Meiotic segregation of human sperm chromosomes in translocation heterozygotes: report of a t(9;10)(q34;q11) and a review of the literature

Meiotic segregation products were studied in sperm from a man who was heterozygous for a reciprocal translocation, t(9;10)(q34;q11). A total of 171 sperm chromosome complements were studied by in vitro fertilization of hamster eggs. All possible 2:2 and 3:1 meiotic segregations were observed with the following frequencies: alternate, 41%; adjacent-1, 48%; adjacent-2, 5%; 3:1, 6%. Within alternate segregations, the number of normal sperm (35) was not significantly different from the number of sperm carrying a balanced form of the translocation (33), as expected. The proportion of sperm with an unbalanced form of the translocation was 60%. There was no evidence for an interchromosomal effect, since the frequencies of numerical (8%) and structural (15%) chromosomal abnormalities (both unrelated to the translocation) were within the normal range of control donors. The literature on a total of 10 translocation heterozygotes studied by sperm chromosome analysis was reviewed.     

Analysis of meiotic segregation in a man heterozygous for a 13;15 Robertsonian translocation and a review of the literature

Summary Meiotic segregation was studied in a male heterozygous for a 13;15 Robertsonian translocation using in vitro sperm penetration of hamster eggs. Sixty-seven sperm chromosome complements were obtained and R-banded. Alternate segregation produced equal numbers of normal (31) and balanced (29) gametes, as was theoretically expected. Incidence of unbalanced complements was 10.4%, and the frequency of abnormalities unrelated to the translocation was 7.4%. This study confirms the predominance of alternate meiotic segregation in Robertsonian translocation carriers. Four sperm studies of Robertsonian translocation have been previously reported. A review of the combined results points out the low incidence of imbalance in the sperm of Robertsonian translocation carrier and the lack of evidence for an interchromosomal effect.     

Cytogenetics of human sperm: meiotic segregation in two translocation carriers.

Meiotic segregation products were studied in sperm from two men heterozygous for the reciprocal translocations t(8;15)(p22;q21) and t(3;16)(p23;q24). A total of 226 and 201 sperm complements, respectively, were analyzed. In each translocation, 63% of complements were unbalanced, and alternate and adjacent 1 percentages were similar. The 3:1 segregation frequencies produced by the two translocations were 3.5% and 5.0%.     

Sperm chromosome analysis of two men heterozygous for reciprocal translocations: t(1;9)(q22;q31) and t(16;19)(q11.1;q13.3).

Sperm chromosome complements were analysed in two men who were heterozygous carriers of reciprocal translocations. A total of 363 sperm were karyotyped after in vitro penetration of hamster oocytes, including 180 sperm from a male with a t(1;9)(q22;q31) and 183 from a male with a t(16;19)(q11.1;q13.3). All possible 2:2 and 3:1 meiotic segregations were observed for both translocations. The frequencies of alternate, adjacent 1, adjacent 2, and 3:1 segregations were 46%, 38%, 13%, and 4% for the t(1;9) and 40%, 28%, 31%, and 1% for the t(16;19), respectively. Within the alternate segregation group, the number of normal sperm was not significantly different from the number of sperm carrying a balanced form of the translocation for either of the translocations, as expected. There was no evidence for an interchromosomal effect of either translocation, since the frequencies of numerical abnormalities unrelated to the translocation were within the normal range observed in sperm from control donors. The percentage of sperm with an unbalanced form of the translocation was 54% for the t(1;9) and 61% for the t(16;19).     

Sperm chromosome complements from two human reciprocal translocation heterozygotes

Summary Using the hamster oocyte/human sperm fusion technique, we studied sperm chromosome complements in two male reciprocal translocation heterozygotes, 46,XY,t(11;17)(p11.2;q12.3) and 46,XY,t(1;11) (p36.3;q13.1). For the t(11;17) carrier, 202 sperm chromosome complements were obtained, but 18 karyotypes were not included in the segregation data because of multiple breaks and rearrangements. The alternate and adjacent I types, adjacent II, and 31 segregations accounted for 38.6%, 32.1%, 26.6%, and 2.7% of the sperm analyzed from the t(11;17) carrier. A total of 575 sperm chromosome complements was obtained using sperm from the t(1;11) heterozygote, and 27 karyotypes were excluded from the segregation data because of multiple breaks and rearrangements. For the t(1;11) carrier, the alternate and adjacent I types, adjacent II, and 31 segregations were responsible for 31.4%, 42.9%, 15.9%, and 8.0% of the analyzed sperm chromosome complements. Chromosomal abnormalities unrelated to the translocation, particularly the conservative estimate of aneuploidy frequency, were within the range observed in normal men. Hence, there was no evidence for an interchromosomal effect causing meiotic nondisjunction, despite the large sample sizes studied.     

Meiotic and sperm chromosome studies in a reciprocal translocation t(1;2)(q32;q36)

Summary Meiotic and sperm chromosomes were studied in a man heterozygous for a reciprocal translocation t(1;2)(q32; q36). Forty-five meiotic metaphase I cells were obtained from semen samples: 86.6% were 22,XY,IV and 13.3% had synaptic anomalies that affected all or some of the bivalents. The quadrivalents observed had a ring configuration (92.3%) or a chain configuration (7.7%). A total of 105 sperm chromosome complements were analyzed: 41% resulted from an alternate segregation, and the percentage of unbalanced sperm was 59%; most of them (71%) resulted from an adjacent 1 segregation. The frequency of anomalies unrelated to the translocation (5.7% numerical and 14.1% structural anomalies) were within the normal range for control donors. There was a good correspondence between the percentage of cells with a ring IV (92.3%) and the proportion of 2:2 segregations (88.6%) and between the percentage of chain IV (7.7%) and the incidence of 3:1 segregations (11.4%).     

Cytogenetic analysis of sperm from a male heterozygous for a 13;14 Robertsonian translocation

Summary Cytogenetic analysis of 121 sperm from a man heterozygous for a t(13;14) Robertsonian translocation was performed using the technique of in vitro penetration of hamster eggs. The frequency of sperm that were chromosomally unbalanced with respect to the translocation was 27%. The frequency of chromosomally normal (36%) and balanced (38%) complements was approximately equal, as theoretically expected. There was no evidence for an interchromosomal effect since the frequency of numerical chromosomal abnormalities (2.5%) and structural chromosomal abnormalities (10.7%) — both unrelated to the translocation — were within the normal range of control donors. The ratio of X-and Y-chromosome bearing sperm was equal, and there was no evidence for preferential segregation of the X chromosome with the translocation.     

Meiotic segregation, recombination, and gamete aneuploidy assessed in a t(1;10)(p22.1;q22.3) reciprocal translocation carrier by three- and four-probe multicolor FISH in sperm.

Meiotic segregation, recombination, and aneuploidy was assessed for sperm from a t(1;10)(p22.1;q22.3) reciprocal translocation carrier, by use of two multicolor FISH methods. The first method utilized three DNA probes (a telomeric and a centromeric probe on chromosome 1 plus a centromeric probe on chromosome 10) to analyze segregation patterns, in sperm, of the chromosomes involved in the translocation. The aggregate frequency of sperm products from alternate and adjacent I segregation was 90.5%, and the total frequency of normal and chromosomally balanced sperm was 48.1%. The frequencies of sperm products from adjacent II segregation and from 3:1 segregation were 4.9% and 3.9%, respectively. Reciprocal sperm products from adjacent I segregation deviated significantly from the expected 1:1 ratio (P < .0001). Our assay allowed us to evaluate recombination events in the interstitial segments at adjacent II segregation. The frequencies of sperm products resulting from interstitial recombination in chromosome 10 were significantly higher than those resulting from interstitial recombination in chromosome 1 (P < .006). No evidence of an interchromosomal effect on aneuploidy was found by use of a second FISH method that simultaneously utilized four chromosome-specific DNA probes to quantify the frequencies of aneuploid sperm for chromosomes X, Y, 18, and 21. However, a significant higher frequency of diploid sperm was detected in the translocation carrier than was detected in chromosomally normal and healthy controls. This study illustrates the advantages of multicolor FISH for assessment of the reproductive risk associated with translocation carriers and for investigation of the mechanisms of meiotic segregation of chromosomes.     

Sperm chromosome analysis of two males heterozygous for a t(2;17)(q35;p13) and t(3;8)(p13;p21) reciprocal translocation

Summary Sperm chromosome complements from two males, one heterozygous for the reciprocal translocation t(2;17)(q35;p13) (n = 18) and one for t(3;8) (p13;p21) (n = 73), were analyzed. Only 2:2 segregations were observed with t(2;17): alternate, 56%; adjacent-I, 33%; adjacent-II, 11%. Both 2:2 and 3:1 meiotic segregations occurred in t(3;8): alternate, 34.2%; adjacent-I, 43.8%; adjacent-II, 20.5% and 3:1, 1.4%. A significant excess of chromosomally normal versus balanced sperm complements was observed with both translocation heterozygotes. The frequencies of other chromosome aberrations unrelated to the translocations were 16.7% for t(2;17) and 8.2% for t(3;8). The ratio of X-bearing to Y-bearing sperm was not different from the theoretically expected ratio of 1:1.     

Analysis of the chromosome complement in outbred mouse sperm fertilizing in vitro

The chromosome complements in a population of mouse sperm from random-bred ICR donors were analyzed at first-cleavage metaphase after in vitro fertilization (IVF) of oocytes from females of the same strain. The sperm were aged as donations occurred within an average of 31 days, either since last mating or at arrival at the animal facility in the case of virgin males. Of a total of 598 sperm complements studied from 22 sexually mature males aged 10–26 weeks old, there was one diploid complement (0.17%). The frequencies of hyperhaploidy and structural aberrations that were studied in 338 complements were 4.4% and 3.6%, respectively, giving an overall frequency of 8.0%. The hyperhaploid complements consisted of n + 1, n + 2, n + 3, and n + 7 counts, while the structural abnormalities were of the chromosome type and included large and small fragments and a possible translocation. This is the highest frequency of sperm chromosome abnormalities reported for mouse sperm obtained from males under physiological conditions and fertilized in vitro or in vivo. Sperm aging, strain, and/or technique differences are among the factors that may be responsible for this high frequency. Since the 8.0% frequency of hyperhaploidy and structural abnormalities is similar to the frequency reported for human sperm after IVF, the outbred murine in vitro fertilization system may be a useful model to study the origin of human sperm chromosome abnormalities.     

The murine Rb(6.16) translocation: evidence for sperm selection and a modulating effect of aging

Summary The segregation products of the Rb(6.16) translocation were studied at first cleavage metaphase. Male mice heterozygous for the translocation mated at 3- and 14-day intervals to superovulated random-bred ICR females. Chromosome preparations of the recovered one-cell embryos were sequentially G- and C-banded and male and female complements analyzed cytogenetically. Of the 309 zygotes analyzed from both mating groups, no unbalanced segregants of the translocation were observed. In the 3-day group there was a highly significant difference (P<0.001) from the expected 1:1 ratio between sperm with normal (70.5%) and balanced segregants (26.2%) of alternate segregation. In the 14-day group the level of significance for the difference was ten times lower (P<0.01) as normal segregants were observed in 56.4% of the sperm and balanced ones in 36.5%. A comparison of the two groups using a 2×2 contingency table showed that segregant type was related to mating frequency (P<0.05). There was a highly significant distortion (P<0.01) of the sex ratio, with 178 Y-bearing and 131 X-bearing sperm in the combined populations. When sex ratio was analyzed according to mating intervals, the distortion was significant (P<0.01) only for the 3-day group. An analysis of the sex ratio according to the segregant type showed no significant deviation from 1:1 for type 1 segregants, which had normal chromosomes, while type 2 segregants, with the translocation, had a deficiency of X-bearing sperm. This deficiency was significant (P<0.05) only for the 3-day population. Analysis of meiotic preparations showed no association between the translocation trivalent and the X-Y bivalent. Our results, obtained under physiological conditions, provide definitive evidence for sperm selection and support previous findings that aging of sperm can modify the effect of selection.     

Meiotic products of two reciprocal translocations studied by multicolor fluorescence in situ hybridization

The sperm products of two male carriers of reciprocal translocations were studied by fluorescence in situ hybridization (FISH) using a combination of three probes for each translocation. One patient carried a t(2;18)(p21;q11.2), the other a t(8;9)(q24.2;q32). The probes selected included a centromeric marker for each chromosome involved in the translocation plus a third probe distal to the translocation breakpoint of one of the translocation chromosomes. This assay identifies alternate, adjacent 1, adjacent 2, and 3:1 types of meiotic products. It allows the identification of recombination events and also estimation of the frequency of diploidy. For the t(2;18), the frequency of normal and balanced sperm and of adjacent 1, adjacent 2, and 3:1 products was 43.6%, 29. 8%, 10.5%, and 12.8%, respectively. Similar segregation patterns had been reported for this donor by direct sperm karyotyping of sperm cells. For the t(8;9), the frequency of normal and balanced sperm and of adjacent 1, adjacent 2, and 3:1 products was 44.4%, 41%, 3.1%, and 9.4%, respectively. The frequency of complementary adjacent 1 products was statistically different in both the t(2;18) (P < 0. 0001) and the t(8;9) (P < 0.0001) carrier. When the number of adjacent 2 products with one translocation chromosome (regardless of normal or derivative) was compared to the number of adjacent 2 products with the second translocation chromosome (again, regardless of normal or derivative), no statistical difference was noted for either the t(2;18) (P = 0.32) or the t(8;9) (P = 0.69). Recombination events within the interstitial segment of chromosome 2 were statistically higher than those seen in chromosome 18 (P < 0. 0001), whereas in chromosomes 8 and 9, recombination in the interstitial segments was similar (P = 0.64). The rate of diploidy was similar in both the t(2;18) (0.5%) and the t(8;9) (0.6%). Thus, FISH provides chromosome information on the sperm products produced by translocation carriers, although it cannot provide an assessment of the full chromosome complement of the spermatozoon.     

Meiotic segregation analysis of a 14;21 Robertsonian translocation carrier by fluorescence in situ hybridization

Meiotic segregation of chromosomes 14 and 21 in sperm from a 14;21 Robertsonian translocation carrier was analyzed with dual-color FISH using two locus-specific DNA probes (Tel 14q and LSI 21). The frequency of normal or chromosomally balanced sperm, resulting from alternate segregation, was 88.42%. The frequency of unbalanced sperm, resulting from adjacent segregation, was 11.25%. These observed frequencies deviated significantly from the theoretical frequencies (33.33% and 66.67%, respectively) based on random chromosome segregation, with sperm resulting from alternate segregation being preferentially produced in the translocation carrier. With respect to the chromosomally unbalanced sperm, the frequency of 21q disomic sperm was 2.45%, which is in agreement with the frequencies of unbalanced fetuses or offspring at the time of amniocentesis or at term (0-4.3%) reported by others. Although the frequency of 14 or 21 nullisomic sperm should be theoretically equal to that of 14q or 21q disomic sperm in both the carrier and controls, the frequency of nullisomic sperm was significantly higher than that of disomic sperm in the carrier (P=0.0009 for chromosome 14, P<0.0001 for chromosome 21) but not in the controls (P=0.091 for chromosome 14, P=0.74 for chromosome 21). This evidence suggests the occurrence of maturation arrest during spermatogenesis of the carrier.     

FISH on sperm heads allows the analysis of chromosome segregation and interchromosomal effects in carriers of structural rearrangements: results in a translocation carrier, t(5;8)(q33;q13)

Using three-color fluorescence in situ hybridization (FISH) with specific DNA probes, we have determined the chromosome segregation pattern of the translocated chromosomes in a human male carrier of a reciprocal translocation, t(5;8)(q33;q13). At the same time, we have assessed the possible interchromosomal effect on pair 21 using dual-color FISH. The segregation results showed that a 45.12% of the spermatozoa analyzed resulted from alternate segregation, 38.31% resulted from adjacent I, 6.97% from adjacent II, and 6.56% from 3:1 segregation. Finally, 1.23% could be either diploid sperm or 4:0 segregation. In both types of adjacent segregations, an excess of products containing short translocated segments (adjacent I) and interstitial regions (adjacent II) were found. Products resulting from the presence of an interstitial chiasma in pair 5 (1.26%), were found much more frequently (P < 0.0001) than those resulting from an interstitial chiasma in pair 8 (0.13%) (evaluated after adjacent II segregation). In 3:1 segregation, the products containing one chromosome were observed more frequently than those containing three chromosomes (P < 0.0001). No evidence of an interchromosomal effect on chromosome 21 was detected, the percentage of disomy 21 being similar to that in the controls (0.23% vs. 0.37%). However, the percentage of diploid sperm (1.18%) was significantly higher (P < 0. 0001) than that in the controls (0.27%). FISH therefore appears to be a useful technique for assessing the percentage of abnormal sperm in translocation carriers. Their application in assisted reproduction centers could offer patients more accurate genetic counseling.     

Chromosome analysis of spermatozoa from a male heterozygous for a 13;14 Robertsonian translocation

Summary Cytogenetic analysis of 78 spermatozoa from a man heterozygous for a t(13;14) Robertsonian translocation was performed. R banding was applied for chromosomal identification. Incidence of normal and balanced complements were respectively 50% and 41.3%. Six unbalanced complements (7.7%) were observed, resulting from adjacent segregation. Although alternate segregation is the most common mode of distribution, the possibility of producing unbalanced zygotes exists. The frequency of abnormalities unrelated to the translocation was 16.5% including 12.8% hypohaploïdy, 2.5% hyperhaploidy, and 1.2% of structural aberrations. An excess of t(13;14) X complements was observed (24 with X versus 14 with Y). This may result from the close association between trivalent (13;14) and X chromosome observed in the pachytene spermatocyte nucleus.     

Cytogenetic analysis of 400 sperm from three translocation heterozygotes

Summary Sperm chromosome complements were studied in three men who carried reciprocal translocations. A total of 400 sperm were karyotyped after in vitro penetration of hamster eggs: 217 sperm from t(2;9) (q21;p22), 164 from t(4;6) (q28;p23) and 19 from t(7;14) (q21;q13). All possible 22 and 31 meiotic segregations were observed for t(2;9) and t(4;6); for t(7;14) only 22 segregations were observed. For alternate segregations, the number of normal sperm was not significantly different from the number of sperm carrying a balanced form of the translocation in any of the translocations, as theoretically expected. The percentage of sperm with an unbalanced form of the translocation was 57% for t(2;9), 54% for t(4;6) and 47% for t(7;14). There was no evidence for an interchromosomal effect in any of the translocations since the frequencies of numerical abnormalities (unrelated to the translocation) were within the normal range of control donors. The frequencies of X- and Y-bearing sperm did not differ significantly from 50%. Results from a total of 17 reciprocal translocations studied by sperm chromosomal analysis were reviewed.     

Evidence for differential maturation of reciprocal sperm segregants in the murine Rb(6.16) translocation heterozygote.

The fertilizing ability of unaged sperm and those aged experimentally in the cauda by surgically ligating the corpus epididymis in males carrying the Rb(6.16) translocation was studied. Chromosomally normal females were inseminated with unaged sperm delivered by males mating at 3-day intervals, and aged sperm were studied after matings on 6-14 postoperative days. The sperm chromosome complement was analyzed in first-cleavage metaphase zygotes after sequential G- and C-banding of the chromosomes. Of 283 metaphasic zygotes in the control group, 183 (or 64.7%) were analyzed and showed a ratio of 2.7:1 for chromosomally normal and balanced segregants of the translocation, deviating significantly (P less than 0.001) from the expected 1:1. The ratio of X- to Y-bearing sperm also deviated from expected (P less than 0.01) mostly due to a significant deficiency (P less than 0.05) of balanced sperm that were X-bearing. Fertilized oocytes were recovered from matings of 10 males on days 6-8 postoperatively, and, of 139 metaphasic one-cell zygotes, 101 (or 72.3%) were analyzed. These showed a Mendelian ratio of 1:1 for normal and balanced segregants. The sex ratio in the aged group (57Y:41X) also showed no deviation from 1:1. The results, which reveal significant physiological distortions for both the segregation and the sex ratios in males heterozygous for the Rb(6.16) translocation, suggest that differential maturation of the translocation-bearing sperm and the chromosomally normal reciprocal exists. The findings further support the concept that sperm chromosomal complement affects their maturation and function, and that factors on chromosome 6 and the X or Y chromosome additively affect sperm function.     

Chromosomal breakage in human spermatozoa, a heterozygous effect of the Bloom syndrome mutation.

The chromosome complements of 662 spermatozoa produced by the three fathers of individuals with Bloom syndrome (BS) were analyzed to determine whether the BS mutation could affect chromosome segregation and the frequency of aneuploidy in sperm. The frequency of numerical abnormalities was not significantly different from that in normal controls studied in our laboratory, but the frequencies of structural abnormalities were significantly increased in two of the men, 14.3% and 15.9%, versus 8.6% in controls. More striking was the increase in these two men of cells with multiple structural abnormalities: 8.1% and 6.7% with multiple abnormalities, versus 2.3% in controls.     

Chromosome segregation into the spermatozoa of two men heterozygous for different reciprocal translocations

Sperm chromosomes from two human males, each heterozygous for a different reciprocal translocation, were examined. Chromosomally normal sperm were found in equal numbers to those carrying the translocation in the balanced form, in both males. Alternate segregation was more common than adjacent segregation in both translocations. Male W. G. had a greater proportion of sperm containing chromosome abnormalities unrelated to the translocation than did J.S., the second made studied. J.S. however, had a greater frequency of chromosomally unbalanced sperm. The great majority of unbalanced sperm in both males was due to adjacent I segregation.     

Chromosome segregation in a man heterozygous for a pericentric inversion, inv(9)(p11q13), analyzed by using sperm karyotyping and two-color fluorescence in situ hybridization on sperm nuclei

Analysis of sperm karyotypes and two-color fluorescent in situ hybridization (FISH) on sperm nuclei were carried out in a man heterozygous for the pericentric inversion inv(9)(p11q13). Sperm chromosome complements were obtained after in vitro fusion of zona-free hamster oocytes and donor sperm. A total of 314 sperm complements was analyzed: 153 (48.7%) carried the inverted chromosome 9 and 161 (51.3%) carried the normal one. None of the sperm complements contained a recombinant chromosome 9, suggesting that no chiasmata were formed in the heterochromatic region. The frequency of structural chromosome aberrations unrelated to the inversion (8.3%) and the frequency of conservative aneuploidy (3.2%) were within the limits observed in our control donors. The proportions of X-bearing (47.3%) and Y-bearing sperm (52.7%) were not significantly different from the expected 1:1 ratio. The percentage of disomy for chromosome 21 was analyzed by two-color FISH in 10 336 sperm nuclei. The disomy rate for chromosome 21 (0.30%) was not significantly different from that found in our controls. These results suggest that the risk for this man of producing chromosomally abnormal offspring or spontaneous abortions was not increased, and do not support the existence of an interchromosomal effect for chromosome 21. Received: 28 October 1996