBC IVF Center for Reproductive Health in the Division of Reproductive Endocrinology and Infertility for their clinical work. This presented study was supported by a grant from Canadian Institutes of Health Research (CIHR) (MOP-53067 to S.M.) and The Hospital for Sick Children Foundation (X9 02-086 to S.M.).
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionAcknowledgementsReferences |
|---|
Anton E, Blanco J, Egozcue J and Vidal F (2004) Sperm FISH studies in seven male carriers of Robertsonian translocation t(13;14)(q10;q10). Hum Reprod 19,45–51.
Baccetti B, Capitani S, Collodel G, Estenoz M, Gambera L and Piomboni P (2002) Infertile spermatozoa in a human carrier of Robertsonian translocation 14;22. Fertil Steril 78,1127–1130.[CrossRef][Web of Science][Medline]
Baschat AA, Kupker W, al Hasani S, Diedrich K and Schwinger E (1996) Results of cytogenetic analysis in men with severe subfertility prior to intracytoplasmic sperm injection. Hum Reprod 11,330–333.
Blanco J, Egozcue J and Vidal F (2000) Interchromosomal effects for chromosome 21 in carriers of structural chromosome reorganizations determined by fluorescence in situ hybridization on sperm nuclei. Hum Genet 106,500–505.[CrossRef][Web of Science][Medline]
Colombero LT, Hariprashad JJ, Tsai MC, Rosenwaks Z and Palermo GD (1999) Incidence of sperm aneuploidy in relation to semen characteristics and assisted reproductive outcome. Fertil Steril 72,90–96.[CrossRef][Web of Science][Medline]
Estop AM, Cieply K, Munne S, Surti U, Wakim A and Feingold E (2000) Is there an interchromosomal effect in reciprocal translocation carriers? Sperm FISH Studies. Hum Genet 106,517–524.[CrossRef][Web of Science][Medline]
Frydman N, Romana S, Le Lorc’h M, Vekemans M, Frydman R and Tachdjian G (2001) Assisting reproduction of infertile men carrying a Robertsonian translocation. Hum Reprod 16,2274–2277.
Gianaroli L, Magli MC, Ferraretti AP, Munne S, Balicchia B, Escudero T and Crippa A (2002) Possible interchromosomal effect in embryos generated by gametes from translocation carriers. Hum Reprod 17,3201–3207.
Honda H, Miharu N, Samura O, He H and Ohama K (2000) Meiotic segregation analysis of a 14;21 Robertsonian translocation carrier by fluorescence in situ hybridization. Hum Genet 106,188–193.[CrossRef][Web of Science][Medline]
Martin RH, Ko E and Hildebrand K (1992) Analysis of sperm chromosome complements from a man heterozygous for a robertsonian translocation 45,XY,t(15q;22q). Am J Med Genet 43,855–857.[CrossRef][Web of Science][Medline]
Mennicke K, Diercks P, Schlieker H, Bals-Pratsch M, al Hasani S, Diedrich K and Schwinger E (1997) Molecular cytogenetic diagnostics in sperm. Int J Androl 20 (Suppl. 3),11–19.
Morel F, Roux C and Bresson JL (2001) FISH analysis of the chromosomal status of spermatozoa from three men with 45,XY,der(13;14)(q10;q10) karyotype. Mol Hum Reprod 7,483–488.
Nielsen J and Wohlert M (1991) Chromosome abnormalities found among 34,910 newborn children: results from a 13-year incidence study in Arhus, Denmark. Hum Genet 87,81–83.[CrossRef][Web of Science][Medline]
Ogawa S, Araki S, Araki Y, Ohno M and Sato I (2000) Chromosome analysis of human spermatozoa from an oligoasthenozoospermic carrier for a 13;14 Robertsonian translocation by their injection into mouse oocytes. Hum Reprod 15,1136–1139.
Pellestor F, Imbert I, Andreo B and Lefort G (2001) Study of the occurrence of interchromosomal effect in spermatozoa of chromosomal rearrangement carriers by fluorescence in-situ hybridization and primed in-situ labelling techniques. Hum Reprod 16,1155–1164.
Rousseaux S, Chevret E, Monteil M, Cozzi J, Pelletier R, Delafontaine D and Sele B (1995) Sperm nuclei analysis of a Robertsonian t(14q21q) carrier, by FISH, using three plasmids and two YAC probes. Hum Genet 96,655–660.[CrossRef][Web of Science][Medline]
Scriven PN, Flinter FA, Braude PR and Ogilvie CM (2001) Robertsonian translocations – reproductive risks and indications for preimplantation genetic diagnosis. Hum Reprod 16,2267–2273.
Shi Q and Martin RH (2001) Aneuploidy in human spermatozoa: FISH analysis in men with constitutional chromosomal abnormalities, and in infertile men. Reproduction 121,655–666.[Abstract]
Sybenga J (1975) Chromosome structural variants. In Sybenga J (ed.) General Cytogenetics. North-Holland, Amsterdam, The Netherlands, pp. 165–212.
Tang SS, Gao H, Robinson WP, Ho Yuen B and Ma S (2004) An association between sex chromosomal aneuploidy in sperm and an abortus with 45,X of paternal origin: possible transmission of chromosomal abnormalities through ICSI. Hum Reprod 19,147–151.
Testart J, Gautier E, Brami C, Rolet F, Sedbon E and Thebault A (1996) Intracytoplasmic sperm injection in infertile patients with structural chromosome abnormalities. Hum Reprod 11,2609–2612.
Therman E, Susman B and Denniston C (1989) The nonrandom participation of human acrocentric chromosomes in Robertsonian translocations. Ann Hum Genet 53,49–65.[Web of Science][Medline]
Vegetti W, Van Assche E, Frias A, Verheyen G, Bianchi MM, Bonduelle M, Liebaers I and Van Steirteghem A (2000) Correlation between semen parameters and sperm aneuploidy rates investigated by fluorescence in-situ hybridization in infertile men. Hum Reprod 15,351–365.
Submitted on September 13, 2005; resubmitted on November 7, 2005; accepted on November 15, 2005.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  G. Kirkpatrick, K. A. Ferguson, H. Gao, S. Tang, V. Chow, B. H. Yuen, and S. Ma A comparison of sperm aneuploidy rates between infertile men with normal and abnormal karyotypes Hum. Reprod., July 1, 2008; 23(7): 1679 - 1683. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R.H. Martin Cytogenetic determinants of male fertility Hum. Reprod. Update, June 4, 2008; (2008) dmn017v1. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Anton, F. Vidal, and J. Blanco Role of sperm FISH studies in the genetic reproductive advice of structural reorganization carriers Hum. Reprod., August 1, 2007; 22(8): 2088 - 2092. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||