New insights on the origin and relevance of aneuploidy in human spermatozoa.

In humans, the most common chromosomal abnormality is aneuploidy. Because the majority of aneuploid conceptuses die during the early stages of embryonic development, an accurate estimate of the frequency of aneuploidy at conception can only be assessed by directly studying the gametes. The vast majority of aneuploidies arise de novo as a result of sporadic chromosome missegregation in paternal or maternal meiosis. In this review, we present the basic current knowledge about the incidence of aneuploidy in human spermatozoa in the general population and in patient populations where elevated levels of sperm aneuploidy are observed. These include infertile patients, patients with abnormal somatic karyotypes, and individuals exposed to certain environmental/lifestyle hazards. The clinical impact of increased levels of aneuploidy is discussed. We then focus on the non-disjunction mechanisms that cause aneuploidy during spermatogenesis and the factors that predispose to non-disjunction in male germ cells followed by an analysis of the sex differences in the incidence of gamete aneuploidy. Recent meiotic studies using multiplex-FISH on three fertile men have revealed that the frequency of conservative aneuploidy of metaphase II spermatocytes is similar to that observed in non-inseminated oocytes of young women. These findings suggest that the differences in the incidence of aneuploidy between spermatozoa and oocytes are not due to differences in chromosome segregation errors but rather to more effective checkpoint mechanisms in spermatogenesis than in oogenesis.

Aneuploidy in human spermatozoa.

We reviewed the frequency and distribution of disomy in spermatozoa obtained by multicolor-FISH analysis on decondensed sperm nuclei in (a) healthy men, (b) fathers of aneuploid offspring of paternal origin and (c) individuals with Klinefelter syndrome and XYY males. In series of healthy men, disomy per autosome is approximately 0.1% but may range from 0.03 (chromosome 8) to 0.47 (chromosome 22). The great majority of authors find that chromosome 21 (0.18%) and the sex chromosomes (0.27%) have significantly elevated frequencies of disomy although these findings are not universal. The total disomy in FISH studies is 2.26% and the estimated aneuploidy (2× disomy) is 4.5%, more than double that seen in sperm karyotypes (1.8%). Increased disomy levels of low orders of magnitude have been reported in spermatozoa of some normal men (stable variants) and in men who have fathered children with Down, Turner and Klinefelter syndromes. These findings suggest that men with a moderately elevated aneuploidy rate may be at a higher risk of fathering paternally derived aneuploid pregnancies. Among lifestyle factors, smoking, alcohol and caffeine have been studied extensively but the compounding effects of the 3 are difficult to separate because they are common lifestyle behaviors. Increases in sex chromosome abnormalities, some autosomal disomies, and in the number of diploid spermatozoa are general features in 47,XXY and 47,XYY males. Aneuploidy of the sex chromosomes is more frequent than aneuploidy of any of the autosomes not only in normal control individuals, but also in patients with sex chromosome abnormalities and fathers of paternally derived Klinefelter, Turner and Down syndromes.

Preimplantation genetic diagnosis for complex chromosome rearrangements.

Complex chromosome rearrangements (CCR) are structural rearrangements that involve more than two breakpoints. The most common ones involve three chromosomes and three breakpoints, but double translocations can also occur. Theoretically, the potential for chromosome imbalance in the gametes of CCR carriers is higher than for simple translocations, and thus they are at an even higher risk of recurrent miscarriage. Preimplantation genetic diagnosis (PGD) has been shown to significantly reduce the risk of repeated pregnancy loss in translocation carriers, and thus it is well indicated for CCR. Here we describe different approaches for PGD of CCRs. After PGD of five couples carriers of CCR, only 6.4% of embryos were found normal or balanced, but the transfer of these six embryos into four couples, resulted in two pregnancies, one with normal non-identical twins, and the other with a balanced karyotype. Thus the implantation rate was 50%, and the pregnancy per retrieval was 40%, with 0% spontaneous abortions and 0% unbalanced offspring. This compares favorably with a prior history of spontaneous abortions, unbalanced offspring, and low fertility.

Is there an interchromosomal effect in reciprocal translocation carriers? Sperm FISH studies.

Chromosome translocations have been known to affect disjunction of chromosomes unrelated to the translocation in the mouse and in Drosophila. However, in humans, an interchromosomal effect in chromosome translocations has not been demonstrated. The availability of techniques that allow the study of nondisjunction in sperm cells has permitted us to evaluate the possibility of an interchromosomal effect in male translocation heterozygotes. In this study, multicolor fluorescence in situ hybridization was used to determine levels of disomy for the clinically relevant chromosomes X, Y, 13, 18, and 21 in 332,858 spermatozoa from nine reciprocal translocation heterozygotes and nine controls with normal karyotypes. The specific translocations studied were as follows: t(10;12)(p26.1;p13.3), t(2;18)(p21;q11.2), t(3;19)(p25;q12), t(5;8)(q33;q13), t(11;22)(q23;q11), t(3;4)(p25;p16), t(8;9) (q24.2;q32), t(10;18)(q24.1;p11.2), and t(4;10)(q33;p12.2). Comparisons of disomy rates between carriers and controls were performed by using the Mann-Whitney test. Our results showed that the rates of sex chromosome hyperhaploidy were similar in controls (0.21%) and in translocation carriers (0.19%). Similarly, the frequencies of disomy for chromosomes 13, 18, and 21 did not differ significantly between controls and carriers (0.05% versus 0.08%, 0.07% versus 0.03%, and 0.14% versus 0.20%, respectively). Sex chromosome nondisjunction was more common than nondisjunction of chromosomes 13 and 18 both in controls (P=0.0057) and in carriers (P=0.0008). Similarly, the rates of chromosome disomy for chromosome 21 were higher than those for chromosomes 13 and 18 in both controls (P=0.0031) and translocation carriers (P=0.0057). In our study, the excess of chromosome 21 disomy versus disomy of the other autosomes was more pronounced in carriers than in controls. Thus, although the difference of disomy 21 between controls and carriers was not statistically significant, it is worthy of attention.

Multicolor fluorescence in situ hybridization analysis of the spermatozoa of a male heterozygous for a reciprocal translocation t(11;22)(q23;q11).

A reciprocal translocation between chromosomes 11 and 22 is a site-specific translocation that has been seen in many families with no common ancestry. This translocation is of particular interest because balanced carriers have a 0.7-3.7% risk of having children with the supernumerary der(22), resulting from a 3:1 segregation. We have used a three color fluorescence in situ hybridization (FISH) with specific DNA probes to determine the chromosome segregation pattern of a male carrier of a translocation t(11;22)(q23;q11). The probes selected included a centromeric marker for chromosome 11, a marker closely linked to the centromere of chromosome 22, and a third probe distal to the translocation breakpoint of chromosome 22. The results showed that 3:1 segregation is preferential in this patient, with 40.1% of spermatozoa belonging to this segregation type. Alternate segregation followed with 27.4% of analyzed spermatozoa; 17.6% resulted from adjacent 1 and 12.5% resulted from adjacent 2 segregation. We detected 0.5% of presumably diploid spermatozoa. Complementary adjacent 1 products were observed at statistically different frequencies (P = 0.02). Complementary adjacent 2 products without recombination in the interstitial segments were also seen at different frequencies (P = 0.002). In 3:1 segregation, the products containing one chromosome were observed more frequently than those with three chromosomes (P = 0.0001). The 24,+der(22) gamete was seen more frequently than all of the other gametes combined which had 24 chromosomes resulting from 3:1 segregation. The results of this study demonstrate that in this t(11;22) carrier, 3:1 segregation is preferential but not exclusive.

Meiotic products of a Klinefelter 47,XXY male as determined by sperm fluorescence in-situ hybridization analysis.

The meiotic segregation of 24 spermatozoa obtained from a 47,XXY male is described. Three-colour fluorescence in-situ hybridization with probes for chromosomes X, Y and 18 was used. Five spermatozoa carried an X chromosome, seven carried a Y, six had an XY gonosomal complement, five were missing the sex chromosome and one spermatozoon was presumably diploid with an XX/1818 complement. Our results support the hypothesis that XXY cells are able to complete meiosis. In this patient, the percentage of spermatozoa with an abnormal number of sex chromosomes increased from 1/6 (17%) among spermatozoa with normal morphology to 11/18 (61%) in spermatozoa with abnormal morphology.

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.

A Ph-negative chronic myeloid leukemia patient with a non-classical BCR-ABL rearrangement characterized by fluorescence in situ hybridization.

A patient with chronic myeloid leukemia (CML), a normal karyotype and a BCR-ABL rearrangement is presented. Southern blot analysis detected the rearrangement, whereas RT-PCR with b2a2 and b3a2 primers did not. Fluorescence in situ hybridization (FISH) with an ABL probe (9q34.2) and an Mbcr probe (22q11) showed ABL and BCR signals on chromosome 22. Subsequent FISH studies with cosmids mapping to 9q34.3 showed normal hybridization patterns to chromosome 9, suggesting an interstitial insertion of ABL containing DNA sequences into chromosome 22 in this patient. The lack of reciprocal translocation sequences was investigated with RT-PCR, primers a1b and c7. The absence of ABL-BCR gene expression in this and other patients described in the literature with this subtype of Ph-negative CML, does not seem to have an impact on the clinical course of the disease.

The meiotic segregation pattern of a reciprocal translocation t(10;12)(q26.1;p13.3) by fluorescence in situ hybridization sperm analysis.

The meiotic segregation of chromosomes 10 and 12 was analyzed in a male heterozygous for a reciprocal translocation, t(10;12)(q26.1;p13.3), using fluorescence in situ hybridization (FISH). Centromeric specific probes that detect alpha satellite sequences of chromosomes 10 and 12 were used. A total of 10,049 spermatozoa were analyzed. The frequencies of alternate/adjacent 1, adjacent 2, and 3:1 modes of segregation were: 84.25, 10.95%, and 4.42%, respectively. Diploidy was present in 0.23% of spermatozoa. Similar segregation patterns have been reported for this donor by direct karyotyping of sperm cells. FISH is a valuable technique for studying meiotic segregation patterns in that larger samples can be studied in a relatively short time. However, it does not provide information on the full chromosome complement of the spermatozoon.

An analysis of human sperm chromosome aneuploidy.

A sperm chromosome analysis of 24 men with normal or balanced karyotypes was carried out to study the frequency of sperm chromosome aneuploidy. A total of 3,446 human sperm complements (36-315 per donor) was analyzed after in vitro penetration of hamster eggs. Two sets of donors were studied at two different centers in the United States (center 1) and Spain (center 2). The frequencies of hyperhaploidy and hypohaploidy in control donors were similar between center 1 (1.9% vs. 7.7%) and center 2 (1.8% vs. 10.3%). In carrier donors there were no significant differences between the two centers in the frequency of hyperhaploidy (0.8% vs. 1.9%), but that of hypohaploidy was significantly higher in center 2 (11.0%) than in center 1 (4.6%). A significant excess of hypohaploid complements, as compared to hyperhaploid complements, was found in both centers in both control and carrier donors. The sex ratio was similar in both centers and did not differ significantly from a 1:1 sex ratio. The larger chromosomes in the complement (1, 2, 3, 4, 5, 7, and 10) presented a significantly lower frequency of hypohaploidy, while some of the smaller chromosomes (13, 19, and 21) showed a higher frequency of hypohaploidy than expected. Chromosome 21 and the sex chromosomes showed an increase in the percentage of hyperhaploidy, as compared to other chromosomes, that was close to statistical significance (P = 0.08). Our results reflect a preferential loss of small chromosomes during slide preparation and suggest that chromosome 21 and the sex chromosomes could be more frequently involved in aneuploidy.

Mechanisms of small ring formation suggested by the molecular characterization of two small accessory ring chromosomes derived from chromosome 4.

Molecular cloning of a microdissected small accessary ring chromosome 4 from a moderately retarded and dysmorphic patient has been performed to identify the origin of the ring chromosome. FISH was performed with cosmids identified with the cloned, microdissected products and with other markers from chromosome 4. The present study clearly demonstrates that the small ring in this patient originates from three discontinuous regions of chromosome 4: 4p13 or 14, the centromere, and 4q31. It is suggested that the origin of the ring chromosome is a ring involving the entire chromosome 4, which has then been involved in breakage and fusion events, as a consequence of DNA replication generating interlocked rings. A second severely retarded and dysmorphic patient also had a small accessary ring derived from chromosome 4. FISH studies of this ring are consistent with an origin from a contiguous region including the centromere to band 4q12. It is apparent that there are at least two mechanisms for the formation of small ring chromosomes. This adds a further complication in any attempt to ascertain common phenotypes between patients known to have morphologically similar markers derived from the same chromosome.

An analysis of human sperm chromosome breakpoints.

Sperm chromosome analysis of 19 sperm donors with either normal or balanced karyotypes was carried out in order to explore the nature of sperm chromosome structural aberrations. A total of 2,389 cells (range 36-298/donor) were karyotyped after in vitro penetration of hamster eggs. The median percentage of sperm structural aberrations was 9.3% (SD +/- 4.7; range 0%-17.8%), with a total of 247 breakpoints, of which 220 could be characterized fully. Two sets of donors were studied in two different centers: center 1 (United States) and center 2 (Spain). The frequencies of nonrejoined and rejoined chromosome-type aberrations were very similar between center 1 and center 2: 83.6% and 10.0%, and 75.0% and 10.3%, respectively. Chromatid-type aberrations were more frequent in center 2 (14.7%) than in center 1 (6.4%) (P = .037). Chromosome 4 had less than the expected number of breakpoints (P < .001). A positive significant correlation was found between sperm breakpoints reported in this study and sites of balanced chromosome de novo rearrangements detected at prenatal diagnosis and reported in the literature (P = .0001).

Segregation analysis of four translocations, t(2;18), t(3;15), t(5;7), and t(10;12), by sperm chromosome studies and a review of the literature.

We examined the meiotic segregation patterns of 444 sperm cells belonging to four reciprocal translocation carriers, t(2;18)(p21;q11.2), t(3;15)(q26.2; q26.1), t(5;7)(q13; p15.1), and t(10;12)(q26.1;p13.3). For the t(2;18) carrier, the frequencies of alternate, adjacent-1, adjacent-2, and 3:1 segregations were 41.9%, 35.2%, 14.4%, and 8.4%, respectively. For the t(3;15) carrier, the segregation pattern was 48% alternate, 36% adjacent-1, 12% adjacent-2, 2% 3:1, and 2% 4:0. One cell was the result of a 4:0 segregation. For the t(5;7) heterozygote, the corresponding segregation frequencies were 40.2%, 26.2%, 16.6%, and 17.0%. This translocation heterozygote showed a higher number of 3:1 segregations than adjacent-2 segregations, which is unusual. The t(10;12) segregations were 61.1%, 26.3%, 6.9%, and 5.6%. The percentages of chromosome abnormalities unrelated to the translocation ranged from 0% to 0.6% for aneuploidy and from 5.5% to 10.9% for structural abnormalities. These frequencies are within the ranges for control donors. Sperm chromosome data from the literature on the segregation of 30 reciprocal translocations were reviewed.

Duplication 20p identified via fluorescent in situ hybridization.

A 3-year-old girl is reported with dup (20p) resulting from 3:1 segregation of a de novo t(20;21). The proposita presented with minor anomalies, developmental delay, a clinical phenotype suggestive of 20p trisomy, and a karyotype with a 21p+ and an additional small marker chromosome. Conventional cytogenetic techniques were not informative for the identification of the origin of the extra material of chromosome 21p nor for the marker chromosome. The 21p+ and marker chromosomes were successfully characterized using fluorescent in situ hybridization (FISH).

Three unrelated cases of paracentric inversions of 1p in individuals with abnormal phenotypes.

Paracentric inversions, involving a rearrangement within one chromosome arm, are rare. Although carriers of balanced paracentric inversions should theoretically not be at risk for abnormal offspring, such cases have been reported. We report on 2 unrelated cases of inherited paracentric inversions of 1p with breakpoints at p32 and p36.1 and p32.3 and p36.22 in individuals with abnormal phenotypes. Another case of 2 abnormal monozygotic twins with a de novo paracentric inversion of 1p with breakpoints at p22 and p34 is presented as well.

Studies on sperm chromatin structure alterations and cytogenetic damage of mouse sperm following in vitro incubation. Studies on in vitro-incubated mouse sperm.

Mouse epididymal sperm incubated in Tyrode's T6 fertilization media were analyzed over time for chromosome damage by two methods. First, cytogenetic analysis was done on paternal pronuclei metaphase chromosomes. After 6 hours incubation 11% of the cells demonstrated chromosome structural abnormalities. Secondly, sperm nuclei were measured by the sperm chromatin structure assay, which is a measure of the susceptibility of sperm DNA to the nuclei demonstrated an increased susceptibility to DNA denaturation, reaching near 100% by 48 hours. Changes in chromatin structure at the molecular level may lead to chromosome breaks seen in pronuclear chromosomes.

Chromosome analysis of human spermatozoa stored in vitro.

Two-hundred-sixty-five chromosome spreads from control human sperm samples capacitated in TEST-yolk buffer at 4 degrees C and 232 chromosome spreads from sperm samples incubated in vitro in Biggers-Whitten-Whittingham (BWW) for 24 h at room temperature prior to capacitation, were studied after fusion of sperm cells with zona-free hamster eggs. Sperm cells were provided by two volunteer donors. The results indicate an increase in chromosome structural abnormalities after in-vitro incubation of the spermatozoa from 1.8 to 7.7% in donor no. 1 and from 4.5 to 12.5% in donor no. 7. Overall, structural abnormalities increased 3.3-fold. The number of aneuploid spermatozoa and the sex ratio did not change significantly. The implications of the use of different media for storing spermatozoa are discussed.

The genetic risks of in vitro fertilization techniques: the use of an animal model.

PURPOSE: The influence of some technical and biological parameters on the genetic characteristics of embryos derived from in vitro fertilization (IVF) techniques was studied. METHOD: Using a murine model, we assessed the effect of gamete manipulation, gamete maturation stage, and maternal age on the chromosome complements of first-cleavage embryos. RESULTS AND CONCLUSIONS: We found a positive correlation between some of these parameters and the incidence of the different chromosome abnormalities studied. Regarding aneuploidy, we observed an influence of maternal age, using both prepubertal and old females. Polyspermy showed a positive correlation with in vitro fertilization, the immaturity and overmaturity of the oocytes employed, and the use of prepubertal females. The appearance of diploid female complements was related to oocyte immaturity and prepubertal females, while diploid male complements were directly related to in vitro fertilization. Premature chromosome condensation (PCC) had a direct relationship with oocyte immaturity and in vitro maturation of the oocyte. Finally, structural abnormalities were associated with the process of sperm aging in vitro.

The segregation of a translocation t(1;4) in two male carriers heterozygous for the translocation.

We examined the meiotic segregation pattern of a t(1;4)(p36.2;q31.3) reciprocal translocation in two male cousins heterozygous for the translocation. The wife of subject 1 had four recognized spontaneous abortions and two carrier daughters, and the wife of subject 2 had three recognized spontaneous abortions and no live-born children. The results showed that subject 1 had an imbalance rate of 54% and subject 2 had an imbalance rate of 61% with respect to the translocation. This was not statistically different (P = 0.3174) and the 95% confidence intervals overlapped for each segregation type. The sex ratio of X- and Y-bearing sperm was not statistically different than the expected 50%. The rate of structural abnormalities was 11.3% in subject 1 and 17.8% in subject 2. Both of these values were above the range of control subjects in our lab, but only subject 2's value fell outside the 95% confidence interval for the control population.