Meiotic behaviour of the sex chromosomes in three patients with sex chromosome anomalies (47,XXY, mosaic 46,XY/47,XXY and 47,XYY) assessed by fluorescence in-situ hybridization

Meiotic studies using multicolour fluorescent in-situ hybridization (FISH) and chromosome painting were carried out in three patients with sex chromosome anomalies (47,XXY; 46,XY/47,XXY and 47,XYY). In the two patients with Klinefelter syndrome, although variable percentages of XXY cells (88.5 and 28.3%) could be found in the pre-meiotic stages, none of the abnormal cells entered meiosis, and all pachytenes were XY. However, the abnormal testicular environment of these patients probably resulted in meiotic I non-disjunction, and a certain proportion of post-reductional cells were XY (18.3 and 1.7%). The fact that none of the spermatozoa were XY also suggests the existence of an arrest at the secondary spermatocyte or the spermatid level. In the XYY patient, most (95.9%) premeiotic cells were XYY. The percentage of XYY pachytenes was 57.9%. The sex chromosomes were either in close proximity (XYY) or the X chromosome was separated from the two Ys (X + YY). A high proportion (42.1%) of post-reductional germ cells were XY. However, only 0.11% of spermatozoa were disomic for the sex chromosomes. In this case, the data suggest the existence of an arrest of the abnormal cells at the primary and the secondary spermatocyte or the spermatid level, giving rise to the continuous elimination of abnormal cells in the germ-cell line along spermatogenesis. The fact that the proportion of diploid spermatozoa was only increased in one of the three cases (XXY) is also suggestive of an arrest of the abnormal cell lines in these patients. The two apparently non-mosaic patients were, in fact, germ-cell mosaics. This suggests that the cytogenetic criteria used to define non-mosaic patients may be inadequate; thus, the risk of intracytoplasmic sperm injection in apparently non-mosaics may be lower than expected.     

Preferential location of sex chromosomes, their aneuploidy in human sperm, and their role in determining sex chromosome aneuploidy in embryos after ICSI

BACKGROUND: In babies born after ICSI procedures, an increase of de-novo sex chromosome abnormalities has been observed. Several hypotheses have been proposed to explain these findings: an increased rate of sex chromosome aneuploidy in sperm of oligozoospermic men, or a preferential location of the sex chromosomes in the sub-acrosomal region of the sperm nucleus which leads to a reduced DNA decondensation of this region. In order to investigate which theory may be more reliable, we studied the localization of sex chromosomes and their aneuploidy rate in sperm in men undergoing ICSI. METHODS: Using fluorescent in-situ hybridization we studied sex chromosome localization and the aneuploidy rate for sex chromosomes and chromosome 18 in 20 oligospermic men undergoing ICSI and in 10 controls. RESULTS: In 40.94 and 52.92% of cases, the X and Y chromosomes respectively were localized in the sub-acrosomal region of the sperm nucleus compared with only 14.29% of cases of chromosome 18 (P < 0.001). An increase of sex chromosome aneuploidy in sperm of oligospermic men was observed; 2.91 versus 0.69% of controls (P < 0.001). CONCLUSIONS: Sex chromosomes are localized preferentially in the sub-acrosomal region of sperm and sex chromosome aneuploidy rate in the sperm of oligozoospermic men is higher than in controls.     

Sex chromosome aneuploidy and anthropometry: A new proportionality assessment using the phantom stratagem

Reported anthropometric data on 121 subjects with 47, XYY, 47,XXY, 47,XXX, and 45,X aneuploidies were compared to those from 578 male and female control subjects by use of a single, unisex reference person (“phantom”). Subjects and controls were geometrically scaled to a standard stature of 170.18 cm, thus eliminating variance due to height. Deviations of anthropometric variables from specified phantom values were expressed as standard z-scores. By comparing z-scores of individual aneuploidy classes with those of their controls, further differences in proportionality came to light. The stratagem disclosed a systematic proportionality pattern between subjects and controls which appeared to be related to each specific sex chromosome aneuploidy. The phantom stratagem for proportional growth assessment appears to merit further use in genetic investigations where individual differences in size and shape confound the analysis of anthropometric data.     

Sperm aneuploidy frequencies analysed before and after chemotherapy in testicular cancer and Hodgkin's lymphoma patients

BACKGROUND Multicolour fluorescent in situ hybridization was utilized to detect sperm aneuploidy for chromosomes 13, 21, X and Y in testicular cancer and Hodgkin's lymphoma chemotherapy patients. METHODS Aneuploidy was assessed before, and 6, 12 and/or 18-24 months after, the initiation of chemotherapy, and compared with age matched controls. 635 396 sperm were scored blindly with 5000 sperm/patient/chromosome/ time point, where sperm was available. (First two phrases have been reversed). RESULTS Comparing testicular cancer and Hodgkin's lymphoma patients to each other and with controls, cancer-specific differences were identified. Hodgkin's lymphoma patients, particularly, exhibited significantly increased aneuploidy frequencies for all chromosomes throughout treatment. At 6 months, all cancer patients showed significantly increased frequencies of XY disomy and nullisomy for chromosomes 13 and 21. In general, aneuploidy frequencies declined to pretreatment levels 18 months after treatment initiation, but increased aneuploidy frequencies persisted in some chromosomes for up to 24 months. CONCLUSIONS Because of elevated aneuploidy frequencies prior to and up to 24 months from the start of chemotherapy, patients should receive genetic counselling about the potentially increased risk of an aneuploid conceptus from sperm cryopreserved prior to chemotherapy, and for conceptions up to 2 years after the initiation of treatment.     

A fluorescent in situ hybridization analysis of the chromosome constitution of ejaculated sperm in a 47, XYY male

Two semen samples from a 47, XXY male were examined using chromosome-specific DNA probes and fluorescent in situ hybridization (FISH) to determine the distribution of sex chromosomes and an autosome (chromosome 17) in the sperm. A motile population of sperm was also prepared from one sample using the swim-up technique to compare the motile and total sperm populations. Chromosomes were localized using single FISH and a biotinylated chromosome 17 probe (TR17), or double FISH using a biotinylated X chromosome probe (TRX) and a digoxigenin-labelled Y chromosome probe (HRY). Labelling efficiencies were 95–98%. Ploidy levels were estimated by measurement against a microscope eyepiece graticule. The overall ratio of X-to Y-bearing sperm was 47% to 48.4% in the neat samples, and 48.4% to 45.3% in the swim-up fraction. Neither of the ratios was significantly different from 1:1. The frequencies of monosomic and disomic (but otherwise haploid sperm) were not different from the frequencies we observed in normal donors. In contrast, the frequencies of both diploid and tetraploid cells were increased in the neat samples of the XYY male. In the swim-up fractions, however, none of these parameters differed from those of ten normal semen donors. These results support the hypothesis that the extra Y chromosome in XYY men is eliminated during spermatogenesis.     

Sperm chromosome analysis in a man heterozygous for a reciprocal translocation 46,XY t(12;20)(q24.3;q11)

Sperm chromosome complements were studied in a man who carried a reciprocal translocation t(12;20)(q24.3;q11). A total of 113 spermatozoa were karyotyped after in-vitro penetration of hamster eggs. 2:2 and 3:1 meiotic segregations were observed with the following frequencies: alternate 47%, adjacent 1 42%, adjacent 2 10%, 3:1 2%. For alternate segregations, the number of normal spermatozoa (25) was not significantly different from the number of spermatozoa carrying a balanced form of the translocation (28), as theoretically expected. The proportion of spermatozoa with an unbalanced form of the translocation was 53%. There was no evidence for an interchromosomal effect since the frequencies of numerical and structural abnormalities (unrelated to the translocation) were within the normal range of control donors.     

Chromosome constitution and apoptosis of immature germ cells present in sperm of two 47,XYY infertile males

BACKGROUND: In order to assess sperm alterations observed in some XYY males, we analysed the chromosome constitution as well as apoptosis expression in germ cells from two oligozoospermic males with high count of immature germ cells in their semen. METHODS: Sex chromosome number and distribution were assessed at pachytene stage by fluorescence in situ hybridization (FISH). Immature germ cells and spermatozoa were examined by FISH and TdT (terminal deoxynucleotidyl transferase)-mediated dUDP nick-end (TUNEL) assay, combined with immunocytochemistry using the proacrosin-specific monoclonal antibody (mAb 4D4). RESULTS: For patients 1 and 2, two Y chromosomes were present in respectively 60.0 and 39.6% of pachytenes. The three sex chromosomes were always in close proximity and partially or totally condensed in a sex body. XYY spermatocytes I escape the pachytene checkpoint and achieve meiosis. Nevertheless, nuclear division and/or cytokinesis were often impaired during meiosis leading to diploid (mainly 47,XYY cells) and tetraploid (94,XXYYYY) meiocytes. The presence of binucleated (23,Y)(24,XY) immature germ cells resulting from cytokinesis failure agree with a preferential segregation of the two Y chromosomes during meiosis I. In addition, 69.6% (patient 1) and 53.12% (patient 2) of post-reductional round germ cells were XY. However, high level of apoptotic round germ cells (94.9% for patient 1 and 93.3% for patient 2) was detected and may explain the moderate increase of hyperhaploid XY spermatozoa. Segregation errors also occurred in the XY cell line responsible for disomic 18 and X, as well as 46,XY diploid spermatozoa. CONCLUSIONS: Our data are in agreement with the persistence of the extra Y chromosome during meiosis in XYY oligozoospermic males responsible for spermatogenesis impairment and a probable elimination via apoptosis of most XYY germ cells not solely during but also after meiosis.     

The association of folate, zinc and antioxidant intake with sperm aneuploidy in healthy non-smoking men

BACKGROUND: Little is known about the effect of paternal nutrition on aneuploidyin sperm. We investigated the association of normal dietaryand supplement intake of folate, zinc and antioxidants (vitaminC, vitamin E and β-carotene) with the frequency of aneuploidyin human sperm. METHODS: Sperm samples from 89 healthy, non-smoking men from a non-clinicalsetting were analysed for aneuploidy using fluorescent in situhybridization with probes for chromosomes X, Y and 21. Dailytotal intake (diet and supplements) for zinc, folate, vitaminC, vitamin E and β-carotene was derived from a food frequencyquestionnaire. Potential confounders were obtained from a self-administeredquestionnaire. RESULTS: After adjusting for covariates, men with high folate intake(>75th percentile) had lower frequencies of sperm with disomiesX, 21, sex nullisomy, and a lower aggregate measure of spermaneuploidy (P 0.04) compared with men with lower intake. Inadjusted continuous analyses, total folate intake was inverselyassociated with aggregate sperm aneuploidy (–3.6% change/100µg folate; 95% CI: –6.3, –0. 8) and resultswere similar for disomies X, 21 and sex nullisomy. No consistentassociations were found between antioxidant or zinc intakesand sperm aneuploidy. CONCLUSIONS: Men with high folate intake had lower overall frequencies ofseveral types of aneuploid sperm.     

Genetics: Constitution of semen samples from XYY and XXY males as analysed by in-situ hybridization

A brightfield microscopical in-situ hybridization (ISH) techniquewas applied to semen samples of two 47,XYY males, one 46,XY/47,XXYmale with fertility problems, and two normal 46,XY men, whoserved as controls. The use of a standardized nuclear DNA decondensationmethod, together with double-target ISH and morphological staining,allowed an accurate study of the sex chromosomal content andmorphology of spermatozoa. In the males carrying an extra sexchromosome, we detected X- and Y-bearing spermatozoa in a ratiowhich did not differ significantly from the .1:1 ratio foundin normal males. Aneuploidy for the sex chromosomes was foundin 15% of the spermatozoa of both XYY males and in 3% of theXXY male. The most striking finding was the relatively low percentageof spermatozoa in these patients, with an average of 65% inthe XYY males and 84% in the XXY male. The other cells representedimmature germ cells (IGC), including spermatogonia and spermatocytesarrested at various stages of spermatogenesis. Apparently, inXYY or XXY men, these IGC are shed into the semen to an increasedextent as compared to normal, fertile men. The sex chromosomeconstitution of these IGC was heterogeneous. However, the findingthat the majority of spermatozoa in semen of 47,XYY and 47,XXYmales carried a single sex chromosome strengthens the hypothesisthat a 46,XY germ cell line must be present, apparently witha proliferative advantage over the 47,XYY or 47,XXY cells.     

Morphologically normal spermatozoa of patients with secretory oligo-astheno-teratozoospermia have an increased aneuploidy rate

BACKGROUND: Normal morphology is a major criterion for selecting spermatozoa to be injected. Given that teratozoospermia is one of the most critical parameters associated with sperm aneuploidy, the purpose of this study was to evaluate the aneuploidy rate of morphologically normal spermatozoa of patients with oligo-astheno-teratozoospermia (OAT). METHODS: Ten patients with secretory OAT and six age-matched normozoospermic men with a normal karyotype were enrolled. After assignment to normal or abnormal category, the location of each spermatozoon was recorded using an electronic microstage locator. Slides were then subjected to triple-colour fluorescence in situ hybridization for chromosomes X, Y and 12. RESULTS: OAT patients had a lower number of morphologically normal and abnormal spermatozoa carrying the X chromosome, compared with normozoospermic men. They also exhibited increased XY and XX disomy rates. Morphologically abnormal spermatozoa from normozoospermic men also had an increased XX disomy rate compared with normally shaped spermatozoa obtained from the same men. The total sperm aneuploidy rate of morphologically abnormal spermatozoa of normozoospermic men was 4.4-fold higher than that of spermatozoa with normal morphology. The total aneuploidy rates of spermatozoa with normal or abnormal head shape from OAT patients were similar to each other and to that of abnormally shaped spermatozoa from normozoospermic men, but they were higher than the rate found in normally shaped spermatozoa of normal men. CONCLUSIONS: Normally shaped spermatozoa of OAT patients have an increased aneuploidy rate.     

Sperm chromosome complements in a man heterozygous for a reciprocal translocation t(2; 3)(q24;p26)

Sperm chromosome complements were studied in a man heterozygousfor a reciprocal translocation t(2; 3)(q24;p26). This man wasidentified during a family study after his sister was investigatedfor amenorrhea. A total of 92 spermatozoa were karyotyped afterin-vitro penetration of hamster eggs. The frequencies of alternate,adjacent 1, adjacent 2 and 3: 1 segregations were 55.4, 36.1,7.2 and 1.2% respectively. For alternate segregations, the numberof normal spermatozoa (n < 25) was not significantly differentfrom the number of spermatozoa carrying a balanced form of thetranslocation (n < 21), as theoretically expected. The proportionof spermatozoa with an unbalanced form of the translocationwas 44.6%. There was no evidence for an interchromosomal effectsince the frequencies of numerical and structural abnormalities(unrelated to the translocation) were within the normal rangeof control donors.     

Correlation between semen parameters and sperm aneuploidy rates investigated by fluorescence in-situ hybridization in infertile men

Spermatozoa from 32 infertile patients and 13 controls with normal semen parameters were analysed using dual and triple colour fluorescence in-situ hybridization (FISH) techniques, in order to investigate the rates of aneuploidy for chromosomes 13, 18, 21, X and Y. The patients were divided into three groups according to their karyotypes or the karyotypes of their offspring: 15 were infertile men with abnormal semen parameters and normal karyotypes (group 1), 13 were infertile men with abnormal karyotypes and normal or abnormal semen (group 2) and four were infertile men with abnormal semen and normal karyotypes but whose wives conceived a child (or a fetus) with a numerical chromosomal abnormality through an intracytoplasmic sperm injection cycle (group 3). Patients with abnormal semen parameters showed a significantly higher aneuploidy rate for the investigated chromosomes in their spermatozoa compared to controls (P < 0.005). Our data suggest the presence of a correlation between poor semen parameters and an increase in aneuploidy rate of chromosomes 13, 18, 21, X and Y in spermatozoa (r = -0.81071, P < 0.002); therefore the risk of a chromosomal aneuploidy in spermatozoa seems to be inversely correlated to sperm concentration and total progressive motility. Patients with abnormal karyotypes showed a higher incidence of diploidy and chromosomal aneuploidies compared to controls (P < 0.002). This strongly suggests the presence of an interchromosomal effect of the cytogenetic rearrangement. Men who fathered a child with an abnormal karyotype through intracytoplasmic sperm injection did not present a higher aneuploidy rate for the investigated chromosomes in spermatozoa compared to patients with infertility due to a similar male factor but showed higher incidence of chromosomal aneuploidy compared to normal controls.     

Some considerations bearing on the doctrine of self-fulfilling prophecy in sex chromosome aneuploidy

The doctrine of self-fulfilling prophecy has been invoked in studies of the effects of sex chromosome aneuploidy on human development as a reason for routine concealment of the diagnosis from affected children and their families. It has been assumed that knowledge of the existence of risk for deviance from normal development automatically creates a self-defeating emotional climate. This communication attempts to delineate both advantageous and deleterious aspects of self-fulfilling prophecy in the medical management of sex chromosome aneuploidy, presents an alternative approach, and reviews the experience in a prospective study of 52 families where a policy of disclosure was followed.     

Abnormalities for chromosomes 13 and 21 detected in spermatozoa from infertile men

Sperm samples from infertile men with oligozoospermia or teratozoospermiawere studied by multicolour fluorescence in-situ hybridization(FISH) using DNA probes for chromosomes 13 and 21. A total of90 809 sperm nuclei from nine infertile men and 182 799 spermnuclei from 18 control donors were analysed. There was a highlysignificant increase in the frequency of spermatozoa disomicfor chromosome 13 in infertile patients (0.28%) compared tocontrol donors (0.13%) (two-tailed Z statistic P <0.0001and for chromosome 21 (0.48% in infertile men versus 0.37% incontrols, P <0.0001). Also there was a significantly increasedfrequency of diploid spermatozoa in infertile men (0.85%) comparedto control donors (0.66%) (P <0.0001). Our previous studieson these same infertile patients demonstrated increased frequenciesof sperm disomy for chromosomes 1 and XY. This suggests thatinfertile men, who are prime candidates for intracytoplasmicsperm injection, may be at a very small increased risk of aneuploidoffspring.     

Sperm aneuploidy in fathers of children with paternally and maternally inherited Klinefelter syndrome

BACKGROUND: It is unclear whether frequency of sperm aneuploidy is associated with risk of fathering children with trisomy. METHODS: We recruited 36 families with a boy with Klinefelter syndrome (KS), interviewed the fathers about their exposures and medical history, received a semen sample from each father, and collected blood samples from the mother, father and child. We applied a multicolour fluorescent in-situ hybridization assay to compare the frequencies of sperm carrying XY aneuploidy and disomies X, Y and 21 in fathers of maternally and paternally inherited KS cases. RESULTS: Inheritance of the extra X chromosome was paternal in 10 and maternal in 26 families. Fathers of paternal KS cases produced higher frequencies of XY sperm (P = 0.02) than fathers of maternal KS cases. After controlling for age, the major confounding variable, the difference between the two groups was no longer significant (P less-than-or-equal 0.2). Also, there were no significant differences between the parental origin groups for disomy X, Y or 21. CONCLUSIONS: Men who fathered a child with a Klinefelter syndrome produced higher frequencies of XY sperm aneuploidy, which is explained, in part, by both paternal age and parent of origin.     

Human male infertility: chromosome anomalies, meiotic disorders, abnormal spermatozoa and recurrent abortion

Human male infertility is often related to chromosome abnormalities.In chromosomally normal infertile males, the rates of chromosome21 and sex chromosome disomy in spermatozoa are increased. Higherincidences of trisomy 21 (seldom of paternal origin) and sexchromosome aneuploidy are also found. XXY and XYY patients produceincreased numbers of XY, XX and YY spermatozoa, indicating anincreased risk of production of XXY, XYY and XXX individuals.Since XXYs can reproduce using intracytoplasmic sperm injection(ICSI), this could explain the slight increase of sex chromosomeanomalies in ICSI series. Carriers of structural reorganizationsproduce unbalanced spermatozoa, and risk having children withduplications and/or deficiencies. In some cases, this risk isconsiderably lower or higher than average. These patients alsoshow increased diploidy, and a higher risk of producing diandrictriploids. Meiotic disorders are frequent in infertile males,and increase with severe oligoasthenozoospemia (OA) and/or highfollicle stimulating hormone (FSH) concentrations. These patientsproduce spermatozoa with autosomal and sex chromosome disomies,and diploid spermatozoa. Their contribution to recurrent abortiondepends on the production of trisomies, monosomies and of triploids.The most frequent sperm chromosome anomaly in infertile malesis diploidy, originated by either meiotic mutations or by acompromised testicular environment.     

Sperm chromosome studies in individuals treated for testicular cancer.

Sperm chromosome studies have shown that patients treated with chemotherapy for testicular cancer have a much higher incidence of chromosome abnormalities than patients treated for other types of cancer or than controls. In two out of four cases, penetration of zona-free hamster eggs was close to zero, indicating that after 2-7 years after treatment the functional capacity of the sperm had not been recuperated. The cytogenetic study of the spermatozoa shows that many of the abnormalities observed corresponded to structural aberrations that may not have a pathogenic effect in the production of abortions or of children with chromosome abnormalities.     

Sperm chromosome complements in a man heterozygous for a reciprocal translocation 46,XY,t(9;13)(q21.1;q21.2) and a review of the literature

Sperm chromosome complements were studied in a man heterozygous for a reciprocal translocation t(9;13)(q21.1;q21.2). A total of 89 spermatozoa were karyotyped after in vitro penetration of hamster eggs. The frequencies of alternate, adjacent 1 and adjacent 2 segregations were 46.9%, 35.8% and 17.3% respectively. For alternate segregation, the number of normal spermatozoa (21) was not significantly different from the number of spermatozoa carrying a balanced form of the translocation (17), as theoretically expected. The proportion of spermatozoa with an unbalanced form of the translocation was 53.1%. There was no evidence for an interchromosomal effect since the frequency of numerical abnormalities (unrelated to the translocation) was within the normal range of control donors. Data from a total of 31 reciprocal translocations studied by sperm chromosomal analysis were reviewed.     

Sperm chromosome studies in an infertile man with partial, complete asynapsis of meiotic bivalents

Meiotic and sperm chromosome studies were carried out in two semen samples from an infertile man with a 46,XY karyotype, oligoasthenoteratozoospermia and abundant exfoliation of spermatogenic cells. Meiotic preparations showed partial, complete asynapsis in a large proportion of metaphase I figures observed, and absence of metaphase II figures, while 24 of the 30 sperm chromosome karyotypes analysed were normal. The remaining sperm karyotypes were as follows: one with structural abnormalities, one with both structural abnormalities and hypohaploidy and four with hypohaploidy. The total frequency of chromosomal abnormalities (6.7%) is similar to that obtained by us in normal men (10.9%). The frequency of spermatozoa with structural abnormalities (6.7%) was not significantly different from that obtained by us in normal men (6.9%). These results suggest that, in some cases, asynaptic spermatogenic cells do not proceed further than metaphase I and only normal germ cells continue spermatogenesis.