Osvaldo Rajmil

Linear increase of structural and numerical chromosome 9 abnormalities in human sperm regarding age.

A simultaneous four-colour fluorescence in situ hybridisation (FISH) assay was used in human sperm in order to search for a paternal age effect on: (1) the incidence of structural aberrations and aneuploidy of chromosome 9, and (2) the sex ratio in both normal spermatozoa and spermatozoa with a numerical or structural abnormality of chromosome 9. The sperm samples were collected from 18 healthy donors, aged 24–74 years (mean 48.8 years old). Specific probes for the subtelomeric 9q region (9qter), centromeric regions of chromosomes 6 and 9, and the satellite III region of the Y chromosome were used for FISH analysis. A total of 190 117 sperms were evaluated with a minimum of 10 000 sperm scored from each donor. A significant linear increase in the overall level of duplications and deletions for the centromeric and subtelomeric regions of chromosome 9 (P≤0.002), chromosome 9 disomy (P<0.0001) as well as diploidy (P<0.0001) was detected in relation to age. The percentage of increase for each 10-year period was 29% for chromosome 9 disomy, 18.8% for diploidy, and ranged from 14.6 to 28% for structural aberrations. Our results indicate a linear increase in structural aberrations and disomy for chromosome 9 in sperm with respect to age.

Linear increase of diploidy in human sperm with age: a four-colour FISH study.

The aim of this study was to determine if donor age is associated with an increased incidence of diploidy and of disomy for the sex chromosomes and for chromosomes 6 and 21. We used simultaneous fluorescence in situ hybridisation (FISH) for chromosomes 6, 21, X and Y in sperm from 18 healthy donors, aged 24–74 years (mean 48.8 years). A total of 194 024 sperm were analysed, with a minimum of 10 000 sperm scored for each donor. Our results indicate a significant increase of the level of diploidy (P=0.002), and a marginal significance of total sex chromosome disomy (P=0.055) with age. No increase was observed for disomies XX, YY, XY, 21 or 6. The percentages of increase for disomy and for diploidy ranged from 0.3 to 17% for each 10-year period. Chromosomes 6 and 21 did not segregate preferentially with the X or Y chromosomes. Our findings show a linear trend association between age and diploidy in human males.

Clinical relevance of Y-linked CNV screening in male infertility: new insights based on the 8-year experience of a diagnostic genetic laboratory

AZF microdeletion screening is routinely performed in the diagnostic work-up for male infertility; however, some issues remain debated. In this study, we provide insights into the sperm concentration cutoff value for routine testing, the predictive value of AZFc deletion for testicular sperm retrieval and the Y-background contribution to the interpopulation variability of deletion frequencies. In the Spanish population, partial AZFc rearrangements have been poorly explored and no data exist on partial duplications. In our study, 27/806 (3.3%) patients carried complete AZF deletions. All were azoo/cryptozoospermic, except for one whose sperm concentration was 2 × 106/ml. In AZFc-deleted men, we observed a lower sperm recovery rate upon conventional TESE (9.1%) compared with the literature (60–80% with microTESE). Haplogroup E was the most represented among non-Spanish and hgr P among Spanish AZF deletion carriers. The analysis of AZFc partial rearrangements included 330 idiopathic infertile patients and 385 controls of Spanish origin. Gr/gr deletion, but not AZFc partial duplications, was significantly associated with spermatogenic impairment. Our data integrated with the literature suggest that: (1) routine AZF microdeletion testing could eventually include only men with ≤2 × 106/ml; (2) classical TESE is associated with low sperm recovery rate in azoospermic AZFc-deleted men, and therefore microTESE should be preferred; (3) Y background could partially explain the differences in deletion frequencies among populations. Finally, our data on gr/gr deletion further support the inclusion of this genetic test in the work-up of infertile men, whereas partial AZFc duplications do not represent a risk for spermatogenic failure in the Spanish population.

Meiotic chromosome abnormalities in fertile men: are they increasing?

OBJECTIVE To determine the basal frequencies of meiotic chromosome abnormalities in fertile men. DESIGN Descriptive design. SETTING Research university laboratory and clinical andrology service. PATIENT(S) Seventeen fertile donors undergoing vasectomy. INTERVENTION(S) Analysis of testicular biopsies. MAIN OUTCOME MEASURE(S) Meiotic chromosome abnormalities in metaphase I spermatocytes. RESULT(S) A total of 1,407 spermatocytes I was analyzed. The main meiotic abnormality was absence or low chiasma number of individual bivalents (23.4%), followed by structural (3.3%) and numerical (0.7%) abnormalities. Sex chromosomes and G-group chromosomes were the most commonly found as univalents at metaphase I. Statistically significant heterogeneity was found for meiotic abnormalities among fertile men, caused by interindividual variation in the level of dissociated sex chromosomes (ranging from 3.2% to 43.7%). The mean total percentage of meiotic abnormalities in spermatocytes I from fertile men was 27.4%, 1.7 times higher than those described a few decades ago in fertile and even in infertile men. CONCLUSION(S) Fertile men are a heterogeneous group for meiotic errors, with individuals showing percentages of meiotic abnormalities as high as 50%. From these findings, caution is recommended when using meiotic studies to diagnose and provide genetic counselling to patients consulting for infertility.

Baseline expression profile of meiotic-specific genes in healthy fertile males

OBJECTIVE To establish the quantitative gene-expression profile of nine meiotic genes involved in synapsis and chromosome cohesion (SYCP1, SPO11, MSH4, MSH5, MLH1, MLH3, PMS2, STAG3, and REC8) in healthy fertile males. DESIGN Prospective study. SETTING Research university laboratory and clinical andrology service. PATIENT(S) Twenty healthy males of proven fertility underwent a vasectomy procedure and four infertile patients with Sertoli cell-only syndrome (SCOS). INTERVENTION(S) Analysis of testicular biopsies from 20 fertile males and four SCOS patients. MAIN OUTCOME MEASURE(S) Quantitative gene expression by real-time polymerase chain reaction in testicular biopsies. RESULT(S) Four of the nine genes under study (PMS2, MLH3, MLH1, and REC8) are expressed in both fertile males and SCOS patients. The remaining five genes are only (SYCP1, SPO11, MSH4, and MSH5) or mainly (STAG3) expressed in fertile males, and thus they could be considered meiotic-specific genes. All genes analyzed are expressed at similar levels among fertile individuals CONCLUSION(S) Gene expression levels reported in this study could be considered the gene expression profile of fertile population, and could be used to compare with the expression pattern of infertile patients. Expression of meiotic-specific genes could be used as a clinical diagnosis tool to ascertain the origin of some cases of idiopathic male infertility and sterility.

Male-to-male transmission of X-linked Alport syndrome in a boy with a 47,XXY karyotype

Alport syndrome (AS) is a genetically heterogeneous renal hereditary disease. Male-to-male transmission has been considered fully indicative of autosomal dominant AS. We report a family with male-to-male transmission of X-linked AS due to an extra X chromosome of paternal origin in the proband. Linkage analysis excluded the autosomal loci and demonstrated segregation with the COL4A5 locus (Xq22.3). Sperm FISH analysis from his father detected an increased XY disomy. Mutation screening of the COL4A5 gene identified a splicing mutation, c.4688G>A. The proband and his paternal grandmother showed random X chromosome inactivation. However, a preferential expression of the aberrantly spliced transcript was detected in the proband when compared to his grandmother. This finding could explain why the AS phenotype of this 47,XXY boy resembles more an affected male than a female carrier. This is the first reported case of concurrence of Alport and Klinefelter syndromes.

ESR1 promoter polymorphism is not associated with nonsyndromic cryptorchidism

The ESR1 promoter microsatellite (TA)n was reported as a potential functional polymorphism. In a case-control study, we were unable to demonstrate any association between (TA)n and nonsyndromic cryptorchidism in Italian and Spanish study populations.

Growth hormone response to growth hormone-releasing hormone stimulation in oligozoospermic patients *

OBJECTIVE To evaluate the GH response to growth hormone-releasing hormone (GH-RH) stimulation in oligozoospermic men. SETTING Outpatient Clinic of Andrology at the Fundación Puigvert and the Department of Clinical Chemistry, Hospital de la Santa Creu i Sant Pau, Barcelona, Spain. PATIENTS Fifteen oligozoospermic patients and 15 normozoospermic fertile men matched for age and body mass index. INTERVENTION Endocrine status was determined by assay of basal levels of gonadotropins (FSH, LH), T, E2, inhibin, and insulin-like growth factor I (IGF-I). Serum GH levels were measured before and after GH-RH administration. RESULTS GH response to GH-RH was significantly greater in patients than in controls. There was a positive correlation between the GH response and IGF-I levels in oligozoospermic patients only. Regression analysis showed a significant negative association of GH peak with inhibin after controlling for IGF-I in oligozoospermic patients. CONCLUSION The results indicate that there is an altered responsiveness of pituitary to GH-RH administration in oligozoospermic patients; this did not appear to be due to the influence of gonadal steroid levels but rather to inhibin or some related peptide.

Association of serum testosterone levels and testicular volume in adult patients

A retrospective observational study was undertaken to gain new insight into the relationship between total testicular volume and levels of serum testosterone, luteinising hormone, follicle‐stimulating hormone, prolactin and clinical variables. A total of 312 men with sexual dysfunction or infertility were divided into groups A and B (156 each) on the basis of basal plasma testosterone ≤5 nmol/L of ≥12 nmol/L respectively. Group A was subclassified in A1 (primary hypogonadism) and A2 (secondary hypogonadism). There were significant differences in total testicular volume between group A (15.33 ± 11.94 ml) and group B (36.74 ± 6.9; p < .001) and also between subgroup A1 (11.07 ± 8.49 ml) and subgroup A2 (23.62 ± 13.04 ml; p < .001). Only 13.5% of patients in group B had a total testicular volume <30 ml. Differences in all studied parameters were found between group A and group B. There were no variations when comparing age, body mass index and testosterone in groups A1 and A2. The use of total testicular volume and body mass index together for predicting testosterone levels yields a sensitivity and specificity of 85.3% and 86.5% respectively. Logistic regression analysis, univariate and multivariate models, using the measurement of total testicular volume resulted in a high capacity to predict testosterone levels.

Whole exome sequencing in non-obstructive azoospermia allows the identification of a high-risk subgroup of infertile men for undiagnosed Fanconi Anemia, a cancer-prone disease

Whole exome sequencing in non-obstructive azoospermia allows the identification of a high-risk subgroup of infertile men for undiagnosed Fanconi Anemia, a cancer-prone disease A. RIERA-ESCAMILLA, C. CHIANESE, D. MORENO-MENDOZA, O. RAJMIL, E. CASAMONTI, E. RUIZ-CASTA~ NE, J. SURRALL ES AND C. KRAUSZ Andrology Department, Fundaci o Puigvert, Universitat Aut onoma de Barcelona, IIB-Sant Pau, Barcelona, Catalonia, Spain; Department of Experimental and Clinical Biomedical Sciences “Mario Serio”, Centre of Excellence DeNothe, University of Florence, Florence, Italy; Genetics Department and Biomedical Research Institute, Hospital de Sant Pau, Center for Biomedical Research on Rare Diseases (CIBERER), and Department of Genetics and Microbiology, Universitat Aut onoma de Barcelona, Barcelona, Catalonia, Spain Background: The etiology of non-obstructive azoospermia (NOA) remains unknown in about 40% of cases and genetic factors are likely to be involved in a large proportion of them. Gene mutations involved in stem cell proliferation and DNA repair may cause isolated NOA or be responsible for syndromic diseases, such as Fanconi Anemia (FA). Although the most frequent presenting symptom in FA is bone marrow failure in childhood, in about 10% of cases the diagnosis is delayed until adulthood and in these late-onset cases the presenting syndrome is frequently a malignant tumor. Methods: An idiopathic NOA patient (index case) with consanguineous parents was subjected to Whole-Exome Sequencing (WES) with the purpose to identify the etiology of NOA. In the second part of the study, two-steps Sanger sequencing of the Fanconi Anemia Complementation Group A gene (FANCA) in the brother of the index case and in 27 selected NOA patients was performed. DEB-induced chromosome breakage test was carried out to confirm the FA diagnosis. Results: Through WES we identified a rare pathogenic homozygous FANCA variant (c.2639G>A) in the index case, affected by NOA due to Sertoli Cell only syndrome (SCOS). The patient’s brother (also affected by NOA) has been found to be a homozygous carrier of the same mutation. The two brothers did not manifest overt anemia, though chromosomal breakage test revealed a reverse somatic mosaicism in the index case and a typical FA picture in the brother. Following this incidental finding of FA, we selected 27 NOA patients with similar testicular phenotype and borderline/mild hematological alterations. Sanger sequencing of the FANCA gene in this selected group of patients allowed the identification of one additional NOA patient with SCOS showing compound heterozygous variants (c.3788_3790delTCT and c.3913C>T). Following our investigation, the three subjects with FANCA mutations are now receiving specific medical attention including strict follow-up by oncohematologists. Conclusion: Our study reports an unexpectedly high frequency of occult FA in a specific subgroup of NOA patients with mild or borderline hematological alterations (2/28; 7.1%). The screening for FANCA mutations in such patients may allow the identification of undiagnosed FA before the appearance of other severe clinical manifestations (cancer, bone marrow failure etc.) of the disease. Our finding highlights the importance to introduce the systematic evaluation of hematological parameters into the routine andrological workup in NOA patients. Moreover, corroborates previous epidemiological observations reporting a higher risk of morbidity (including cancer) and a lower life expectancy in infertile men in respect to fertile, normozoospermic men. Based on our data, we propose a novel genetic link between idiopathic NOA and a chronic, cancer-prone disease. Funding: Instituto Carlos III (FIS/FEDER: PI14/01250; PI17/01822) and Ente Cassa di Risparmio di Firenze.