Decreased protein tyrosine phosphorylation and membrane fluidity in spermatozoa from infertile men with varicocele.

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.

Human sperm subpopulations: relationship between functional quality and protein tyrosine phosphorylation.

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.

Male antisperm antibodies: association with a modified sperm stress test and lipid peroxidation.

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.

Serum inhibin B may be a reliable marker of the presence of testicular spermatozoa in patients with nonobstructive azoospermia.

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.

Effects of long-term in vitro incubation of human spermatozoa: functional parameters and catalase effect.

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.

Endometrial dating and determination of the window of implantation in healthy fertile women.

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.

Modified sperm stress test: a simple assay that predicts sperm-related abnormal in-vitro fertilization.

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.

Occurrence of GABA and GABA receptors in human spermatozoa.

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.

Development of an objective, semiautomated method for assessment of sperm motility.

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.

Development of an objective and manual technique to study the human sperm morphology.

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.

Comparison between swim-up and glass bead column techniques for the separation of human spermatozoa.

The glass bead column filtration method was compared with the swim-up technique regarding spermatozoa recovery in normal and pathological human semen. The general characteristics of the semen and the spermatic ATP concentration were measured in 67 patients. Samples processed with glass bead columns provided a good amount of motile forms which however, showed less velocity and linearity than in the case of using the swim-up technique. Additionally, the ATP determination in spermatozoa was included.

Computer assisted measurement in normal and pathological human semen, fresh and post swim-up technique.

A total of 429 semen samples were studied. Two hundred and twenty-nine samples with normal characteristics were processed following a basic procedure; the remaining 200 were normal and pathological samples that were analyzed pre and post swim-up. Semen specimens were allowed to liquefy for 30 minutes and sperm count, motility, velocity and linearity were determined using the Cellsoft Automatic Semen Analyzer. In normal patients, a significant increase of motility, velocity and linearity (p less than 0.001) post swim-up, was observed. Sperm recovery in this group was 13.9 +/- 1% of the whole motile sperm population. In the polyzoospermic group, recovery of motile spermatozoa post swim-up was significantly decreased as compared with the normal group (p less than 0.001). In the asthenozoospermic group (plus either hypospermia or hyperspermia, or with more than 30% of motile spermatozoa or less than 30% of motility), no variations of velocity or linearity as compared to the normal group were observed. In all the pathological groups studied, a significant increase in velocity and linearity (p less than 0.001) post swim-up, was observed.

Effect of lipid peroxidation upon human spermatic adenosinetriphosphate (ATP). Relationship with motility, velocity and linearity of the spermatozoa.

The aim of the present paper was to study the effect of forced lipid peroxidation upon human spermatozoa and its relation with motility, velocity, linearity and ATP content. Forty-three pools of semen samples were studied one hour after liquefaction. After washing, normal and pathological spermatozoa presented no variations in motility, velocity or linearity with respect to basal values. Forced peroxidation produced a significant decrease of motility, velocity and linearity (p less than 0.02) with respect to control. Spermatic ATP was not significantly different in control vs forced peroxidation groups in normozoospermic samples. In contrast, a significant decrease of ATP between control and peroxidation groups was observed in asthenozoospermia at 120 minutes of incubation (p less than 0.02). In the asthenozoospermic group, a significant LPO-p increase was observed in comparison with the normal group.

Adenosine 5 triphosphate (ATP) content and acrosin activity in polyzoospermic subjects.

The aim of this paper was to measure the concentration of spermatic ATP and acrosin in a group of patients with polyzoospermia, and to compare these values with those found in a group of normozoospermic men. The general characteristics of the semen, the ATP and the acrosin, were measured in a group of 46 men with polyzoospermia, who consulted in our clinic for sterility. The results were compared with those of 50 normozoospermic men in the control group. Statistically, no significant difference was found between the volume, motility and morphology of the samples in both groups; however, the concentration of spermatic ATP and acrosin decreased significantly in a group of polyzoospermic patients. This could be due to a functional defect of the spermatozoa in this group of patients.

Adenosine 5'-triphosphate (ATP) concentration and acrosin activity in human spermatozoa. Their relation with the sperm penetration assay (SPA).

Fertility of men depends on the quality of semen. The aim of the present paper is to determine both the acrosin activity by radioimmunoassay and ATP concentration by bioluminescence in human spermatozoa, and evaluate these results in those samples with normal or low sperm penetration according to SPA test. Ejaculates obtained from 42 untreated men, were studied one hour after the obtention. These materials were divided into two groups:20 human semen samples with "in vitro" potentiality to penetrate zone-free hamster ova, between 15% to 98% and 22 human semen with SPA test between 0% to 14%. When we compare the group with normal penetration response vs that group with low or absent penetration ones, a significant decrease of ATP and acrosin concentrations was observed (P less than 0.001). Nevertheless no significant difference was observed in relation with percentage of motility, volume (ml), sperm concentration (10(6)/ml), percent of quick progressive spermatozoa and number of gametes capable of migrating into the medium layer (10(6)), between the group with low or absent penetration test against that one with normal zona-free hamster egg test.

Changes in sialic acid concentration in human saliva during the menstrual cycle.

Salivary sialic acid concentration was studied daily in 20 fertile women during the menstrual cycle. The Warren method was used and plasma LH and BBT were also determined. The sialic acid concentrations in saliva throughout the cycle showed a peak 4 or 5 days before the LH preovulation surge. This maximal peak showed a mean of 10.89 +/- 1.11 mg%. This method may be useful for predicting the pre- and postovulatory fertile period.

Adenosine 5'-triphosphate (ATP) activity in normal and pathological human semen. Relationship with round cells of the ejaculate.

The aim of the present paper is deleted to determine ATP activity in the human seminal cells by bioluminescence and to investigate the relationship between these values and the number of round cells either peroxidase positive or peroxidase negative. We studied 244 untreated men, divided into six groups. Our mean value of ATP activity in normal semen was 20.02 +/- 0.65 n moles per 10(8) spermatozoa. In asthenozoospermic or oligoasthenozoospermic patients with less than 1,000,000 round cells per ml, the concentration of ATP was significantly lower than normal (P less than 0.001) or (P less than 0.05). Semen with normal characteristics or samples from asthenozoospermic or oligoasthenozoospermic patients but with more than 1,000,000 round cells, per ml and with predominance of peroxidase negative cells, had an ATP concentration higher than normal subjects (P less than 0.001). We believe that the knowledge of the quantity and quality of round cells of the ejaculate is important to interpret the seminal values of ATP.