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

Teratozoospermia is characterized by the presence of spermatozoa with abnormal morphology in sperm. This condition is frequently associated with infertility and intracytoplasmic sperm injection (ICSI) is frequently used as the treatment of choice. However, the use of ICSI has created consequential debate concerning the genetic risk for the offspring. Fluorescence in situ hybridization technique (FISH), allowing the specific identification of human chromosomes in sperm nuclei, has been used to study chromosome abnormalities in sperm from men with teratozoospermia and a normal karyotype. In this review, we present studies that have tried to determine if men with a normal blood karyotype but suffering from teratozoospermia present a higher aneuploidy frequency. The literature is limited to three forms of teratozoospermia. The first group consists of "polymorphic teratozoospermia", where a majority of spermatozoa display more than one type of abnormality. In this case, only a slight increase in aneuploidy frequency is observed, which cannot be differentiated from the results observed in oligo-astheno-teratozoospermia (OAT). The second group, named "globozoospermia", is characterized by round spermatic heads, absence of acrosome and disorganization of mid-piece and tail. In this case, some studies have shown a significant, but moderate, increase in the aneuploidy frequency for acrocentrics and sex chromosomes. The aneuploidy frequency remains low, also ICSI can be proposed to these patients, but few successes occur. The third group consists of "enlarged head teratozoospermia", where almost all spermatozoa have an enlarged head, multiple tail and abnormal acrosome. In this case a very high level of missegregation is observed, leading to nearly 100% aneuploidy. In this particular group, ICSI must be refuted, and patients have to be redirected to other possibilities, like sperm donation.     

Syndrome de klinefelter et conseil génétique

Klinefelter’s syndrome is a common sex chromosomal aberration generally characterized by hypergonadotrophic hypogonadism and azoospermia. However, spermatogenesis impairment is variable and severe oligozoospermia can be found in some men, particularly those exhibiting a mosaic karyotype 47,XXY/ 46,XY. New reproductive technologies, such as intracytoplasmic sperm injection (ICSI), allow Klinefelter patients to have a progeny, even those who are azoospermic after testicular sperm recovery. The question therefore arises of whether or not there is a genetic risk for pregnancies from affected fathers. Sperm karyotyping, by in vitro penetration of zona-free hamster eggs or by fluorescence in-situ hybridization (FISH), is a method of choice for measuring aneuploidy rate in spermatozoa of patients carrying gonosomal abnormalities. A theoretical model would predict a high level of 24,XX and/or 24,XY disomic sperm cells in Klinefelter patients if 47,XXY spermatogonia were able to complete meiosis and achieve spermatogenesis. Interestingly, current observations show that the rate of abnormal spermatozoa in these patients is low, around 1–2%, which indicates that only 46,XY spermatogonia can produce mature sperm cells and that oligozoospermic Klinefelter patients probably carry a 47,XXY / 46,XY mosaicism, at least at the testicular level. However, this low but statistically significant level of disomic spermatozoa emphasizes the fact that their spermatogenesis occurs in a compromised environment which could increase the risk of meiotic errors. Therefore, the possible occurrence of autosomal aneuploidies in children born from Klinefelter fathers leads to the following recommendations: a) individual analysis by FISH of the sperm aneuploidy rate in each Klinefelter patient candidate for ICSI; b) proposal of fetal karyotyping after amniocentesis in pregnancies obtained by this technique.     

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.     

La sélection des spermatozoïdes à fort grossissement permet-elle une diminution de la fréquence des aneuploïdies spermatiques?

Severe male infertility concerns two categories of men. Men with abnormal karyotype, who represent 2 to 14% of infertile men and who can produce sperm cells carrying unbalanced chromosomes related to the patients initial chromosomal reorganization inducing a variable risk of transmission of the abnormality to their conceptus. The second category is men with a normal karyotype but an increased rate of spermatic aneuploidy in a context of severe oligo- and/or asthenozoospermia and men from couples in implantation failure. ICSI is the standard Assisted Medical Reproductive technique for most of these 2 categories despite the obvious increased chromosomal risk. This raises the question of how to morphologically identify sperm cells with abnormal chromosome content during ICSI ? Unfortunately, no relationship has yet been found between sperm morphology in the ICSI sperm fraction (×200) and their chromosome content. Nevertheless, since the end of the 1990s, Bartoov’s team has developed MSOME (Motile Sperm Organelle Morphology Examination) consisting of high-power examination of sperm cells up to × 12,250. This technique was indicated for cases of repeated ICSI failures and appeared to increase pregnancy rates. But was this improvement due to better selection of the chromosomal content of sperm cells to be injected? The present study addressed this question by estimating the value of MSOME in the selection of euploid sperm cells in 2 groups of patients known to have an increased rate of sperm aneuploidy. Group 1 was composed of 2 patients with normal karyotype who presented a macrocephalic sperm syndrome with more than 99% of aneuploid sperm. Group 2 was composed of 11 patients with abnormal karyotype: 6 patients with reciprocal translocation and 5 patients with Robertsonian translocation. The purpose of this study was to compare spermatozoa aneuploidy rates in fresh semen, to those obtained after ICSI selection (×200) and MSOME selection (×6000). Three specific steps of the protocol were (1) all sperm cells selected in MSOME were “top sperm cells“ (2) fixation of selected sperm cell (average loss of 15% during FISH washes) (3) FISH results were validated by two different examiners. FISH analysis of X, Y and 18 chromosomes showed that MSOME eliminates polyploid and diploid sperm cells in patients with macrocephalic sperm syndrome, but the 6 sperm cells selected were all haploid and aneuploid. FISH analysis of X, Y and 18 chromosomes of all other patients did not show any influence of the selection method on the aneuploidy rate. For the 5 subjects with a Robertsonian translocation, the global results of FISH analysis paradoxically showed a significant decrease of the euploidy rate in MSOME selection. The global results of FISH analysis for the 6 patients with mutual reciprocal translocations, showed that the various mutual translocations were not modified between whole sperm and the 2 selection methods. On the other hand, a significant decrease of adjacent 1 and 2 segregation frequency was observed between whole sperm and MSOME selection, associated with a significant increase of 3:1 segregation frequency suggesting that the segregations which modify the structure of chromosomes, for example adjacent 1 and 2 segregations, would induce visible morphological modifications selected by MSOME. We hypothesized that the efficacy of spermatic apoptosis could be modulated by morphology but also by the chromosome contents of the sperm cell. In conclusion, MSOME does not provide any guarantee of the normal chromosome contents of the TOP selected sperm cell. However, these results obtained in a small series of patients suggest that MSOME can eliminate some chromosome abnormalities (adj1 and 2) which would alter sperm nuclear structures.     

Increased Frequency of Aneuploidy in Long-Lived Spermatozoa

Aneuploidy commonly causes spontaneous abortions, stillbirths, and aneuploid births in humans. Notably, the majority of sex chromosome aneuploidies in live births have a paternal origin. An increased frequency of aneuploidy is also associated with male infertility. However, the dynamics and behavior of aneuploid spermatozoa during fertilization in humans have not been studied in detail. Therefore, we compared the frequency of aneuploidy and euploidy in live spermatozoa from normozoospermic men over a 3-day period. To assess the dynamics and behavior of aneuploid spermatozoa, we simultaneously evaluated sperm viability using the hypo-osmotic swelling test and sperm aneuploidy using fluorescence in situ hybridization. Whereas the frequency of viable euploid spermatozoa significantly decreased over 3 days, the frequency of viable spermatozoa with aneuploidy interestingly showed a time-dependent increase. In addition, spermatozoa with abnormal sex chromosomes survived longer. To compared with spermatozoa with other swelling patterns, those with tail-tip swelling patterns had a lower frequency of aneuploidy at all time points. This study revealed the novel finding that the frequency of aneuploid spermatozoa with fertilization capability significantly increased compared to that of euploid spermatozoa over 3 days, suggesting that aneuploid spermatozoa can survive longer than euploid spermatozoa and have a greater chance of fertilizing oocytes.     

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.     

Detection of sex chromosome aneuploidy in dog spermatozoa by triple color fluorescence in situ hybridization

With the development of a direct visualization of sex chromosome in a single sperm by fluorescence in situ hybridization (FISH) technique, the frequency of aberration (aneuploidy) in spermatozoa in several mammals has been investigated. However, there is no report in the incidence of X-Y aneuploidy in the sperm population of dogs. Therefore, in this study, the aneuploidy in dog spermatozoa was examined by multicolor FISH using specific molecular probes for canine sex chromosomes and autosome. Semen from eight male Labrador retrievers was used as specimen. For decondensation of sperm nuclei, the specimen was treated with 1 M NaOH for 4 minutes at room temperature. Probes for chromosomes X, Y, and 1, labeled with SpectrumGreen, Cy3 and Cy5, respectively, were hybridized with decondensed spermatozoa. Fluorescence in situ hybridization signals in sperm heads were clearly detected in each specimen, regardless of the sperm donor. The FISH signal of at least one of the three probes was detected in all sperm heads examined. There was no significant difference between the theoretical ratio (50:50) and the observed ratio of X and Y chromosomes in spermatozoa of all the eight dogs. Mean percentage of sex chromosome aneuploidy was 0.127% (ranged between 0% and 0.316%). This percentage of canine sex chromosome aneuploidy was lower than the one reported in cattle, horses, river buffalo, and goats sperm, but higher than that observed in mice and sheep.     

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

The aim of aneuploidy evaluation in spermatozoa from patients presenting spermatogenesis defects is to identify a relationship between meiotic errors and quantitative or qualitative alterations of spermatogenesis. During the past ten years, the use of fluorescence in situ hybridization (FISH) has permitted the determination of the frequency of numerical chromosome aberrations in different clinical situations. It has been established that infertile males with reduced sperm count and a normal constitutional karyotype have a significantly high risk of aneuploidy in their spermatozoa particularly regarding sex chromosomes. Concerning sperm motility, the data are more controversial. However, patients of severe asthenozoospermia induced by specific morphological deformities involving sperm flagella have a significantly high risk of producing aneuploid spermatozoa.     

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     

Frequency and distribution of chromosome abnormalities in human spermatozoa

This study reviews the frequency and distribution of numerical and structural chromosomal abnormalities in spermatozoa from normal men obtained by the human-hamster system and by multicolor-FISH analysis on decondensed sperm nuclei. Results from large sperm karyotyping series analyzed by chromosome banding techniques and results from multicolor FISH in sperm nuclei (of at least 10(4) spermatozoa per donor and per probe) were reviewed in order to establish baseline values of the sperm chromosome abnormalities in normal men. In karyotyping studies, the mean disomy frequency in human sperm is 0.03% for each of the autosomes, and 0.11% for the sex chromosomes, lower than those reported in sperm nuclei by FISH studies using a similar methodology (0.09% and 0.26%, respectively). Both types of studies coincide in that chromosome 21 and sex chromosomes have a greater tendency to suffer segregation errors than the rest of the autosomes. The mean incidence of diploidy, only available from multicolor FISH in sperm nuclei, is 0.19%. Inter-donor differences observed for disomy and diploidy frequencies among FISH studies of decondensed sperm nuclei using a similar methodology could reflect real differences among normal men, but they could also reflect the subjective application of the scoring criteria among laboratories. The mean frequency of structural aberrations in sperm karyotypes is 6.6%, including all chromosome types of abnormalities. Chromosome 9 shows a high susceptibility to be broken and 50% of the breakpoints are located in 9q, between the centromere and the 9qh+ region. Structural chromosome aberrations for chromosomes 1 and 9 have also been analyzed in human sperm nuclei by multicolor FISH. Unfortunately, this assay does not allow to determine the specific type of structural aberrations observed in sperm nuclei. An association between advancing donor age and increased frequency of numerical and structural chromosome abnormalities has been reported in spermatozoa of normal men.     

Pesticides and the induction of aneuploidy in human sperm

Pesticides are some of the most frequently released toxic chemicals into the environment. Exposure to them has been associated with reproductive dysfunction, but the knowledge of the genotoxic risks of these substances is still limited. In vitro and in vivo, many pesticides are shown to induce aneuploidy. Analysis of sperm chromosomes by fluorescence in situ hybridization (FISH) with chromosome-specific probes has obtained increasing popularity in genetic toxicology. Sperm-FISH studies on men exposed to pesticides have yielded conflicting results: in men exposed to multiple pesticides during spraying no increased disomy frequencies in sperm were observed, although one study reported an increased rate of sex chromosome nullisomy. In contrast the two studies conducted in pesticide factories showed increased frequencies of sperm aneuploidy in exposed men compared to controls. The available data indicates that at least some of the commonly used pesticides are capable of inducing aneuploidy in human sperm when the exposure level is high enough.     

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.     

Processing and selection of surgically-retrieved sperm for ICSI: a review

Although the technique of intracytoplasmic sperm injection (ICSI) has been a revolution in the alleviation of male infertility, the use of testicular sperm for ICSI was a formerly unseen breakthrough in the treatment of the azoospermic man with primary testicular failure. At the clinical level, different procedures of testicular sperm retrieval (conventional TESE, micro-TESE, FNA/TESA, MESA, PESA) are being performed, the choice is mainly based on the cause of azoospermia (obstructive versus non-obstructive) and the surgeon’s skills. At the level of the IVF laboratory, mechanical procedures to harvest the sperm from the tissue may be combined with enzymatic treatment in order to increase the sperm recovery rates. A number of techniques have been developed for viable sperm selection in males with only immotile testicular sperm available. However, large, well-designed studies on the benefit and safety of one over the other technique are lacking. Despite all the available methods and combinations of laboratory procedures which have a common goal to maximize sperm recovery from testicular samples, a large proportion of NOA patients fail to father a genetically own child. Advanced technology application may improve recovery rates by detection of the testicular foci with active spermatogenesis and/or identification of the rare individual sperm in the testicular suspensions. On the other hand, in vitro spermatogenesis or sperm production from embryonic stem cells or induced pluripotent stem cells might be future options. The present review summarizes the available strategies which aim to maximize sperm recovery from surgically retrieved samples.     

Génotoxicité des chimiothérapies et radiothérapies: Quelles sont les conséquences pour le spermatozoïde humain?

The prognosis of cancer in young men of childbearing potential has been considerably improved over recent decades as a result of therapeutic progress. Chemotherapy and radiotherapy have well known effects on spermatogenesis. Apart from quantitative and qualitative impairment of spermatogenesis, animal studies have also demonstrated nuclear lesions (aneuploidy, presence of adducts, DNA fragmentation, etc.) and sometimes lesions affecting the F1 and F2 generations. Chromosomal studies of human spermatozoa after radiotherapy have demonstrated an increased frequency of chromosomal anomalies. The first studies concerning the effects of chemotherapy used the heterospecific fertilization technique to demonstrate spermatozoal chromosomal anomalies. More recently, thefluorescence in situ hybridization (FISH) technique has been used to study several chromosomes on a large number of spermatozoa. The results of various studies based on small sample sizes vary as a function of the therapeutic protocol administered and the time of sperm collection in relation to the end of treatment. We studied 5 patients who provided a semen sample 6 to 17 months after completing the BOE chemotherapy protocol (Bleomycin, Etoposide, Cisplatin). We demonstrated an increased rate of aneuploid and diploid spermatozoa. The results of our study and those reported by R. Martin et al. [45, 47] suggest the possibility of a transient effect of chemotherapy on gamete chromosomes. Other studies, conducted in the context of Hodgkin’s disease, have demonstrated the transient nature of the aneuploidy effect. Apart from the harmful action on chromosomes, treatments could also damage spermatozoal DNA. Studies conducted on larger sample sizes and using other methods of analysis therefore appear to be essential. In the meantime, it appears preferable to systematically propose semen cryopreservation before treatment and to provide very cautious advice to patients desiring a pregnancy soon after completion of treatment.     

Azoospermies non obstructives; facteurs prédictifs du prélèvement testiculaire et risques de la fécondation assistée

Surgical sperm retrieval has revolutionized the treatment of azoospermia. Intracytoplasmic sperm injection (ICSI) allows naturally infertile men to have children by allowing defective sperm cells to fertilize oocytes. These techniques, applied without any preliminary animal experimentation, raised an enormous enthusiasm and are performed on a large-scale. To increase the efficiency of these treatments, the clinicians are now trying, without success, to identify factors predictive of success allowing better patient selection and counselling of couples dependent on these techniques in order to avoid useless and harmful interventions. Animal research, conducted after introduction of these techniques, has raised serious doubts about the safety of ICSI and the legitimacy of using defective spermatozoa from genetically high-risk patients. Some studies have also emphasized the unusual frequency of obstetric and neonatal problems as well as rare diseases and malignancies in ICSI-born children and ART-born children. However, these disturbing findings are not specifically related to the ICSI procedure, as demonstrated by well-conducted large-scale follow-up studies in ICSI-born children. This paradox raises a lively debate. ICSI-children follow-up studies should continue until sound data taking into account the genetic and all other parental background are obtained. In conclusion, non obstructive azoospermic patients should be informed of the limits of sperm retrieval and genetic screening tools as well as all risks common to ICSI and ART.     

Increase of ICSI efficiency with hyaluronic acid binding sperm for low aneuploidy frequency in pig

The present study was designed to evaluate the ability of hyaluronic acid binding sperm (HABS) in increasing the efficiency of intracytoplasmic sperm injection (ICSI) in terms of the production of chromosomally normal porcine embryos. Porcine embryos were produced by in vitro fertilization (IVF), ICSI and ICSI using hyaluronic acid binding sperm (ICSI-HABS). Chromosome aneuploidy in sperm and embryos was evaluated using chromosome 1 submetacentric probe for fluorescence in situ hybridization (FISH) analysis. No significant differences were observed in the blastocysts rates (18.6, 23.6 and 23.8%) and cell numbers (61.8+/-12.5, 55.5+/-7.3 and 59.3+/-9.6) among embryos derived from IVF, ICSI, and ICSI-HABS. However, the frequency of normal diploidy in ICSI-HABS (75.5%) was significantly higher (P<0.05) than that in IVF (57.0%) and ICSI (68.2%). Embryos from ICSI-HABS showed significantly lower chromosome abnormality rate (P<0.05). Both ICSI and IVF embryos showed higher rates of polyploidy, and hence chromosomally abnormal embryos, in comparison to ICSI-HABS embryos. In addition, we investigated the chromosomal complement of porcine spermatozoa by FISH. The rate of chromosome number abnormality in porcine sperm was found to be 6.25% (70/1120). Thus, we conclude that the use of hyaluronic acid binding sperm is superior to morphological sperm selection for ICSI in producing chromosomally normal embryos and increasing the ICSI efficiency by lowering the aneuploidy frequency. Our results indicate that the selection of normal sperm with hyaluronic acid binding assay might help to reduce the early embryonic mortality due to chromosomal aneuploidy thereby increasing the success rate of embryo transfer technology in pigs.     

Germ-cell nondisjunction in testes biopsies of men with idiopathic infertility.

Intracytoplasmic sperm injection (ICSI) has been used in combination with testicular sperm extraction to achieve pregnancies in couples with severe male-factor infertility, yet many of the underlying genetic mechanisms remain largely unknown. To investigate nondisjunction in mitotic and meiotic germ cells, we performed three-color FISH to detect numeric chromosome aberrations in testicular tissue samples from infertile men confirmed to have impaired spermatogenesis of unknown cause. FISH was employed to determine the rate of sex-chromosome aneuploidy in germ cells. Nuclei were distinguished as haploid or diploid, respectively. The overall incidence of sex-chromosome aneuploidy in germ cells was found to be significantly higher (P<.00001) in all three abnormal histopathologic patterns (range 39.0%-43.5%) as compared with normal controls (29.1%). The relative ratio of normal to aneuploid nuclei in the diploid cells of patients with impaired spermatogenesis was approximately 1.0, a >300% decrease when compared with the 4.42 ratio detected in patients with normal spermatogenesis. These results provide direct evidence of an increased incidence of sex-chromosome aneuploidy observed in germ cells of men with severely impaired spermatogenesis who might be candidates for ICSI with sperm obtained directly from the testis. The incidence of aneuploidy was significantly greater among the diploid nuclei, which suggests that chromosome instability is a result of altered genetic control during mitotic cell division and proliferation during spermatogenesis.     

Apport de l’exploration cytogénétique et ultrastructurale dans le pronostic de fertilité des sujets globozoospermiques

Globozoospermia is a severe form of teratozoospermia characterized by round-headed sperms with absence or presence of a rudimentary acrosome. The objective of this study is to analyze sperm from six patients with globozoospermia syndrome and report the results of 11 intracytoplasmic sperm injection (ICSI) attempts. The investigation of these issues was carried out by studying the sperm aneuploidy rate by fluorescent in situ hybridization (sperm-FISH) for chromosomes X, Yand 18. The rate of DNA fragmentation was studied by using the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) technique and a detailed ultrastructural morphology study of the sperm using transmission electron microscopy. Eleven ICSI attempts were performed in patients with low fertilization rate, (9.37%) and pregnancy did not occur. This study confirmed the variability of sperm phenotypes observed in this syndrome and the low fertilization rates after IVF-ICSI regardless of the phenotype.     

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.     

Long‐term proliferation and characterization of human spermatogonial stem cells obtained from obstructive and non‐obstructive azoospermia under exogenous feeder‐free culture conditions

Objectives: The aim of the present study was to improve efficiency of isolation and to optimize proliferative potential of human spermatogonial stem cells (SSCs) obtained from obstructive azoospermic (OA) and non‐obstructive azoospermic (NOA) patients, and further, to characterize these cells for potential use in infertility treatment or study of reproductive biology. Materials and methods: We have applied a cell‐sorting method, using collagen and magnetic activated cell separation to overcome obstacles, developing a collection system, and simple long‐term proliferation system, that yields large numbers of high‐purity SSCs from obstructive OA and NOA patients. Results: SSCs derived from OA and NOA patients proliferated and maintained their characteristics for more than 12 passages (>6 months) in vitro. Moreover, the population of cells positive for the SSC‐specific markers GFRα‐1 and integrin α6, increased to more than 80% at passage 8. Conclusion: These finding may support the idea that in vitro propagation of SSCs could be a useful tool for infertility treatment and study of reproductive biology.