Comparison of chromosomal abnormalities in hamster egg and human sperm pronuclei

One thousand human sperm and hamster egg haploid karyotypes were analyzed at the pronuclear stage after in vitro penetration. The frequency of abnormalities in human sperm was 8.5%, with 5.2% aneuploidy and 3.3% structural abnormalities. The hamster egg complements had an abnormality rate of 3.8%, with 3.3% aneuploidy and 0.5% structural abnormalities. In both human and hamster complements, chromosome abnormalities were observed in all chromosome groups, demonstrating that all chromosomes are susceptible to nondisjunction, not just acrocentric or small chromosomes. There is an intriguing difference between the frequency of hyperhaploid and hypohaploid complements in human sperm and hamster eggs. In the human complements, 2.4% were hyperhaploid and 2.7% hypohaploid. This is very close to the theoretical 1 to 1 ratio expected from nondisjunction. The hamster egg complements had more hypohaploid (2.2%) than hyperhaploid (0.9%) complements, despite identical treatment. Higher rates of hypohaploidy are generally ascribed to artificial loss of chromosomes, but may in fact reflect a predisposition of oocytes to anaphase lag during meiosis. The frequency of abnormalities (both numerical and structural) is higher in human complements than in hamster. This may reflect an innate propensity for meiotic chromosome abnormalities in humans or may result from greater exposure of humans to mutagenic agents.     

Cytogenetic analysis of Q-banded pronuclear chromosomes in fertilized Syrian hamster eggs

The frequency and type of chromosome abnormalities were analyzed in 917 female pronuclei in Syrian hamster eggs fertilized by human sperm. Analysis at this stage allows detection of errors which have occurred during meiosis I and II. The chromosomes were Q-banded to identify individual chromosomes and detect subtle alterations. Thirty-three (3.6%) of the hamster egg complements were abnormal: 19 (2.1%) were hypohaploid, seven (0.76%) were hyperhaploid, two (0.2%) had double aneuploidy, and five (0.5%) had a structural chromosome abnormality. Since there were significantly more hypohaploid than hyperhaploid complements, a conservative estimate of aneuploidy can be derived by doubling the frequency of hyperhaploid complements. Thus a minimal estimate of aneuploidy (single, 1.5%, and double, 0.2%) is 1.7% and a minimal estimate of the total frequency of abnormalities is 2.2%. All chromosome groups were represented among the aneuploid complements suggesting that all chromosomes are susceptible to non-disjunction.     

Effect of cryopreservation on the frequency of chromosomal abnormalities and sex ratio in human sperm

The effects of cryopreservation on the frequency and type of chromosomal abnormalities in human sperm were investigated. Employing a technique that enables direct visualization of human sperm chromosomes following in vitro penetration of hamster oocytes, sperm samples from 10 normal men were examined before and after freezing in liquid nitrogen. A total of 1,960 sperm karyotypes were analyzed, 1,132 before freezing and 828 after freezing. There was no significant difference in the frequency of structural chromosomal anomalies (10.5% prefreeze vs. 8.5% postfreeze), but there was a significant decrease in the frequency of numerical abnormalities (5.2% prefreeze vs. 3.0% postfreeze). However, there was a large excess of hypohaploid complements compared with hyperhaploid complements, suggesting that the hypohaploid complements were caused by technical artefact. A conservative estimate of aneuploidy, derived by doubling the hyperhaploid frequencies, did not differ before (0.4%) and after (0.4%) freezing. There was no evidence for interdonor variability in response to sperm cryopreservation for total chromosomal abnormalities, structural abnormalities, and sex ratios. The sex ratios were also not affected by cryopreservation and did not differ significantly from the theoretical 50%. It is concluded that cryopreservation does not affect the frequencies of chromosomal abnormalities or alter the sex ratio in human sperm, provided that an adequate cryoprotective buffer and freezing system is employed.     

The effect of age on the frequency of sperm chromosomal abnormalities in normal men.

It has been suggested that advanced paternal age (independent of maternal age) is associated with an increased incidence of trisomy. However, studies of human liveborn offspring and of data from prenatal diagnosis have yielded conflicting results. To investigate this possible paternal age effect, we have studied sperm chromosome complements from 30 normal men of proven fertility stratified by age, with five males in each of six age categories (20-24, 25-29, 30-34, 35-39, 40-44, and 45+ years). Sperm chromosome complements were visualized after penetration of golden-hamster oocytes. A minimum of 30 complements were analyzed for each male. The analysis was performed blindly, without knowledge of the donor's age. The mean frequency of sperm chromosomal abnormalities in the individual men was 10.4% with means of 4.7% for numerical abnormalities and 6.2% for structural abnormalities. There was no relationship between age and the frequency of numerical abnormalities in sperm. Since there was a significant difference between the frequency of hyperhaploid and hypohaploid complements, these two types of numerical abnormalities were analyzed separately. There was no correlation between the frequency of hypohaploid complements and age. There was a significant negative correlation between age and the frequency of hyperhaploid complements. For structural abnormalities, there was a highly significant positive correlation with age. Thus, our results do not support the hypothesis of an increased risk of trisomy with paternal age.     

The frequency of aneuploidy among individual chromosomes in 6,821 human sperm chromosome complements

The human sperm/hamster egg fusion technique has been used to analyse 6,821 human sperm chromosome complements from 98 men to determine if all chromosomes are equally likely to be involved in aneuploid events or if some chromosomes are particularly susceptible to nondisjunction. The frequency of hypohaploidy and hyperhaploidy was compared among different chromosome groups and individual chromosomes. In general, hypohaploid sperm complements were more frequent than hyperhaploid complements. The distribution of chromosome loss in the hypohaploid complements indicated that significantly fewer of the large chromosomes and significantly more of the small chromosomes were lost, suggesting that technical loss predominantly affects small chromosomes. Among the autosomes, the observed frequency of hyperhaploid sperm equalled the expected frequency (assuming an equal frequency of nondisjunction for all chromosomes) for all chromosome groups. Among individual autosomes, only chromosome 9 showed an increased frequency of hyperhaploidy. The sex chromosomes also showed a significant increase in the frequency of hyperhaploidy. These results are consistent with studies of spontaneous abortions and liveborns demonstrating that aneuploidy for the sex chromosomes is caused by paternal meiotic error more commonly than aneuploidy for the autosomes.     

Chromosomal analysis of unfertilized human oocytes

Unfertilized human oocytes were obtained from women in an in-vitro fertilization programme. The women had a mean age of 29.4 years (range 24-35 years). Chromosomal complements could be analysed in 50 oocytes. Q-banding of the chromosomes facilitated identification of individual chromosomes: 34 oocytes (68%) had the normal haploid chromosomal complement, 14 complements were hypohaploid (28%), 1 complement was hyperhaploid (2%) and 2 had structural abnormalities (4%). (One oocyte had numerical and structural abnormalities). The 16 abnormal oocytes were obtained from 15 different women. A conservative estimate of aneuploidy in this sample is 4%; however, the frequency of aneuploidy may be higher if there is a predisposition to chromosome loss during oogenesis. This study provides information on the largest series of karyotyped unfertilized human oocytes published to date.     

Cytogenetics of human oocytes,zygotes, and embryos after in vitro fertilization

Summary Chromosome errors, inherited or arising de novo during gametogenesis and transmitted at fertilization to the conceptus, may be a major cause of embryonic mortality. The in vitro fertilization and embryo transfer (IVF/ET) procedure provides extra material — oo-cytes, zygotes, and embryos — to investigate the contribution of chromosomal abnormality to implantation failure. This paper reviews the results of cytogenetic studies on such material. Estimates from a total of 1120 oocytes from 11 studies give an overall proportion of chromosomal abnormality of 35%. Single and multiple nullisomies and disomies are found, involving nonrandom chromosome gain or loss. Hypohaploid complements are more frequent than hyperhaploid complements. The higher rate of chromosome loss of hypohaploid karyotypes was found to be largely artifactual. The estimated overall frequency of aneuploidy is 13%. In embryos the level of chromosomal abnormality is 23%–40%. Errors of fertilization are responsible for a substantial number of triploid embryos, many of which develop into mosaics. Factors extrinsic to the conceptus, such as infertility, advanced maternal age, and ovarian hyperstimulation, may increase the level of chromosomal abnormality. More refined methods for accurately recognizing and selecting chromosomally normal embryos for transfer are needed to improve the success rate of this reproductive technology.     

The chromosome constitution of 1000 human spermatozoa

Summary Chromosomal analysis of 1000 spermatozoa from 33 normal men was performed using in vitro fertilization of zonafree golden hamster eggs. The frequency of abnormal sperm complements was 8.5%: 5.2% were aneuploid and 3.3% had a structural chromosome abnormality. The frequencies of hyperhaploid (2.4%) and hypohaploid (2.7%) sperm complements were not significantly different and all chromosome groups were represented among the aneuploid complements. The majority (22/33) of structurally abnormal complements had a chromosome break. The percentages of X and Y-bearing sperm were 53.9% and 46.1%, which is significantly different from the expected one to one ratio.     

A cytogenetic investigation of the effects of cryopreservation on human sperm.

The effects of cryopreservation on the frequency and type of chromosome abnormalities in human sperm have been investigated for the first time. With a technique which enables direct visualization of human sperm chromosomes following in vitro penetration of hamster oocytes, sperm samples from 13 normal men were examined before and after being frozen in liquid nitrogen. The overall abnormality frequencies of 17.8% for fresh semen and 13.4% for previously frozen semen were not significantly different (chi 2(1) df = 3.04, p = 0.08). When specific abnormality types were analyzed, only the category of hypohaploidy was significantly different (chi 2(1) df = 6.75, p = 0.009) before (7.5%) and after (3.4%) freezing. Hypohaploidy was significantly higher than hyperhaploidy both prefreeze and postfreeze, and chromosome loss was random. Because the observed excess of hypohaploid cells may be attributable to technical artifact, the aneuploidy levels were estimated by doubling the number of hyperhaploid cells. Neither the adjusted numerical abnormality frequencies (1% prefreeze vs. 0% postfreeze) nor the overall abnormality frequencies (11.8% prefreeze vs. 10.4% postfreeze) were significantly different. The types and distributions of karyotypically abnormal sperm complements (numerical, structural, or combined) observed before and after freezing were not different. Interdonor variability in sperm chromosome abnormality frequencies and a possible donor-dependent response to cryopreservation were suggested by the data. The sex ratios were not affected by cryopreservation and did not differ significantly from the theoretical 50%. It is concluded that cryopreservation does not affect the type or frequencies of chromosome abnormalities or alter the sex ratio in human sperm.     

Is there a relationship between sperm chromosome abnormalities and sperm morphology?

This review explores the relationship between sperm chromosomal constitution and morphology. With the advent of techniques for obtaining information on the chromosome complements of spermatozoa, this relationship has been studied in fertile men and in men with a high frequency of chromosomal abnormalities. Using human sperm karyotype analysis, no relationship between sperm chromosome abnormalities and morphology was found in fertile men, translocation carriers or post-radiotherapy cancer patients. Fluorescence in situ hybridization (FISH) analysis has not generally revealed a specific association between morphologically abnormal sperm and sperm chromosome abnormalities, but has indicated that teratozoospermia, like other forms of abnormal semen profiles (aesthenozoospermia, oligozoospermia) is associated with a modest increase in the frequency of sperm chromosome abnormalities. However, FISH studies on some infertile men and mouse strains have suggested that certain types of morphologically abnormal spermatozoa, such as macrocephalic multitailed spermatozoa, are associated with a very significantly increased frequency of aneuploidy. Thus, there may be an association between sperm morphology and aneuploidy in infertile men with specific abnormalities.     

An increased frequency of human sperm chromosomal abnormalities after radiotherapy

13 cancer patients were studied before radiotherapy (RT) and at regular intervals after RT to determine the effect of RT on chromosomal abnormalities in sperm. The men were 19-47 years old and received testicular radiation doses of 0.4-5.0 Gray. Human pronuclear sperm chromosomes were analysed after penetration of zona-pellucida-free hamster eggs. Unfortunately the hamster egg penetration rates were exceedingly low, both before and after RT and this limited the number of sperm chromosome complements which could be analysed. Before RT, the frequency of abnormal sperm chromosome complements was 0% (0/9). After RT, the majority of men were azoospermic for 24 months but complements could be analysed from 4 men. In the first 12 months the frequency of abnormalities was 13% (1/8) and at 24 months it was 13% (7/55). By 36 months after RT, most men had recovered sperm production and the frequency of abnormalities in 8 men was 21% (18/86), which is significantly higher than the rate in control donors (8.5%). For individual men the range was 6-67%, and there was a significant correlation between testicular radiation dose and the frequency of sperm chromosomal abnormalities. The frequencies of both numerical and structural abnormalities were significantly increased after RT. This is the first evidence that radiation may increase the frequency of chromosomal abnormalities in human gametes.     

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.     

Chromosome analysis of human sperm

Summary A modified technique has been developed for the visualization of the chromosomes in human sperm. The cytogenetic analysis of 129 G-banded human sperm metaphases of 6 normal donors showed an incidence of structural and numerical chromosome abnormalities of 7.8%. Two out of 129 spermatozoa were aneuploid (1.6%). The frequency of sperms with chromatid-type aberrations was 2.3% (3/129). Chromosome-type aberrations were found in 5 out of 129 (3.9%) spermatozoa. X to Y ratio did not differ significantly from the expected one-to-one ratio. Twenty-six sperm complements from a patient 18–20 months after testes exposure to 30 Gy were examined. A significant increase of numerical and structural chromosome abnormalities was not observed. Chromatidtype aberrations were found in two sperm complements (7.7%) and chromosome-type aberrations in one sperm complement (3.9%). The cytogenetic analysis of 15 human sperms from a cancer patient 26 months after chemotherapy showed an increased frequency of aberrant sperm complements (33.4%). One chromatid-type (6.7%), three chromosometype aberrations (20.0%) and one (6.7%) hyperploid sperm complement could be observed. The sample size is still too small to answer the question whether chemical mutagens may increase the frequency of chromosomal abnormalities in human sperm.     

Cytogenetic studies in motile sperm from normal men

Summary Most studies on human sperm chromosomes from normal men involve the heterologous fertilization of zona free hamster eggs by unselected human sperm. In this work, we have performed cytogenetic studies of highly motile sperm, selected by a swim-up method. A total of 505 motile human sperm complements from three normal donors was analysed. The total frequency of sperm with chromosomal abnormalities (10.9%; 6.9% structural aberrations and 4.0% aneuploidy) and the sex ratio (50.4% X49.6% Y) were similar to those obtained from whole semen samples. Our results indicate that the selection of motile sperm does not imply chromosomal selection.     

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.     

Human sperm chromosome complements in chemotherapy patients and infertile men

Our studies of human sperm karyotypes and interphase sperm analyzed by fluorescence in situ hybridization (FISH) have both yielded estimates of disomy frequencies of approximately 0.1% per chromosome with an overall aneuploidy frequency in human sperm of approximately 5%–6%. However, the distribution of aneuploidy in sperm is not even, as our data from sperm karyotypes and multicolour FISH analyses both demonstrate a significant increase in the frequency of aneuploidy for chromosome 21 and the sex chromosomes. We have studied men at increased risk of sperm chromosomal abnormalities including cancer patients and infertility patients. Testicular cancer patients were studied before and 2–13 years after chemotherapy (CT) with BEP (bleomycin, etoposide, cisplatin). Sperm karyotype analysis on 788 sperm demonstrated no significant difference in the frequency of numerical or structural chromosomal abnormalities post-CT vs pre-CT. Similarly, multicolour FISH analysis for chromosomes 1, 12, XX, YY and XY in 161,097 sperm did not detect any significant differences in the frequencies of disomy before and after treatment. However, recent evidence has suggested a significant increase in the frequency of disomy and diploidy during CT. We have found that infertile men, who would be candidates for intracytoplasmic sperm injection, have an increased frequency of chromosomally abnormal sperm karyotypes. Also, FISH analysis for chromosomes 1, 12, 13, 21, XX, YY and XY in 255,613 sperm demonstrated a significant increase in chromosomes 1, 13, 21, and XY disomy in infertile men compared with control donors. Received: 4 July 1998; in revised form: 7 September 1998 / Accepted: 8 September 1998     

Chromosome aberrations in 450 sperm complements from eight controls and lack of increase after chemotherapy in two patients

Summary Four hundred fifty sperm complements from eight controls were analyzed. A conservative estimate of aneuploidy was 1.8% with a hyperhaploid rate of 0.9% (4/450). The overall frequency of structural aberrations was 8.9% (40/450). The proportion of X-bearing (47.5%) and Y-bearing (52.5%) sperm did not differ significantly. Sperm complements were analyzed from a cancer patient 9 months after poly chemotherapy (n = 63) and from a patient being treated with Imurek (azathioprine) (n = 30). There was no significant increase in the incidence of numerical and structural chromosome aberrations in the sperm of either patient. The percentages of X-bearing and Y-bearing sperm were not significantly different from the expected 50%.     

Chromosome abnormalities in sperm from infertile men with normal somatic karyotypes: oligozoospermia

Recently, intracytoplasmic sperm injection (ICSI) has been extremely successful for the treatment of male infertility. However, transmission of cytogenetic defects to offspring is a great concern. There are two types of cytogenetic problems in patients seeking ICSI; one is the transmission of genetic defects from patients with constitutional chromosomal abnormalities and the second is the generation of de novo defects in infertile men. Generally about 5.1% of infertile men have chromosomal abnormalities. Among such infertile men, men with severe spermatogenesis defects, including oligozoospermia and azoospermia, are subjects for ICSI. Therefore it is very important to obtain cytogenetic information in these infertile patients. Furthermore, oligozoospermic men with a normal somatic karyotype also have increased frequencies of sperm chromosome abnormalities. Oligozoospermia is usually associated with other sperm alterations, for example oligoasthenozoospermia, oligoteratozoospemia and oligoasthenoteratozoospermia. In this review, the relationship between sperm concentration and sperm aneuploidy frequencies has been analyzed. The inverse correlation between the frequency of sperm aneuploidy and concentration has been reported in extensive studies. Especially in severe oligozoospermia, a significantly higher frequency of sex chromosome aneuploidy has been observed and this has been corroborated in recent clinical outcome data of ICSI.     

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.     

Aneuploidy frequency in sperm of fertile men

Analysis of sperm aneuploidy in 11 healthy men using two-or three-color FISH permitted to determine the average frequency of disomy for chromosomes 13 and 21 (0.11% and 0.2%, respectively), disomy for chromosome 18 (0.05%) and to reveal gonosomal aneuploidy variants and their frequency. The frequency of XX disomy was 0.04%; XY, 0.17%; YY, 0.06%; and gonosomal nullisomy, 0.29%. We assessed the frequency of meiotic nondisjunction of 13, 21, 18, X, and Y chromosomes and the frequency of XX, XY, and YY diploid spermatozoa. The XY variant prevailed in gonosomal aneuploidy and diploidy and was associated with abnormal chromosomal segregation in meiotic anaphase I. The contribution of human sperm chromosomal imbalance to early embryonic lethality and to some forms of chromosomal abnormalities in the off-spring is discussed.