Identificación de espermatozoides humanos en muestras contaminadas con levaduras / Identification of human spermatozoids in samples contaminated with yeast

RESUMEN La violencia sexual es un problema mundial, y representa un delito grave que frecuentemente es difícil imputar, ante la ausencia de testigos. La valoración de las muestras de semen enviadas al laboratorio, para la detección de espermatozoides, es de gran importancia para encontrar al agresor; sin embargo, la evidencia no es sencilla, por la presencia de otros organismos y la labilidad espermática. El objetivo del presente estudio fue identificar espermatozoides humanos en muestras contaminadas con levaduras. Se aplicaron diferentes técnicas y recursos microscópicos. Se seleccionaron 46 muestras de semen fresco. Se dividieron en dos grupos: G1 normo u oligozoos-pérmicas (n = 28) y G2 azoospérmicas (n = 18). Cada muestra se diluyó 1:1 con solución fisiológica y se agregaron 0.05 mL de suspensión de levaduras Candida albicans. Como control, se procesaron testigos puros de espermatozoides (CE) y levaduras (CL). Las muestras se analizaron con microscopio óptico (MO), contraste de fase (CF), de luz polarizada (LP) y microscopio fluorescente (MF) con diferentes técnicas de tinción. El tinte Papanicolaou mostró mayor eficiencia que el tinte hematoxilina verde brillante (P < 0.05) para identificar espermatozoides con microscopio óptico. La detección de elementos refringentes mostró mayor correlación con la presencia de espermatozoides (CF: r = 0.966; P < 0.001), que la presencia de elementos birrefringentes (LP: r = 0.737; P < 0.001) y elementos fluorescentes (MF: r = 0.487; P < 0.05). La microscopía confocal con tinción de blanco de calcofluor-azul de Evans y naranja de acridina permitió la identificación de contaminación por levaduras. El empleo de técnicas microscópicas auxiliadas con técnicas de tinción permite confirmar la presencia de espermato zoides enteros o fragmentados en muestras de semen contaminadas con levaduras.

ABSTRACT Sexual violence is a global problem, and represents a serious felony which is often difficult to impute due to the absence of witnesses. The evaluation of semen samples sent to the laboratory, for the detection of spermatozoa is of great importance to find the aggressor. However the evidence is not simple due to the presence of other organisms and sperm lability. The objective of the present study was to identify human spermatozoa in samples contaminated with yeast. Different techniques and microscopic resources were applied and 46 samples of fresh semen were selected. They were divided into two groups: G1 normo or oligozoos-perms (n = 28) and G2 azoosperms (n = 18). Each sample was diluted 1:1 with physiological solution and 0.05 mL of yeast suspension Candida albicans was added. As controls, pure sperm (CE) and pure yeast (CL) were processed. The samples were analyzed with optical microscopy (MO), phase contrast (CF), polarized light (LP) and fluorescent microscope (MF) with different staining techniques. Papanicolaou dye showed greater efficiency than bright hematoxylin-green dye (P < 0.01) to identify spermatozoa with an optical microscope. The presence of refringent elements showed a higher correlation with the presence of spermatozoa (LP: r = 0.966; P < 0.001), that the presence of birrefringent elements (LP: r = 0.737; P < 0.001) and fluorescent elements (MF: r = 0.487, P < 0.05). Confocal microscopy with Evans blue calco-fluor white stain and acridine orange allowed the identification of yeast contamination. The use of microscopic techniques aided by staining techniques confirms the presence of whole or fragmented spermatozoa in semen samples contaminated with yeasts.

Infertilidad masculina. Exposición laboral a factores ambientales y su efecto sobre la calidad seminal / Male infertility. Labor exposure to environmental factors and their effect on seminal quality / Infertilidad masculina. Exposição laboral a fatores ambientales e efeito sobre a qualidade seminal

Resumen:Utilizando datos Pub Med Medline, Science Direct, Endocrine Society, Sociedad Argentina de Andrología se realizó una revisión bibliográfica para estudiar e interpretar el efecto de factores ambientales como pesticidas, tóxicos, solventes, calor y radiación electromagnética sobre la reproducción. Muchos se comportan como disruptores endocrinos alterando la homeostasis y provocando desequilibrio hormonal durante el desarrollo y en la descendencia. El monocultivo de transgénicos pulverizados con venenos impacta negativamente en la salud reproductiva masculina. La temperatura elevada por exposición a fuentes que irradian calor o permanecer muchas horas sentado, altera la espermatogénesis. Los artículos electrónicos emiten radiaciones perjudiciales incrementando la exposición ambiental a campos electromagnéticos que alteran la función testicular. Los varones expuestos incrementan el riesgo de infertilidad presentando membrana disfuncional, mayor fragmentación del ADN y alteraciones en morfología, movilidad y concentración espermáticas. Estos efectores del entorno laboral deben ser evaluados en la infertilidad masculina.

Abstract:Using data Pub Med Medline, Science Direct, Endocrine Society, Argentine Society of Andrology, a bibliographic review was made to study and interpret the effect of environmental factors such as pesticides, toxic, solvents, heat and electromagnetic radiation on reproduction. Most of these factors behave as endocrine disruptors altering the homeostasis and causing hormonal unbalance during development and offspring. Monoculture of transgenic sprayed with poisons negatively impacts on male reproductive health. Elevated temperature by exposure to sources that radiate heat or remain seated for many hours alters the spermatogenesis. Electronic articles emit harmful radiation by increasing environmental exposure to electromagnetic fields that testicular function. Exposed men increase the risk of infertility by presenting dysfunctional membrane, increased DNA fragmentation and alterations in sperm morphology, motility and concentration. These effectors in the work environment must be evaluated in male infertility.

Resumo: Utilização de dados Pub Med Medline, Science Direct, Sociedade Endócrina, Sociedade Argentina de Andrologia se realizó uma revisão bibliográfica para estudar e interpretar o efeito de fatores ambientales como pesticidas, tóxicos, solventes, calor e radiação eletromagnética sobre a reprodução. Muitos se comportan como disruptores endocrinos alterando a homeostase e provocando desequilibrio hormonal durante o desenvolvimento e na descida. O monocultivo de transgênicos pulverizados com venenos impacta negativamente na saúde reprodutiva masculina. A temperatura elevada por um desperdício de calor e calor irradia o tempo de espera, altera a espermatogénesis. Os artigos eletrônicos emitem radiações perjudiciales incrementando a análise ambiental a campos eletromagnéticos que alteram a função testicular. Los varones expuestos incrementan o risco de infertilidade apresentando membrana disfuncional, maior fragmentação do ADN e alterações em morfología, movilidade e concentração espermáticas. Estes exercícios do ambiente devem ser avaliados na infertilidade masculina.

Importancia de la evaluación del estrés oxidativo en el semen humano / Importance of the assessment of oxidative stress in human semen

Utilizando bases de datos Medline, Science Direct, Endocrine Society y Sociedad Argentina de Andrología se realizó una revisión bibliográfica para estudiar e interpretar el efecto del estrés oxidativo (EO) en el proceso reproductivo. Los seres vivos que utilizan oxígeno para obtener energía son liberadores de especies reactivas de oxígeno (ERO). Existen situaciones andrológicas, factores medioambientales y compuestos químicos que incrementan las citoquinas proinflamatorias, generan EO alterando la regulación del proceso espermatogénico. Los espermatozoides son susceptibles al daño oxidativo, debido al elevado contenido de ácidos grasos poliinsaturados (AGPI) en su membrana y el escaso citoplasma que limita el contenido de enzimas antioxidantes. El EO induce daño peroxidativo en la membrana espermática y fragmentación del ADN en los genomas nucleares y mitocondriales. El test estrés espermático modificado (MOST) estima la resistencia espermática a la lipoperoxidación. El incremento de ERO en hombres con trastornos reproductivos y su correlación con alteraciones seminales revelan la importancia de la evaluación del EO en el estudio integral del hombre que consulta por infertilidad.

Data from Medline, Science Direct, Endocrine Society and organisms of information of Argentina Society of Andrology were used to study and interpret the effect of oxidative stress (OS) in the reproductive process. The living things use the oxygen for energy they release reactive oxygen species (ROS). There andrology situations enviromental factors, chemical agents that increase of proinflammatory cytokines, OS generated by altering the regulation of spermatogenesis process. The spermatozoids due to high concentration of polyunsaturated fatty acids (PFA) in its membrane are sensitive to the oxidative damage induced for ROS and it also contain low levels of antioxidant enzymes for their limited cytoplasm. OS induced peroxidative damage in sperm membrane and DNA fragmentation in the nuclear and mitochondrial genomes. The modified sperm stress test (MOST) estimates sperm resistance to lipoperoxidation. The ROS increased in men with reproductive disorders and its correlation with seminal alterations reveal the importance of the evaluation of OS in the study of man consulting for infertility.

Evaluation of urinary N-acetyl-beta-D-glucosaminidase as a marker of early renal damage in patients with type 2 diabetes mellitus.

OBJECTIVE: To evaluate the clinical usefulness of urinary N-acetyl-beta-D-glucosaminidase (NAG) excretion for the detection of early tubular damage in type 2 diabetes mellitus (T2DM). SUBJECTS AND METHODS: Thirty six patients with T2DM were divided into two groups based on urinary albumin to creatinine ratio (ACR): normoalbuminuria (ACR <30 mg/g; n=19) and microalbuminuria (ACR =30-300 mg/g; n=17). The following parameters were determined in both groups: urinary NAG and albumin, serum and urine creatinine, fasting plasma glucose and glycated hemoglobin (HbA1c). RESULTS: Urinary NAG levels [Units/g creatinine; median (range)] were significantly increased in microalbuminuria group [17.0 (5.9 - 23.3)] compared to normoalbuminuria group [4.4 (1.5 - 9.2)] (P<0.001). No differences between groups were observed in fasting glucose, HbA1c, serum creatinine levels and estimated glomerular filtration rates (eGFR). Urinary NAG positively correlated with ACR (r=0.628; p<0.0001), while no significant association was observed between NAG and glycemia, HbA1c, serum creatinine and eGFR. CONCLUSIONS: The increase of urinary NAG at the microalbuminuria stage of diabetic nephropathy (DN) suggests that tubular dysfunction is already present in this period. The significant positive association between urinary NAG excretion and ACR indicates the possible clinical application of urinary NAG as a complementary marker for early detection of DN in T2DM.

Evaluation of urinary N-acetyl-beta-D-glucosaminidase as a marker of early renal damage in patients with type 2 diabetes mellitus / Avaliação da N-acetil-beta-D-glucosaminidase urinária como marcador de dano renal precoce em pacientes com diabetes melito tipo 2

Objective To evaluate the clinical usefulness of urinary N-acetyl-beta-D-glucosaminidase (NAG) excretion for the detection of early tubular damage in type 2 diabetes mellitus (T2DM). Subjects and methods Thirty six patients with T2DM were divided into two groups based on urinary albumin to creatinine ratio (ACR): normoalbuminuria (ACR <30 mg/g; n=19) and microalbuminuria (ACR =30‐300 mg/g; n=17). The following parameters were determined in both groups: urinary NAG and albumin, serum and urine creatinine, fasting plasma glucose and glycated hemoglobin (HbA1c). Results Urinary NAG levels [Units/g creatinine; median (range)] were significantly increased in microalbuminuria group [17.0 (5.9 - 23.3)] compared to normoalbuminuria group [4.4 (1.5 - 9.2)] (P<0.001). No differences between groups were observed in fasting glucose, HbA1c, serum creatinine levels and estimated glomerular filtration rates (eGFR). Urinary NAG positively correlated with ACR (r=0.628; p<0.0001), while no significant association was observed between NAG and glycemia, HbA1c, serum creatinine and eGFR. Conclusions The increase of urinary NAG at the microalbuminuria stage of diabetic nephropathy (DN) suggests that tubular dysfunction is already present in this period. The significant positive association between urinary NAG excretion and ACR indicates the possible clinical application of urinary NAG as a complementary marker for early detection of DN in T2DM. .

Objetivo Avaliar a utilidade clínica da excreção urinária da N-acetil-beta-D-glucosaminidase (NAG) para a detecção de dano tubular precoce no diabetes melito tipo 2 (DM2). Sujeitos e métodos Foram estudados trinta e seis pacientes com DM2 que se dividiram em dois grupos com base na excreção urinária de albumina (EUA): normoalbuminúrico (EUA <30 mg/g de creatinina; n=19) e microalbuminúrico (EUA =30‐300 mg/g de creatinina; n=17). Em ambos os grupos foram determinados os seguintes parâmetros: NAG e albumina urinária, creatinina sérica e urinária, glicemia de jejum e hemoglobina glicada (HbA1c). Resultados Os níveis de NAG urinária [unidades/g de creatinina; mediana (intervalo interquartílico)] foram significativamente maiores no grupo microalbuminúrico [17,0 (5,9 - 23,3)] em comparação com o grupo normoalbuminúrico [4,4 (1,5 - 9,2)] (p<0,001). Não se observaram diferenças significativas entre os dois grupos nos níveis de glicemia de jejum, HbA1c, creatinina sérica e taxa de filtração glomerular estimada (TFGe). A NAG urinária se correlacionou positivamente com o EUA (r=0,628, p<0,0001), não sendo observada associação significativa da NAG com glicemia, HbA1c, creatinina sérica e TFGe. Conclusões O aumento da NAG urinária na fase de microalbuminúria da nefropatia diabética (ND) sugere que a disfunção tubular já está presente nesse período. A associação positiva significativa entre a excreção urinária da NAG e EUA indica a possível aplicação clínica da NAG urinária como marcador complementar para a detecção precoce da ND no DM2. .

El efecto de los agroquímicos en la espermatogénesis / The effect of the agrochemicals in the spermatogenesis

Se relacionó la exposición ocupacional a agroquímicos con parámetros seminales vinculados con la espermatogénesis. En 104 muestras seminales de pacientes con infertilidad idiopática y 20 a 45 años de edad del Servicio de Reproducción Hospital Centenario (Rosario, Argentina), se efectuó espermograma y estudios funcionales según normas OMS (1999). Se agruparon en G1 (n=42) trabajadores expuestos a agroquímicos y G2 (n=62) hombres sin riesgo espermatogénico. La concentración espermática se determinó en cámara de Neubauer, con Papanicolaou se evaluó la concentración de células germinales y la morfología espermática. Se observó en concentración espermática (espermatozoides.10(6)/ml) G1: 21,1±7,1; G2: 41,6±9,2; morfología ( por ciento espermatozoides normales) G1: 5,2±1,3; G2: 8,2±3,6 y células germinales.10(6)/ml en G1: 0,91±0,51; G2: 0,32±0,21. La concentración espermática fue menor en G1, las alteraciones morfológicas y células germinales mayores en G1. Estos resultados indican que la exposición a agroquímicos altera la espermatogénesis y es un factor a considerar cuando se estudia infertilidad masculina.

The occupational exposition to agrochemicals was related with sperm parameters linked with spermatogenesis. In 104 semen samples of patients with idiopatic infertility of the Reproduction Service of Centenario Hospital (Rosario, Argentina), sperm study and functional tests according to WHO (1999) have been carried out. Two groups were formed: G1 (n=42) workers exposed to agrochemicals; G2 (n=62) men with no spermatogenic risk. The sperm concentration was determined in Neubauer camera, the concentration of germinal cells and the sperm morphology were evaluated with Papanicolaou. The results were: sperm concentration (spermatozoids 10(6)/ml) G1: 21.1 ± 7.1; G2: 41.6 ± 9.2; morphology ( percent normal spermatozoids) G1: 5.2 ± 1.3; G2: 8.2 ± 3.6; and germinal cells 10(6)/ml in G1: 0.91 ± 0.51; G2: 0.32 ± 0.21. The sperm concentration was lower in G1, major morphological changes and germinal cells in G1. This results show that the exposition to agrochemicals alters the spermatogenesis, it is a factor to consider in the male infertility.

Estres oxidativ y su efecto sobre calidad seminal / The oxidative stress effect on semen quality

La membrana espermática tiene ácidos grasos insaturados que la tornan vulnerable al ataque de sustancias oxígeno reactivas. Los espermatozoides poseen sistemas protectores, pero un desbalance entre pro y antioxidantes produce “estrés oxidativo". Objetivo: estudiar en semen de hombres infértiles, el efecto del estrés oxidativo sobre la membrana y núcleo espermático. Se analizaron muestras seminales de 142 hombres infértiles. Se efectuó espermograma, se seleccionaron 83 muestras sin aglutinación ni hiperviscosidad y con concentración espermática mayor a 5 x 10 6 /ml. Se estudió la membrana espermática con Test Hipoosmótico, la condensación cromatínica con Azul de Anilina y el ADN con Naranja de Acridina. Para estrés oxidativo se aplicó el Test MOST (movilidad traslativa final/movilidad traslativa inicial) que evalúa la pérdida de movilidad de los espermatozoides luego de ser incubados por 4 hs. en baño de agua a 40 °C. Se agruparon las muestras en G1: MOST mayor o igual a 0.40 (normal) y G2: MOST menor a 0.40 (anormal). El análisis estadístico demostró diferencia significativa (p<0.003) en las 3 pruebas funcionales. El estrés oxidativo altera estructuras espermáticas esenciales.

The spermatozoids have in its plasmatic membrane high concentrations of unsaturated fatty acids, vulnerable to the attack of the reactive oxygen substances. The male gamete has protective systems, the distortion between pro and antioxidizers produces “oxidative stress". Objective: to study samples of semen from infertile men, the effect of oxidative stress on the sperm membrane and the nucleus. 142 semen samples were analyzed from infertile men. The sperm study was evaluated according to OMS and 83 samples without agglutination and hiperviscosity with concentration of spermatozoids more of 5 x 10 6 /ml were chosen. The sperm membrane was studied with the Hipoosmotic Test, the maturity of chromatina with blue aniline and the nuclear AND with acridine orange. The EO was evaluated with the MOST test (motility end/motility initial) measuring the loss of motility of the spermatozoids incubated at 40ºC in a water bath for 4 hours, separating the samples in : G1 : MOST major or equal to 0.40 (normal) G2 : MOST under to 0.40 ( abnormal). The statistic analysis of the three tests showed significant difference (p<0.003). The oxidative stress affects essential structures.

[Effect of tobacco consumption on the spermatogenesis in males with idiopathic infertility]. / Efecto del tabaquismo sobre la espermatogenesis en hombres con infertilidad idiopatica.

OBJECTIVES: Our objective was to relate the tobacco with seminal spermatogenic parameters such as sperm morphology, concentration of spermatozoids and germinal cells in samples of semen from men with idiopatic infertility. METHODS: A prospective study was carried out on a population of 131 men with idiopatic infertility that attended the Reproduction Service of Centenario Hospital in Rosario from may 2004 to june 2006. Sperm study according to WHO was carried out evaluating germinal cells and sperm morphology with Papannicolaou. The concentration of spermatozoids was determined by means of a subjective method with Neubauer camera. The studied population was divided in the three groups: G1: smokers more of 20 cigarettes/day, G2: smokers under 20 cigarettes/day, G3 non smokers. The smokers had had the habit for over a year. RESULTS: Results were analyzed with the student's t-test. Statistically significant differences between G1 vs G3 (p<0.001) and G2 vs G3 (p<0.005) were found for the three variables. No significant difference was found between the groups of smokers G1 vs G2 (p>0.1). CONCLUSIONS: The results show that tobacco alters sperm concentration and morphology with an increase of immature forms, demonstrating an altered spermatogenesis process. The consumption of tobacco should be evaluated to carry out the integral study of infertile man.

[Modified sperm stress test. Immune response and functionality of the human spermatozoid membrane]. / Estrés espermático modificado. Respuesta inmune y funcionalidad de la membrana del espermatozoide humano.

OBJECTIVE: Our purpose was to study, in semen samples of infertile patients, the relationship between the Modified Sperm Stress Test (MOST) and the presence of antisperm antibodies (ASA), macrophages concentration and the Hypoosmotic Swelling Test (HOST). METHODS: Semen samples from 42 men undergoing evaluation for infertility were examined according to WHO criteria. Twenty-five of them, whithout clumping non hyperviscosity, were selected. The MOST test was applied according to the author's original technique. ASA were determined with a direct mixed agglutination test, TAC II, developed and validate by our group. Macrophage concentration was evaluated with Neutral Red stain, and functional integrity of the sperm membrane with the Hypoosmotic Swelling Test using an hypoosmotic solution of 150 mOm/ml composed of equal parts of fructose and sodium citrate. RESULTS: The Chi square test was applied to the observational data obtaining the following results: There was a statistically significant association between the presence of ASA and altered MOST (p<0.001). In all samples with ASA, abnormal MOST values were obtained (MOST<0.39). Besides, there is a statistically significant association exists between the increased concentration of macrophages and abnormal MOST (p<0.01); and altered HOST was positively correlated with abnormal MOST (p<0.02). CONCLUSION: Results clearly demonstrate the high predictive power of MOST like a test of sperm resistance to the forced lipoperoxidation, offering conditions to become a good predictor of sperm performance. Understanding the sperm resistance to the ROS and their harmfull effects on the sperm functions, a proportion of infertile men can be succesfully treated.

[Macrophages in human semen]. / Macrófagos en semen humano.

OBJECTIVES: To investigate the presence of macrophages in human semen samples and the function they carry out in the seminal fluid. Their presence was studied in relation to spermatic morphology, percentage of spermatozoids with native DNA, and presence of antispermatic antibodies. METHODS AND RESULTS: The work was performed with semen samples from 31 unfertile males from 63 couples in which the "female factor" was ruled out as the cause of infertility. Sperm study according to WHO (1992) was carried out in all samples, in addition to: DNA study with acridine orange as fluorocrom, macrophage concentration by neutral red in a Neubauer camera, and detection of antispermatic antibodies with a mixed agglutination test (TAC II) (validated with Mar Screen-Fertility technologies). Sperm morphology was evaluated by Papanicolaou test. 19/31 selected sperm samples (61.3%) showed increased concentration of macrophages, 13 of them (41.9%) with denaturalized DNA, and 8 (25.8%) abnormal morphology. Six samples showed increased macrophage concentration and predominance of native DNA, whereas 11 samples showed increased macrophages and abnormal morphology. Among 18 (58.1%) samples showing antispermatic antibodies 14 (77.7%) had an increased concentration of macrophages. Statistical analysis resulted in a high correlation between macrophage concentration and increased percentage of spermatozoids with denaturalized DNA (p < 0.05). An increased concentration of macrophages is associated with the presence of antispermatic antibodies (p < 0.05). There was not evidence of significant association between concentration of macrophages and percentage of morphologically normal spermatozoids (p < 0.05). CONCLUSIONS: We can conclude that macrophages are present in human semen and participate in immunovigilance contributing to improve the seminal quality.