RESUMO
Varicocele is a prevalent pathology among infertile men. The mechanisms linking this condition to infertility, however, are poorly understood. Our previous work showed a relationship between sperm functional quality and the ability of spermatozoa to respond to capacitating conditions with increased membrane fluidity and protein tyrosine phosphorylation. Given the reported association between varicocele, oxidative stress, and sperm dysfunction, we hypothesized that spermatozoa from infertile patients with varicocele might have a combined defect at the level of membrane fluidity and protein tyrosine phosphorylation. Semen samples from infertile patients with and without grade II/III left varicocele were evaluated for motion parameters (computer-assisted semen analysis [CASA]), hyperactivation (CASA), incidence and intensity of protein tyrosine phosphorylation (phosphotyrosine immunofluorescence and western blotting), and membrane fluidity (Laurdan fluorometry), before and after a capacitating incubation (6 hr at 37 degrees C in Ham's F10/BSA, 5% CO(2)). Spermatozoa from varicocele samples presented a decreased response to the capacitating challenge, showing significantly lower motility, hyperactivation, incidence and intensity of tyrosine phosphorylation, and membrane fluidity. The findings reported in this article indicate that the sperm dysfunction associated to infertile varicocele coexists with decreased sperm plasma membrane fluidity and tyrosine phosphorylation. These deficiencies represent potential new pathophysiological mechanisms underlying varicocele-related infertility.
Assuntos
Infertilidade Masculina/etiologia , Fluidez de Membrana/fisiologia , Proteínas Tirosina Quinases/metabolismo , Espermatozoides/metabolismo , Varicocele/complicações , Adulto , Membrana Celular/fisiologia , Humanos , Masculino , Fosforilação , Motilidade dos EspermatozoidesRESUMO
BACKGROUND: Human semen is composed of a heterogeneous population of sperm with varying degrees of structural and functional differentiation and normality, which result in subpopulations of different quality. METHODS: Using a discontinuous Percoll gradient, we separated three subsets of sperm [(45%; L45), (65%; L65) and (90%; L90) fractions] from normozoospermic human semen samples from healthy donors and proceeded to characterize their morphology, motility and hyperactivation, as well as their ability to undergo tyrosine phosphorylation under capacitating conditions. RESULTS: As expected, sperm isolated from the lowest density layer (L45) showed the poorest quality, displaying the smallest percentage of morphologically normal and motile sperm. During a capacitating incubation, this subset of cells also showed deficient capacity to undergo hyperactivation and protein tyrosine phosphorylation. Conversely, sperm isolated from the other layers (L65 and L90) showed a time-dependent progressive increment in tyrosine phosphorylation, establishing statistically significant differences with sperm from L45. The tyrosine phosphorylation deficiency of L45 sperm could be overcome when sperm from that fraction were stimulated with activators of the cAMP-dependent kinase (PKA) pathway (dbcAMP + pentoxifylline), pointing to the sperm's plasma membrane as the main site of such deficiency. CONCLUSIONS: Poor quality sperm isolated from a Percoll gradient display an intrinsic tyrosine phosphorylation deficiency, possibly caused by a plasma membrane defect, which is associated with their inability to undergo normal capacitation and, ultimately, acquire optimal fertilizing potential.
Assuntos
Espermatozoides/citologia , Espermatozoides/fisiologia , Tirosina/metabolismo , Bucladesina/farmacologia , Centrifugação com Gradiente de Concentração , Coloides , Humanos , Masculino , Movimento (Física) , Pentoxifilina/farmacologia , Inibidores de Fosfodiesterase/farmacologia , Fosforilação/efeitos dos fármacos , Povidona , Valores de Referência , Dióxido de Silício , Capacitação Espermática , Motilidade dos Espermatozoides , Espermatozoides/metabolismoRESUMO
We previously reported a modified sperm stress test (MOST), low scores (< 0.39) in which were associated with sperm-related abnormal in vitro fertilization. Preliminary observations suggested that the presence of male sperm antibodies (ASA) could give low MOST scores. It was therefore decided to undertake a study to verify this possible association and also to ascertain if such a relationship was causal in nature. Six hundred and fifty semen samples from patients consulting for infertility were assessed for basic seminal characteristics, motion parameters (CASA), ASA and MOST. Thirty-nine samples (6%) were ASA-positive. Samples with and without ASA showed similar characteristics, except for percentage of normal forms and MOST scores (0.35 +/- 0.03 vs. 0.67 +/- 0.01, P < 0.001, for ASA-positive and -negative, respectively). There was a strong statistical association between presence of ASA and low MOST scores (P < 0.0001). One-hundred per cent of ASA-positive samples displayed low MOST scores. To verify the nature of this relationship, we incubated ASA-free spermatozoa with ASA-positive and -negative (control) sera. Despite an increase in the percentage of ASA-bearing spermatozoa in those aliquots incubated with ASA-positive serum, their original (pre-incubation) MOST scores remained unchanged. Furthermore, the rate of lipid peroxidation, indirectly reflected in MOST scores, was not different in the aliquots incubated with ASA. In conclusion, there seems to be a strong association between presence of ASA and low MOST values in semen samples of infertile patients; however, the relationship does not appear to be causal.
Assuntos
Autoanticorpos/análise , Infertilidade Masculina/imunologia , Peroxidação de Lipídeos , Sêmen/imunologia , Espermatozoides/imunologia , Humanos , Infertilidade Masculina/metabolismo , Masculino , Sêmen/metabolismo , Espermatozoides/metabolismoRESUMO
OBJECTIVE: To establish the predictive value of serum inhibin B levels as an indicator of the presence of testicular spermatozoa in nonobstructive azoospermia, compared with the traditional serum FSH marker. DESIGN: Prospective study. SETTING: Private high-complexity reproductive center with university affiliation. PATIENT(S): Seventy-eight patients with nonobstructive azoospermia, 15 patients with obstructive azoospermia, and 10 fertile volunteers. INTERVENTION(S): Blood samples, testicular sperm extraction, percutaneous epididymal sperm aspiration, and semen collection. MAIN OUTCOME MEASURE(S): Serum levels of inhibin B and FSH and presence of spermatozoa on TESE, PESA, or regular semen analysis. RESULT(S): Patients with nonobstructive azoospermia has significantly higher levels of serum FSH and significantly lower levels of inhibin B. Mean inhibin B serum levels were significantly higher in patients with nonobstructive azoospermia who had spermatozoa on TESE than in those in whom no spermatozoa were found (89.31 +/- 73.24 pg/mL vs. 19.23 +/- 22.34 pg/mL), but mean FSH serum levels did not have similar predictive power (21.37 +/- 12.92 IU/mL vs. 19.27 +/- 10.28 IU/mL). The cut-off level of inhibin B separating both groups, as determined by the receiver-operating characteristic curves, was >53 pg/mL. CONCLUSION(S): Serum inhibin B level seems to be more accurate than serum FSH level in prediction of the presence of testicular spermatozoa in patients with nonobstructive azoospermia.
Assuntos
Inibinas/sangue , Oligospermia/sangue , Testículo/fisiologia , Biomarcadores/sangue , Biópsia , Criopreservação , Feminino , Hormônio Foliculoestimulante/sangue , Humanos , Inibinas/fisiologia , Cariotipagem , Masculino , Oligospermia/diagnóstico , Valor Preditivo dos Testes , Gravidez , Estudos Prospectivos , Curva ROC , Preservação do Sêmen , Espermatozoides/fisiologia , Estatísticas não Paramétricas , Testículo/cirurgiaRESUMO
Prolonged incubation of human spermatozoa can have deleterious effects on sperm function. The aim of this paper was to describe the effects of a prolonged in vitro incubation, under similar conditions to those employed in human assisted reproduction, on various sperm functional parameters, and to investigate the effect of an antioxidant (catalase) on this system. Freshly collected ejaculates from 20 healthy donors were studied. Samples were divided into two aliquots: the first was incubated with Ham's F10 containing 3.5% HAS, and the second was incubated in the same medium plus catalase (100 units ml-1). All experiments were carried out with spermatozoa isolated using the swim-up technique. Spermatozoa recovered from the supernatant after 1 h (T1) of incubation in 5% CO2 in air at 37 degrees C, and after 5 h (T6), 23 h (T24) and 47 h (T48), were evaluated for concentration, motion parameters including hyperactivation (computer-assisted analysis), viability, ATP concentration, reactive oxygen species (ROS) generation, DNA integrity (acridine orange), and acrosome reaction (AR). The major alteration observed in sperm function during the prolonged in vitro incubation was a reduction in the number of motile spermatozoa, together with an impairment in the quality of sperm movement. ROS levels increased with the incubation time. No substantial modifications of sperm viability, chromatin condensation and AR inducibility were observed. The addition of catalase to the medium, while keeping ROS values within baseline levels, did not prevent the loss of motility or the corresponding increase in ATP.
Assuntos
Catalase/farmacologia , Espermatozoides/fisiologia , Reação Acrossômica , Trifosfato de Adenosina/metabolismo , DNA/análise , Humanos , Peróxido de Hidrogênio/metabolismo , Masculino , Espécies Reativas de Oxigênio/metabolismo , Contagem de Espermatozoides , Motilidade dos Espermatozoides , Espermatozoides/efeitos dos fármacos , Acetato de Tetradecanoilforbol/farmacologia , Fatores de TempoRESUMO
OBJECTIVE: To reassess endometrial morphological criteria of normality identifying the best morphological and molecular "implantation window" indicators in normal women. DESIGN: Prospective clinical study. SETTING: Assisted reproductive unit. PATIENT(S): Fourteen healthy volunteers. INTERVENTION(S): Blood sampling for LH, E(2), and progesterone (P4) determinations. Daily vaginal ultrasounds. Two endometrial biopsies per volunteer, 7 days apart, during luteal phase. MAIN OUTCOME MEASURE(S): Endometrial dating, pinopodes formation, immunohistochemical determination of integrins (alphavbeta3, alpha4beta1), leukemia inhibitory factor (LIF), interleukin-1 receptor type I (IL-1R tI), mouse ascites Golgi (MAG), the transmembrane mucin (MUC-1), and P4 receptor expression. RESULT(S): In 26 of 28 biopsies observers agreed; in two biopsies there was a discrepancy (difference of 72 hours). With use of LH peak, 24 of 26 samples were in phase, and 2 were 3 days behind. Pinopodes appeared on days 20-21 and persisted through day 28 in small groups or larger areas. beta3 Integrin was highly expressed in luminal and glandular epithelium from day 22 through 28; 48 hours thereafter pinopodes appeared. alpha4 Subunit exhibited luminal epithelium reaction positivity on days 22-23 and glands on days 18-23. LIF and IL-1R tI showed weak, erratic expression. MAG antibodies showed luminal epithelium expression up to day 22 and glands up to day 25. MUC-1 showed positivity during the whole luteal phase. P4 receptors were positive through day 20 and at the end of the luteal phase. CONCLUSION(S): The three most cited markers that frame the window of implantation do not correlate in our material. Pinopodes are present from day 20 on; beta3 and alpha4 integrin subunits indicate a window opening on days 22-23.
Assuntos
Implantação do Embrião , Endométrio/fisiologia , Interleucina-6 , Ciclo Menstrual/fisiologia , Adulto , Endométrio/anatomia & histologia , Estradiol/sangue , Feminino , Hormônio Foliculoestimulante/sangue , Complexo de Golgi/imunologia , Inibidores do Crescimento/metabolismo , Humanos , Imuno-Histoquímica/métodos , Integrina alfa4beta1 , Integrinas/metabolismo , Fator Inibidor de Leucemia , Hormônio Luteinizante/sangue , Linfocinas/metabolismo , Microscopia Eletrônica de Varredura , Mucinas/metabolismo , Estudos Prospectivos , Receptores de Interleucina-1/metabolismo , Receptores Tipo I de Interleucina-1 , Receptores de Retorno de Linfócitos/metabolismo , Receptores de Progesterona/metabolismo , Receptores de Vitronectina/metabolismo , Valores de ReferênciaRESUMO
Loss of sperm motility is associated with the process of sperm senescence and occurs at different rates within a given normal or abnormal sperm population. Reactive oxygen species attack cell membrane phospholipids, generating fatty acid peroxides and other degradation products, that also have deleterious effects on sperm motility and fertilizing ability. The objective of this investigation was to study a modification of the original sperm stress test (MOST), changing the culture medium to one offering transitional metals and shortening the total test time, to ascertain whether it can predict fertilization under these laboratory conditions. A total of 41 semen samples was obtained from patients undergoing in-vitro fertilization (IVF) at our institution. Semen samples were grouped into those producing total fertilization rates (FR) within normal limits (>50%) and those showing low total FR (<50%). The normal FR group had a significantly greater MOST mean value than the low FR group (0.71 versus 0.44). Furthermore, there was a statistically significant correlation between the MOST score and ungrouped fertilization rates (r = 0.53, P = 0.0004). Diagnostic statistics for MOST ratio values predicting <50% FR showed an optimal threshold of 0.39. Collectively, sensitivity, specificity, positive predictive value and negative predictive value have their largest values at this threshold. Taking into account the above mentioned threshold figures, there is a significant association between MOST and FR categories (P = 0.0009). In conclusion, MOST is a simple assay that has significant predictive value for sperm related IVF abnormalities.
Assuntos
Fertilização in vitro , Motilidade dos Espermatozoides , Adulto , Feminino , Humanos , Masculino , Estresse Oxidativo , Valor Preditivo dos TestesRESUMO
Gamma-aminobutyric acid (GABA) concentrations in seminal plasma and washed spermatozoa from normal donors were assessed by a sensitive radioreceptor assay, and were detectable in both fractions. Specific binding of [3H]-muscimol was shown to be dependent on protein concentration, temperature and incubation time. [3H]-muscimol specific binding to human sperm membranes was significantly inhibited by the GABA type A receptor (GABA(A)) antagonist, bicuculline, and by the GABA(A) agonists, muscimol and isoguvacine, but not by the GABA type B receptor (GABA(B)) agonist baclofen. Scatchard analysis of [3H]-muscimol binding yielded a linear plot consistent with a single population of binding sites with a dissociation constant in the low nanomolar range. Incubation with GABA at a high micromolar concentration for 3 h under capacitating conditions resulted in an increase in the percentage of spermatozoa showing hyperactivated motility as assessed by computerized motility analyser. However, low micromolar concentrations of the GABA(A) agonist, muscimol, were sufficient to significantly increase sperm hyperactivity. These results suggest that the effect of GABA on human sperm motility might be mediated through a specific GABA(A) receptor.
Assuntos
Receptores de GABA/metabolismo , Espermatozoides/metabolismo , Ácido gama-Aminobutírico/análise , Ácido gama-Aminobutírico/metabolismo , Bicuculina/farmacologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Antagonistas GABAérgicos/farmacologia , Humanos , Ácidos Isonicotínicos/farmacologia , Masculino , Muscimol/metabolismo , Receptores de GABA/análise , Sêmen/química , Sêmen/metabolismo , Motilidade dos Espermatozoides , Espermatozoides/efeitos dos fármacos , Trítio , Ácido gama-Aminobutírico/farmacologiaRESUMO
Study of sperm motility is associated to the development of precise and economical systems of evaluation. The purpose of this work was to develop an Objective Semi-Automated Method (MOSA) to evaluate the sperm motility. Human semen samples were registered by video-microscopy. The same videofilms were analysed with the MOSA, the subjective method and the automated CellSoft method. The percentages of motile and immotile sperms were equivalent with the three methods. The percentages of rapidly and slowly motile sperms were similar both with the MOSA and the subjective method. The curvilinear and linear velocities as well as the linearity values obtained with the MOSA were different to those obtained with the CellSoft, although such differences would be biologically acceptable. MOSA is an inexpensive, objective and precise method that does not require trained technicians and allows evaluation of several parameters of sperm motility.
Assuntos
Motilidade dos Espermatozoides , Desenho de Equipamento , Estudos de Avaliação como Assunto , Humanos , Processamento de Imagem Assistida por Computador , MasculinoRESUMO
The aim of the present paper is to present results of sperm morphology using an objective and manual technique by video image. Experiment 1:252 spermatozoa heads were measured in a microscope and in a monitor by each of three independent observers. The results allowed the calibration of an acetate overlay according to the WHO guideline and following the strict criteria. Experiment 2: 10 morphology slides from normal and abnormal patients were studied. These slides were evaluated by three independent observers, each counting at least 200 cells using the calibrate acetate overlay. In the first experiment the calculation of the regression out-put was: constant: 0.24, standard error of Yc: 0.04, R squared: 0.96, X coefficient: 0.36, and standard error of the coefficient: 0.03. In the second experiment, it can be seen that the differences among the operators are not statistically significant and therefore the experiment is independent from the operator. In conclusion, the methodology developed in this paper for the evaluation of morphology would be a good tool for the evaluation of human sperm morphology.