Efficacy of MLPA for detection of Y-chromosome microdeletions in infertile Brazilian patients.

PURPOSE: Worldwide publications follow the gold standard method-the polymerase chain reaction (PCR)-for detecting Y-chromosome microdeletions; however, markers are frequently variable between the studies. Can we detect the deletions by another molecular method with more genomic coverage? The Y chromosome harbors several different genes responsible for testicular development and spermatogenesis, and its repetitive conformation predisposes it to complex rearrangements that have clinical impact. Our aim was to evaluate a molecular diagnostic method, the Multiplex Ligand Probe-dependent Amplification (MLPA), which is also a valuable ancillary method for the identification of deletions, duplications, and rearrangements in a single and faster reaction, leading to a better comprehension of patients' phenotypes, and should be considered a useful tool for detection of Y chromosome deletions. METHODS: This is a study of diagnostic accuracy (transversal prospective study) conducted to investigate Y-chromosome deletions in 84 individuals through PCR and MLPA methods. Forty-three infertile men (azoospermic and oligozoospermic) and 41 controls (40 fertile men and 1 normal karyotyped woman) were analyzed by PCR and MLPA techniques. RESULTS: We diagnosed seven (7) deletions (16.2%) by PCR and 9 with MLPA (21%). In addition, we found five (5) duplications and a suggestive mosaic. CONCLUSION: Our results demonstrate that MLPA technique is valuable in the investigation of microdeletions and microduplications. Besides deletions, duplications can cause instability of chromosome genes, possibly leading to infertility. Both studied techniques provide an advantageous diagnostic strategy, thus enabling a better genetic counseling.

Recategorisation of body mass index to achieve andrological predictive power: a study in more than 20 000 patients.

The aim of this study was to recategorise body mass index (BMI) in order to classify patients according to their risk of semen abnormalities. Patients (n=20563) presenting at an andrology laboratory were classified into five groups according to BMI: underweight (BMI <20kg m-2), normal weight (BMI 20-24.9kg m-2), overweight (BMI 25-29.9kg m-2), obese (BMI 30-39.9kg m-2) and morbidly obese (BMI >40kg m-2). Semen quality was evaluated to determine: (1) differences between groups using analysis of variance (ANOVA); (2) the chances of semen abnormalities (using generalised linear models, Chi-squared tests and odds ratios); (3) reference BMI values with andrological predictive power (multivariate conglomerate analyses and multivariate analysis of variance (MANOVA)); and (4) expected values of abnormalities for each new group resulting from BMI recategorisation. Morbidly obese and underweight patients exhibited the highest decrease in semen quality and had higher chances of semen abnormalities. The smallest number of sperm abnormalities was found at a BMI of 27kg m-2. Four reference values were identified, recategorising BMI into four groups according to their risk of semen abnormalities (from lowest to highest risk): Group1,BMI between 20 and 32kg m-2; Group2, BMI <20 and BMI >32-37kg m-2; Group3, BMI >37-42kg m-2; and Group4, BMI >42kg m-2. A BMI <20 or >32kg m-2 is negatively associated with semen quality; these negative associations on semen quality increase from a BMI >37kg m-2 and increase even further for BMI >42kg m-2. The BMI recategorisation in this study has andrological predictive power.

Male cytogenetic evaluation prior to assisted reproduction procedures performed in Mar del Plata, Argentina.

OBJECTIVE: This paper aimed to estimate the frequency of occurrence and the types of chromosomal abnormalities found in 141 infertile men with abnormal semen parameters. METHODS: the frequency and type of chromosomal abnormalities were determined with male mitotic karyotype analysis from peripheral blood through chromosome banding techniques before assisted reproduction procedures. RESULTS: In this series of 141 infertile men, 19 (13%) had chromosomal anomalies and 35 (25%) had polymorphic variants. The main chromosome abnormalities were reciprocal translocations and marker chromosomes in mosaic. CONCLUSIONS: These results stress the relevance of cytogenetic studies for infertile males as a diagnostic tool and a valuable input in genetic counseling.

Genetic screening and evaluation for chromosomal abnormalities of infertile males in Jilin Province, China.

Chromosomal abnormality is the most common genetic cause of male infertility, particularly in cases of azoospermia, oligozoospermia, and recurrent spontaneous abortion. Chromosomal rearrangement may interrupt an important gene or exert position effects. The functionality of genes at specific breakpoints, perhaps with a specific role in spermatogenesis, may be altered by such rearrangements. Structural chromosome abnormalities are furthermore known to increase the risk of pregnancy loss. In this study, we aimed to assess chromosomal defects in infertile men from Jilin Province, China, by genetic screening and to evaluate the relationship between structural chromosome abnormalities and male infertility. The prevalence of chromosomal abnormalities among the study participants (receiving genetic counseling in Jilin Province, China) was 10.55%. The most common chromosome abnormality was Klinefelter syndrome, and the study findings suggested that azoospermia and oligospermia may result from structural chromosomal abnormalities. Chromosome 1 was shown to be most commonly involved in male infertility and balanced chromosomal translocation was identified as one of the causes of recurrent spontaneous abortion. Chromosomes 4, 7, and 10 were the most commonly involved chromosomes in male partners of women experiencing repeated abortion.

Decline in semen quality among infertile men in Brazil during the past 10 years.

PURPOSE: To investigate whether the semen quality of men undergoing conventional semen analysis is deteriorating over time. MATERIALS AND METHODS: We analyzed and compared the sperm count, motility and morphology of 2300 semen samples provided by males undergoing conventional seminal analysis, from years 2000 to 2002 and 2010 to 2012. The incidences of severe oligozoospermia and azoospermia over time were also compared. RESULTS: A total of 764 sperm samples were analyzed in 2000-2002 and 1536 in 2010-2012. Over time, the mean sperm concentration/ml decreased significantly from 61.7 million in 2000-2002 to 26.7 million in 2010-2012 (R2 = 11.4%, p < 0.001), the total sperm concentration decreased significantly from 183.0 million to 82.8 million (R2 = 11.3%, p < 0.001), and the percentage of normal forms decreased significantly from 4.6% to 2.7% (R2 = 9.8%, p < 0.001). The incidence of severe oligozoospermia significantly increased from 15.7% to 30.3% (OR: 1.09, p < 0.001) and the incidence of azoospermia increased from 4.9% to 8.5% (OR: 1.06, p = 0.001). CONCLUSIONS: This study demonstrated a significant time-related decline in semen quality of infertile patients. This finding might have implications on fertility and emphasizes the need for further studies addressing subject's life-style in order to find and reduce the causative agents. Future prospective and multicenter studies including representative samples of the general population are needed to confirm whether semen quality is really declining.

Decline in semen quality among infertile men in Brazil during the past 10 years

ABSTRACTPurpose:To investigate whether the semen quality of men undergoing conventional semen analysis is deteriorating over time.Materials and Methods:We analyzed and compared the sperm count, motility and morphology of 2300 semen samples provided by males undergoing conventional seminal analysis, from years 2000 to 2002 and 2010 to 2012. The incidences of severe oligozoospermia and azoospermia over time were also compared.Results:A total of 764 sperm samples were analyzed in 2000-2002 and 1536 in 20102012. Over time, the mean sperm concentration/ml decreased significantly from 61.7 million in 2000-2002 to 26.7 million in 2010-2012 (R2=11.4%, p<0.001), the total sperm concentration decreased significantly from 183.0 million to 82.8 million (R2=11.3%, p<0.001), and the percentage of normal forms decreased significantly from 4.6% to 2.7% (R2=9.8%, p<0.001). The incidence of severe oligozoospermia significantly increased from 15.7% to 30.3% (OR: 1.09, p<0.001) and the incidence of azoospermia increased from 4.9% to 8.5% (OR: 1.06, p=0.001).Conclusions:This study demonstrated a significant time-related decline in semen quality of infertile patients. This finding might have implications on fertility and emphasizes the need for further studies addressing subject's life-style in order to find and reduce the causative agents. Future prospective and multicenter studies including representative samples of the general population are needed to confirm whether semen quality is really declining.

Variants in endothelial nitric oxide synthase (eNOS) gene in idiopathic infertile Brazilian men.

PURPOSE: In recent years, considerable concern has been expressed about the deleterious effects of reactive oxygen species (ROS) on sperm function, because ROS at high levels is potentially detrimental to sperm function and quality. Nitric oxide (NO) is a powerful anti-oxidant present in seminal plasma. The aim of the study was to analyze the distribution of the of endothelial nitric oxide synthase (eNOS) gene (T-786C, G894T, e 4a/b) polymorphisms in idiopathic infertile Brazilian men and evaluate the possible role of these polymorphisms in sperm count. METHODS: A case-control study was performed comprising 208 infertile men [n=74 with non-obstructive azoospermia and n=134 with severe oligozoospermia] and 201 fertile men as controls. Genotyping of eNOS polymorphisms was performed by real time (T-786C and G894T) and conventional PCR (4a/b). The results were analyzed statistically and a p-value<0.05 was considered significant. RESULTS: According to the sperm count, relatively similar eNOS polymorphism genotypes and allele frequencies were found among the groups. Combined genotypes of the eNOS polymorphisms did not identify a haplotype associated with idiopathic infertility, even when the patients were separated in non-obstructive azoospermia or severe oligozoospermia. CONCLUSION: In conclusion, the findings demonstrate that, in Brazilian population studied, genetic variations, T-786C, G894T, and e 4a/b, of the eNOS gene are not associated with male infertility.

Alteraciones del espermiograma en pacientes asistidos en la Unidad de Biología de la Reproducción de la Universidad de Chile / Alterations in the semen analysis of patients attending the reproductive biology unit at the University of Chile

En Chile, 10 a 15 por ciento de las parejas son consideradas como infértiles y el factor masculino es responsable en un 50 por ciento de los casos. El espermiograma, es un examen fundamental para el diagnóstico inicial de parejas infértiles. Objetivo: Determinar cambios en cuatro parámetros del espermiograma de mayor valor diagnóstico, según edad, estableciendo el parámetro alterado de mayor frecuencia. Métodos: Se realizo un estudio descriptivo retrospectivo de una muestra de 100 pacientes atendidos por problemas de fertilidad entre los años 2004 y 2009, clasificándolos en cuatro grupos etarios. Resultados: Al evaluar la concentración espermática, el 33 por ciento presenta: 5 baja concentración. El 86 por ciento de los pacientes presento astenozoospermia. El 81 por ciento de los pacientes presento anormalidad en la morfología espermática. La viabilidad espermática fue anormal en el 8 por ciento de los pacientes, siendo significativamente más alto en el grupo etario de mayor edad. Conclusiones: Los parámetros estudiados muestran un alto porcentaje de anormalidad en la población en estudio. Al comparar entre grupos, el grupo de mayor edad (sobre los 47 &los) presenta un aumento significativo del- porcentaje de alteraciones en morfología, motilidad y viabilidad respecto a los otros grupos etarios, estableciéndose la edad como un factor negativo en la calidad espermática. La movilidad corresponde al parámetro mas frecuentemente alterado seguido por la morfología espermática a medida que el varón consultante envejece.

In Chile, 10 to 15 percent of the couples are considered as infertile. Since the male factor is responsible of 50 percent of the cases, spermogram is an essential test for initial diagnosis of the infertile couple. Objective: To analyze the frequency of change in four spermogram parameters -according to age- to determine their diagnostic value. Method: A descriptive retrospective study of spermogram data from 100 patients -subdivided in four age groups- analyzed in our Unit for fertility problems between 2004 and 2009 was performed. Results: In sperm count, 33 percenr showed an abnormally low concentration. An 86 percent of the patients has astenozoospermia. 81 percent of the patients showed abnormal sperm morphology. Sperm viability was subnormal in 8 percent of the patients, being significantly higher in the oldest group. Conclusions: The seminal parameters analyzed revealed a high percentage of anomalies in the studied population. The oldest group had significant percentages of anomalies in sperm motility, morphology and viability, thus corroborating that age is a negative factor that affects semen quality. Sperm motility was the most frequently altered parameter followed by sperm morphology in the population under study.

Polymorphisms of estrogen receptors alpha and beta in idiopathic, infertile Brazilian men: a case-control study.

Estrogen plays an important role in the human reproductive system and its action is mediated mainly by two specific receptors: α (ERα) and ß (ERß). There are polymorphic variants in both ER genes, and studies showed their association with reproductive outcomes. We aimed to determine the distribution of ERα and ERß gene polymorphisms in idiopathic, infertile Brazilian patients in a case-control study comprising 187 idiopathic, infertile Brazilian men with nonobstructive azoospermia (NOA, n = 78) or severe oligozoospermia (SO, n = 109) and 216 fertile men. Detection of ERα (PvuII and XbaI) and ERß (AluI and RsaI) gene polymorphisms were performed using TaqMan PCR. The results were analyzed statically, and a P-value < 0.05 was considered significant. Single-marker analysis revealed that neither PvuII nor XbaI polymorphisms of the ERα gene were associated either with NOA group (P = 0.662 and P = 0.527, respectively) or SO group (P = 0.777 and P = 1.0, respectively). Regarding ERß polymorphisms, no statistical difference was observed between the AluI polymorphism and NOA group compared to controls (P = 1.0) or between SO group and controls (P = 0.423). We found similar results with the RsaI polymorphism. Statistical analysis did not reveal a difference between NOA (P = 0.740) and SO (P = 0.920) groups compared to controls. Combined genotypes of ERα and ERß polymorphisms did not identify a haplotype associated with idiopathic infertility. Thus, in the Brazilian population, genetic variations in both estrogen receptors alpha (PvuII and XbaI) and beta (AluI and RsaI) were not relevant to idiopathic infertility.

Embryos generated using testicular spermatozoa have higher developmental potential than those obtained using epididymal spermatozoa in men with obstructive azoospermia.

OBJECTIVE: To determine whether the injection of testicular spermatozoa results in more viable embryos (higher implantation rate) than injection of epididymal spermatozoa in cases of obstructive azoospermia. DESIGN: Retrospective analysis of 265 cases of testicular sperm aspiration (TESA) and percutaneous sperm aspiration (PESA), including 185 cases of obstructive azoospermia. SETTING: Private Infertility clinic. PATIENT(S): None, charts review. INTERVENTION(S): None, charts review. MAIN OUTCOME MEASURE(S): Clinical pregnancy rate (PR), implantation rate. RESULT(S): Although fertilization rates were higher in the PESA group, implantation rates were significantly better in the TESA group. There was also a trend to higher ongoing PR and lower miscarriage rates in TESA cases. CONCLUSION(S): In cases of obstructive azoospermia, embryos generated using testicular spermatozoa have higher developmental potential than those obtained using epididymal spermatozoa.

Asthenozoospermia: analysis of a large population.

Spermatozoa travel a long distance to meet and fertilize the oocyte, so sperm motility is a requisite for normal fertilization. Asthenozoospermia, or low sperm motility, is a common cause of human male infertility. This is a retrospective study (1992-1999) to document the prevalence of this pathology in infertile men and to clarify the probable factors associated to its etiology. The prevalence was 18.71% for asthenozoospermia and 63.13% for asthenozoospermia associated with oligo- and/or teratozoo-spermia; thus, 81.84% of the studied samples had altered motility. Leukocytospermia, the ratio of germ cells/sperm, anti-sperm antibodies, consistency, biochemical markers of accessory sex glands, and sperm response after swim-up were studied in normospermic (N), asthenozoospermic (A), and combined asthenozoospermic (C) samples. No significant difference was found in the frequency of leukocytospermia among groups. The rate of germ cells/(spermatozoa + germ cells) between C and N (p < .01) and C and A (p < .01) was statistically different, while no difference was found on comparing N and A. MAR-test over 40% was found in 6% of the A samples and 7.6% of the C, while no positive values were observed in the N group. The percentage of hyperviscous samples was higher in the low sperm motility samples than in the normal group. Data on concentration of the biochemical markers seem to be decreased in asthenozoospermia. Pure and combined asthenozoo-spermia showed different behavior in sperm recovery after swim-up. Two different asthenozoospermias could be defined: the pure one where sperm environment is involved (immunological factor, hyperviscosity, and secretory gland function) and the combined, where the testis is comprised.

[Frequency and etiology of azoospermia in the study of infertile couples]. / Frecuencia y etiología de azoospermia en el estudio de parejas infértiles.

UNLABELLED: One out of ten couples seek some kind of treatment for infertility of which males are infertile between 30-50%. Azoospermia has been reported in the range of 10 to 20% of cases. MATERIAL AND METHODS: We reviewed 331 cases seen at the clinic of Reproductive Biology of the Hospital Juárez de México, SSA, from January 1993 through February 2000. 66 patients were found with azoospermia (19.93%). Studies performed were: seminogram, testicular and transrectal ultrasonography, cariotype, deferentovesiculography and testicular biopsy. Radioimmunoassay of gonadotropins (FSH, LH) and testosterone was also practiced. RESULTS: Twelve patients (18.18%) were excluded either for desertion or incompletion of studies. The etiology of azoospermia of remainder 54 cases was: secretory (85.19%) of which 41.3% was idiopathic, 10.9% had germ cells aplasia, 10.9% varichocele, 10.9% Klinefelter syndrome. Secondary secreting azoospermia was found in two cases with Kallman's syndrome one patient had excretory and 7 obstructive azoospermia. CONCLUSIONS: Azoospermia was diagnosed in 19.93% (n = 54) with a mean age of 30 years. 85.19% had primary secretory azoospermia with FSH & LH hypergonadotropism in 85.8 and 56.4% respectively (p < 0.05). Obstructive azoospermia was seen in 12.96% and only one case (1.85%) presented excretory azoospermia.

Microdeleçöes do cromossoma Y em homens azoospérmicos e oligoospérmicos severos / Y-Chromosome microdeletions in azoospermic and severel oligozoospermic men

O fator masculino é responsável por aproximadamente metade dos casos de infertilidade do casal. Em mais de 60 por cento dos casos a origem da funçäo testicular diminuída näo é conhecida, podendo haver muitas anomalias genéticas näo identificadas. Microdeleçöes do braço longo do cromossoma Y säo associadas com falência da espermatogênese e tem sido usado para definir três regiöes do Yq (AZFa, AZFb e AZFc) que estäo correntemente deletados em homens inférteis. Em torno de 10 a 15 por cento dos azoospérmicos e 5 a 10 por cento dos homens severemente oligospérmicos tem microdeleçöes do Yq. Técnicas de reproduçäo assistida, principalmente Injeçäo Intra Citoplasmática de Esperma em associaçäo com retirada de esperma testicular, representam uma eficiente terapia para estes pacientes. Os autores fazem uma revisäo atual das microdeleçöes do cromossoma Y e suas conseqüências na fertilidade masculina.

Semen quality of workers occupationally exposed to hydrocarbons.

OBJECTIVE: To determine whether occupational exposure of men to hydrocarbons has adverse effects on the quality of their semen. DESIGN: Comparative study. SETTING: The rubber industry in Mexico City. PATIENT(S): Forty-eight workers who were exposed to hydrocarbons for 2-24 years and 42 unexposed workers. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Environmental hydrocarbon concentrations were determined by continuous air monitoring in all areas of the factory. Analyses of semen samples were performed in accordance with World Health Organization criteria. RESULT(S): Hydrocarbon concentrations were as follows: ethylbenzene, 220.7-234 mg/m3; benzene, 31.9-47.8 mg/m3; toluene, 189.7-212.5 mg/m3; and xylene, 47-56.4 mg/m3. The number of subjects with ejaculates that had normal characteristics was greater in the unexposed group (76%) than in the exposed group (17%). More abnormal characteristics were found in the semen of exposed workers than unexposed workers, including alterations in viscosity, liquefaction capacity, sperm count, sperm motility, and the proportion of sperm with normal morphology. Some abnormal characteristics correlated with the number of years of exposure to the hydrocarbons. CONCLUSION(S): Damage to the spermatogenic process resulting from hydrocarbon exposure was demonstrated by an increased rate of abnormalities in the semen of exposed workers compared with unexposed workers. This information may be useful for conducting future analyses of reproductive risks related to exposure to high concentrations of hydrocarbons.

Síndrome de Klinefelter: presentación de 18 casos / Klinefelterïs syndrome: presentation of 18 cases

Se revisan los aspectos clínicos y de laboratorio de 18 pacientes con síndrome de Klinefelter. El síndrome de Klinefelter es la causa congénita más frecuente de hipogonadismo hipergonadotropo y afecta a 1 de cada 500 varones. El diagnóstico se sospecha en un adolescente que falla en progresar en forma normal durante la pubertad y cuyos testículos son pequeños y firmes. El diagnóstico se confirma al encontrar gonadotrofinas elevadas y en en el cariotipo un cromosoma X extra. El diagnóstico precoz permite instaurar un tratamiento a los 11 o 12 años, permitiendo que la pubertad progrese en armonía, con la de sus pares y evita el riesgo de osteoporosis a futuro. En adultos, el tratamiento con enantato de testosterona o parches transdérmicos ejerce efectos positivos en la masa ósea, muscular y en la libido

Use of clomiphene citrate in the treatment of men with high sperm chromatin stability.

OBJECTIVE: To determine whether improvements of the seminal vesicle function after a 5-day course with clomiphene citrate (CC) may reduce the prevalence of men with high sperm chromatin stability under conditions of sodium dodecyl sulfate (SDS)-ethylenediaminetetraacetic acid (EDTA). DESIGN: A prospective study. SETTING: Andrology laboratory at the Instituto de Investigaciones de la Altura, Universidad Peruana Cayetano Heredia, Lima, Peru. PATIENT(S): Forty-one male partners of infertile couples attending the andrology laboratory. INTERVENTION(S): Clomiphene citrate was administered orally twice a day. Men were treated with CC at 100 mg daily for 5 days. Blood and semen samples were collected before treatment and 24 hours after the last administration. MAIN OUTCOME MEASURE(S): Serum testosterone, seminal fructose, sperm motility, sperm chromatin stability after SDS and EDTA, and prevalence of high sperm chromatin stability. RESULT(S): The percentage of stable sperm after SDS-EDTA correlated inversely with the basal corrected concentration of seminal fructose (-1.77 +/- 0.89, beta +/- SE). High sperm chromatin stability was observed in 53.8% of the study population and in 66.7% of patients with hypofunction of the seminal vesicles. In those men whose seminal vesicle function improved after treatment with CC, the prevalence of high sperm chromatin stability was reduced from 67% to 25% (chi2 = 5.34). Logistic regression analysis showed that the higher the basal corrected seminal fructose levels and the higher the basal serum testosterone levels, the lower the probability of nonresponse of the sperm chromatin stability to treatment with CC (0.54 +/- 0.15, odds ratio +/- SE for corrected fructose; and 0.50 +/- 0.15, odds ratio +/- SE for serum testosterone). CONCLUSION(S): Hypofunction of the seminal vesicles was associated with high sperm chromatin stability, and this high sperm chromatin stability under SDS-EDTA conditions may be reduced by treatment with CC.

[Vasovasostomies performed in 60 cases from June 1990 to January 1994]. / Vasovasostomías efectuadas en 60 casos, desde junio de 1990 a enero de 1994.

60 vasovasostomies were performed between june 1990 and january 1994, following the modified one layer technique, under microscopic magnification. All the patients who requested the surgical procedure were included in the present study despite their age or time after vasectomy, in order to have a non selected group. Follow up was given with sperm counts processed under similar standards and performed at 2, 4 and 6 months after the surgical procedure. Our overall experience seems to confirm that strong correlation does not exist between the surgical technique used (one layer vs two layer), but the lapse of time passed between the vasectomy and the reversal is critical. In our series a pregnancy rate of 56.6% was achieved, with a patency rate of 86.7%.

Vasovasostomías efectuadas en 60 casos, desde junio de 1990 a enero de 1994 / Vasovasostomies performed in 60 cases from June 1990 to January 1994

60 vasovasostomies were performed between june 1990 and january 1994, following the modified one layer technique, under microscopic magnification. All the patients who requested the surgical procedure were included in the present study despite their age or time after vasectomy, in order to have a non selected group. Follow up was given with sperm counts processed under similar standards and performed at 2, 4 and 6 months after the surgical procedure. Our overall experience seems to confirm that strong correlation does not exist between the surgical technique used (one layer vs two layer), but the lapse of time passed between the vasectomy and the reversal is critical. In our series a pregnancy rate of 56.6% was achieved, with a patency rate of 86.7%