Zaida Sarrate

Genetic Analysis of Sperm and Implications of Severe Male Infertility—A Review

The use of fluorescence in situ hybridization (FISH) on decondensed sperm heads has allowed to analyse the chromosome constitution of spermatozoa in different populations. In controls, the mean incidence of disomy (including all chromosomes) is about 6.7 per cent; diploidy increases with age, and some individuals may show a special tendency to nondisjunction. Carriers of numerical sex chromosome anomalies show a low incidence of sex chromosome disomies (2.54-7.69 per cent), and the need to screen ICSI candidates for these conditions has to be reconsidered. Carriers of inversions produce from 0 to 54.3 per cent abnormal sperm. Carriers of Robertsonian translocations produce from 3.4 to 36.0 per cent abnormal sperm, and carriers of reciprocal translocations produce from 47.5 to 81.0 per cent abnormal spermatozoa. However, carriers of translocations usually produce more abnormal embryos than expected from these figures. This may be partly related to interchromosomal effects induced by some structural reorganizations. Males with oligoasthenozoospermia, low motility and/or high FSH concentrations show frequent synaptic anomalies, resulting in the production of aneuploid and diploid sperm. Testicular sperm show extremely high rates of chromosomal abnormalities. The risk of recurrent abortion is increased by the presence of chromosome abnormalities in sperm.

Meiotic abnormalities in infertile males

Meiotic anomalies, as reviewed here, are synaptic chromosome abnormalities, limited to germ cells that cannot be detected through the study of the karyotype. Although the importance of synaptic errors has been underestimated for many years, their presence is related to many cases of human male infertility. Synaptic anomalies can be studied by immunostaining of synaptonemal complexes (SCs), but in this case their frequency is probably underestimated due to the phenomenon of synaptic adjustment. They can also be studied in classic meiotic preparations, which, from a clinical point of view, is still the best approach, especially if multiplex fluorescence in situ hybridization is at hand to solve difficult cases. Sperm chromosome FISH studies also provide indirect evidence of their presence. Synaptic anomalies can affect the rate of recombination of all bivalents, produce achiasmate small univalents, partially achiasmate medium-sized or large bivalents, or affect all bivalents in the cell. The frequency is variable, interindividually and intraindividually. The baseline incidence of synaptic anomalies is 6–8%, which may be increased to 17.6% in males with a severe oligozoospermia, and to 27% in normozoospermic males with one or more previous IVF failures. The clinical consequences are the production of abnormal spermatozoa that will produce a higher number of chromosomally abnormal embryos. The indications for a meiotic study in testicular biopsy are provided.

Role of sperm fluorescent in situ hybridization studies in infertile patients: indications, study approach, and clinical relevance

OBJECTIVE To determine the group of infertile patients that would benefit from sperm fluorescent in situ hybridization (FISH) analysis, the number of chromosomes to be analyzed, and the diagnostic interpretation of the results obtained. DESIGN A retrospective study of sperm FISH analyses. SETTING Universitat Autònoma de Barcelona. PATIENT(S) Three hundred nineteen infertile men. INTERVENTION(S) Semen samples were processed for FISH. MAIN OUTCOME MEASURE(S) The frequencies of chromosomal abnormalities for chromosomes 13, 18, 21, X, and Y were compared to the seminogram, the somatic and meiotic karyotype, and the age. RESULT(S) The highest percentages of patients with an increased rate of sperm chromosome abnormalities were found in the oligozoospermic (50%), oligoasthenozoospermic (33.3%), and oligoasthenoteratozoospermic (21%) individuals. Low sperm count was the only parameter correlated with the percentage of chromosome abnormalities. The 14% of the individuals with a normal somatic karyotype had an increased rate of sperm chromosome abnormalities. This percentage was higher in the group with an altered somatic karyotype (36%) and in patients with meiotic abnormalities (26%). CONCLUSION(S) Sperm FISH studies are indicated when the oligo condition is present and in individuals with an abnormal somatic or meiotic karyotype. The analysis of chromosomes 21, X, and Y is enough to identify at-risk individuals. Significant differences in the rates of chromosome abnormalities should be taken into consideration regardless of the numerical value.

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring. For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice. To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21. This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Hidden mosaicism in patients with Klinefelter's syndrome: implications for genetic reproductive counselling

BACKGROUND Most individuals with Klinefelters syndrome (KS) are azoospermic but residual foci of spermatogenesis have been observed in some patients. However, no consistent predictive factors for testicular sperm extraction success have been established and mosaicism could be a factor to investigate. In this study, we have assessed the degree of mosaicism in somatic and germinal tissues in KS, the meiotic competence of 47,XXY germ cells and the aneuploidy rate of post-reductional cells. METHODS Five patients with KS previously diagnosed as pure 47,XXY have been studied. Samples from four donors were processed as controls. The chromosome constitution of lymphocytes, buccal mucosa and testicular tissue was assessed by interphase fluorescence in situ hybridization for chromosomes X, Y and 18. In meiotic figures, sex chromosome number and pairing was confirmed. RESULTS 46,XY cell lines were observed in all patients and tissues analysed. The degree of mosaicism (mean ± SD) differed among tissues (lowest in lymphocytes: 4.8 ± 2.5%; highest in Sertoli cells: 42.3 ± 11.1%). Meiotic figures were found in three cases (KS1, KS2 and KS5), all of them showed an XY complement. Hyperhaploid post-reductional cells were found in all patients (range: 3.3-36.4%) and increased rates versus controls (P< 0.05) were observed. CONCLUSIONS Diagnosis of homogeneous KS based on lymphocyte karyotyping should be contrasted in other tissues. Mucosa cells could help to better approximate the degree of germ cell mosaicism. Our results indicate that 47,XXY germ cells are not meiotically competent. Increased post-reductional aneuploidy rate is related to meiotic errors in 46,XY cells. Appropriate genetic counselling is recommended in KS.

FISH on sperm: spot-counting to stop counting? Not yet

OBJECTIVE To evaluate the reliability and applicability of the spot-counting system (Cytovision Spot AX workstation) which offers an alternative to the tedious manual analysis of sperm fluorescence in situ hybridization (FISH). DESIGN Manual and automatic analyses were performed and compared. SETTING Universitat Autònoma de Barcelona. PATIENT(S) Twenty-four men who asked for information on infertility showing different seminal parameters. INTERVENTION(S) A semen sample for each patient was collected and prepared for FISH. MAIN OUTCOME MEASURE(S) A dual-color FISH using specific probes for chromosomes 13 and 21 and a triple-color FISH with centromeric probes for chromosomes 18, X, and Y were used (Vysis). Standard FISH analysis was carried out. Automatic analysis was subsequently performed using a Spot AX system. RESULT(S) Overall, we performed 120 comparisons. In 116 out of 120 (96.67%), the percentage of anomalies reported using manual counting fell within the incidence detected using the automatic system. In the remaining comparisons, statistical differences were detected (4 out of 120; 3.33%). Time consumed by the automatic analysis was always higher than the manual one, being influenced by the characteristics of the preparations. CONCLUSION(S) The spot-counting system has potential, but before the service is ready to be offered, we still need to overcome some limitations associated with it.

Meiotic abnormalities in metaphase I human spermatocytes from infertile males: frequencies, chromosomes involved, and the relationships with polymorphic karyotype and seminal parameters

The aim of this study was to look in depth at the relationship between meiotic anomalies and male infertility, such as the determination of the chromosomes involved or the correlation with patient features. For this purpose, a total of 31 testicular tissue samples from individuals consulting for fertility problems were analyzed. Metaphase I cells were evaluated using a sequential methodology combining Leishman stained procedures and multiplex fluorescence in situ hybridization protocols. The number of chromosomal units and chiasmata count per bivalent were established and a hierarchical cluster analysis of the individuals was performed. The relationship of the seminogram and the karyotype over recombination were evaluated using Poisson regression models. Results obtained in this study show a significant percentage of infertile individuals with altered meiotic behavior, mostly specified as a reduction in chiasmata count in medium and large chromosomes, the presence of univalents, and the observation of tetraploid metaphases. Moreover, the number and the type of anomalies were found to be different between cells of the same individual, suggesting the coexistence of cell lines with normal meiotic behavior and cell lines with abnormalities. In addition, chromosomal abnormalities in metaphase I are significantly associated with oligozoospermia and/or polymorphic karyotype variants.

Apoptosis mediated by phosphatidylserine externalization in the elimination of aneuploid germ cells during human spermatogenesis

It has been described that aneuploidies trigger cell cycle checkpoints leading to apoptosis. The aim of this study was to assess the relationship between the presence of chromosomal abnormalities and apoptosis in germ cells and in Sertoli cells. Fourteen diagnostic testicular biopsies from infertile patients were processed following a sequential methodology, which included enzymatic disaggregation, apoptotic staining, cell sorting, cell fixation, and fluorescent in situ hybridization analysis. The chromosome constitution of germ cells (interphase pre‐meiotic germ cells, meiotic figures, post‐reductional germ cells, and spermatozoa) and Sertoli cells was evaluated in non‐sorted and flow‐sorted cell populations (apoptotic and viable). The mean percentage of aneuploidy was compared between the three fractions in each cell type using a Kruskal–Wallis test. If significant results were obtained, a two‐by‐two Chi‐squared test was performed. There were significant differences between the apoptotic fraction and the viable and non‐sorted fractions in the pre‐meiotic germ cells (p < 0.01). In the remaining cell types, no association between the presence of aneuploidy and apoptotic processes was observed, even in the case of post‐reductional germ cells in which we detected the highest rates of aneuploidy regardless of the fraction analyzed. From our data, it can be inferred that most of the aneuploid post‐reductional germ cells are efficiently removed from the testicular epithelium without differentiating into spermatozoa. Our results suggest that the elimination of aneuploid testicular epithelial cells is triggered by different mechanisms. Accordingly, the cellular elimination of aneuploid germ cells beyond the blood–testis barrier does not involve phosphatidylserine externalization.

Chromosome size, morphology, and gene density determine bivalent positioning in metaphase I human spermatocytes

OBJECTIVE To determine whether there is a preferential bivalent distribution pattern in metaphase I human spermatocytes and to analyze whether this positioning is influenced by chiasmata count, chromosome size, gene density, acrocentric morphology, and heterochromatic blocks. DESIGN Proximity frequencies of bivalents were evaluated with the analysis of meiotic preparations combining sequentially standard techniques and multiplex fluorescence in situ hybridization. SETTING University. PATIENT(S) Twenty-five men consulting for fertility problems. INTERVENTION(S) Unilateral testicular biopsies. MAIN OUTCOME MEASURE(S) Proximity analyses were performed for each bivalent considering as nearby bivalents those that were part of the first ring around the bivalent studied. Data were analyzed using Poisson regression models, multidimensional scaling, and cluster analysis. RESULT(S) Some bivalents have a preferential relative position. Significant associations among bivalents related to chromosome size, high gene density, and acrocentric morphology were observed. Chiasmata count and heterochromatic blocks were nonconditioning parameters of the bivalent organization. CONCLUSION(S) This study demonstrates that distribution in metaphase I is nonrandom and influenced by chromosome size, gene density, and acrocentric chromosome morphology. Results support that some features defining chromosome territories are maintained during meiosis.

Unpaired sex chromosomes in metaphase I human spermatocytes locally modify autosomal bivalents positioning

pachytene stage to round spermatids. Furthermore, it has been reported that sex-chromosome univalency is relatively common in human MI spermatocytes (from infertile males).10 The pairing features of these chromosomes (restricted to the short pseudoautosomal regions) promote the presence of univalents and explain the high incidence of sex-chromosome aneuploidies in human spermatozoa from infertile patients.11 Nevertheless, the influence of univalency in the territorial organization of human spermatocytes has not been ascertained. In this study, we have determined the effect of the presence of sex-chromosome univalents on the territorial organization of MI human spermatocytes from infertile patients with a normal somatic karyotype (46,XY). Chromosome proximity analysis was performed in a total of 1944 chromosome units from 81 MI,24,X,Y(MI bearing the X and Y chromosomes as univalents). MI spermatocytes were obtained from testicular biopsies from twenty-six 46,XY infertile men (range 1–10 MI per individual). Biopsies were obtained for diagnostic purposes to evaluate the incidence of meiotic chromosomal abnormalities in spermatocytes at MI,10 and surplus material was used for this study. The study was approved by the Institutional Review Board of the collaborating centers (Centre de Reproducció Assistida Fecunmed, Fundació Puigvert, Institut Marquès, Institut Universitari Dexeus and Instituto de Reproducción CEFER). In every instance, all of the patients signed their informed consent about participation in the study and the centers provided all data while preserving patient anonymity. Testicular biopsies were mechanically disaggregated, and cell suspensions were fixed and dropped onto dry slides.10 Chromosome preparations were processed following a protocol that combine Leishman staining (PanReac AppliChem, Castellar del Vallès, Spain) and multiplex fluorescence in situ hybridization (M-FISH, Spectra VysionTM Assay Protocol, Vysis Inc., Downers Grove, IL, USA) procedures (Figure 1).10 Briefly, MI images from Leishman-stained slides were captured and coordinates were noted. Slides were further processed for M-FISH analysis, which enabled unequivocal chromosome identification based on a distinctive color pattern. MI cells were re-located and re-analyzed after the FISH procedure. Proximity analysis was performed as it has been previously described.2 Briefly, in each cell, nearby chromosome units (including bivalents and univalents) were determined for each unit contrasting Dear Editor, In interphase nuclei, chromosomes occupy specific localizations called chromosome territories (CTs). Distribution of CTs has been related to chromosome size and gene density and is known to influence gene expression.1 This pattern of organization appears to be a widespread nuclear feature, both in somatic and germ cells. In spermatogenic cells, we have previously revealed a somatic-like distribution of bivalents at metaphase I (MI) with a nonrandom bivalent disposition and a preferential association between bivalents according to chromosome size, morphology, and gene density.2 Other studies have revealed preferential proximity of chromosomes 15 and 22 to the sex-chromosomes bivalent in MI.3 Moreover, a nonrandom chromosome distribution has also been reported in sperm nuclei.4 Although the functionality of CTs in male germ cells is not fully understood, it has been proposed that it has a role in the regulation of gene expression during spermatogenesis and early embryo development.5 Accordingly, alterations in CTs in germ cells could have implications for genome regulation leading to male infertility. Our research group has provided the first evidence of alteration in CTs in the presence of Robertsonian translocations (rob) in MI human spermatocytes.6 The impaired fertility in rob carriers is mainly due to the production of chromosomally abnormal spermatozoa because of the regular segregation of the chromosomes involved in the reorganization and the occurrence of interchromosomal effects.7 However, we have also revealed in rob carriers that, the trivalent configuration of the reorganized chromosomes disturbs the overall chromosome positioning at MI.6 Thus, the disruption of MI CTs could be an additional contributory factor to infertility associated with this kind of chromosomal reorganization.6 Interestingly, in human spermatogenesis, the XY chromosomes organize in the sex vesicle at prophase I,8 and in the postmeiotic sex chromatin (PMSC) body in spermatids.9 In terms of gene expression and CTs organization, sex chromosomes configure a silent compartment where chromosomes appear inactivated from the LETTER TO THE EDITOR