Juan C. Calamera

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.

Use of Synthetic Luteinizing Hormone-Releasing Hormone in Treatment of Oligospermic Men: A Preliminary Report

Synthetic luteinizing hormone-releasing hormone (LH-RH) was administered to four normogonadotropic, oligospermic men (24 to 39 years of age) who had no endocrinologic, urologic, or associated vascular disease, to assess its possible therapeutic value in male infertility. Previous testicular biopsies of these subjects indicated alteration of spermiogenesis only. LH-RH (mean dose, 500 mug/day) was administered intramuscularly for 100 to 135 days. Each patient had at least two sperm count before starting therapy and had one every 20 to 30 days during and for two to five months after treatment. The sperm count, semen volume, sperm motility and morphology, and seminal plasma concentrations of fructose and citric acid were studied in each semen sample. In three of the four patients, urinary LH and FSH excretion and plasma testosterone levels were also measured. The sperm count increased clearly in two subjects 30 to 80 days after therapy started; the response was small in the third subject and negative in the fourth. The remaining parameters followed variable courses. Libido increased in all subjects. In the post-treatment period, the two patients who had shown the best response during treatment experienced a new and abrupt increase in the sperm count which remained well above initial values at the end of follow-up. LH-RH appears to be of value in the treatment of certain types of oligospermia, but several issues remain unsettled.

High Cholesterol Content and Decreased Membrane Fluidity in Human Spermatozoa Are Associated With Protein Tyrosine Phosphorylation and Functional Deficiencies

Poor-quality sperm show reduced capacity to undergo capacitation-induced protein tyrosine phosphorylation and hyperactivation. Given that these deficiencies can be overcome by membrane-permeant stimulators of the cAMP-dependent kinase system, we hypothesize that the main defect underlying these deficiencies resides on the sperm plasma membrane. Spermatozoa from semen samples obtained from 15 consenting healthy donors were separated in 2 subpopulations, L45 (first interface) and L90 (pellet), using a 45:65:90 ISolate gradient centrifugation method. These sperm fractions were studied before and after a 6-hour capacitating incubation for sperm motion parameters (computer-assisted analysis), including hyperactivation, protein tyrosine phosphorylation (immunofluorescence), membrane fluidity (Laurdan fluorescence), and sterol and phospholipid content (high-performance thin-layer chromatography). In summary, data indicate that L45 (poor-motility) spermatozoa present an excess of cholesterol and desmosterol, which impairs the normal increase in membrane fluidity during capacitation and its consequent activation of protein tyrosine phosphorylation and hypermotility. Therefore, a defect in membrane composition and dynamics is underlying human sperm biochemical and functional deficiencies related to inadequate capacitation.

Calcium requirements for human sperm function in vitro

OBJECTIVE To determine extracellular calcium (Ca(2+)) requirements for the maintenance of human sperm function in vitro. DESIGN Prospective study. SETTING Basic research laboratory. PATIENT(S) Normozoospermic volunteers provided fresh semen samples; follicular fluid (human FF) and oocytes were collected from women undergoing IVF-ET. INTERVENTION(S) Spermatozoa were incubated for </=18 hours in media containing different CaCl(2) concentrations (maximum, 2.5 mM [control]). MAIN OUTCOME MEASURES Protein tyrosine phosphorylation patterns, development of hyperactivated motility, induction of the acrosome reaction (AR) in response to human FF, and sperm interaction with homologous zona pellucida (ZP). RESULT(S) Cells maintained for 18 hours in medium containing >/=0.1 mM of Ca(2+) were able to undergo the AR when exposed to human FF in the presence of 2.5 mM of Ca(2+). Calcium concentrations of >/=0.22 mM were sufficient to reach protein tyrosine phosphorylation levels and hyperactivated motility values similar to those of controls. Higher Ca(2+) concentrations (>/=0.58 mM) were required to produce maximum human FF-induced AR in previously capacitated cells and to obtain an adequate sperm-ZP binding. CONCLUSION(S) Different steps of the fertilization process have distinctive Ca(2+) requirements. Whereas 0.22 mM of Ca(2+) is sufficient for the development of some capacitation-related events, human FF-induced AR and sperm-ZP interaction require 0.58 mM of this cation.

CAPACITATION-ASSOCIATED CHANGES IN MEMBRANE FLUIDITY IN ASTHENOZOOSPERMIC HUMAN SPERMATOZOA

The fertilizing potential of human spermatozoa relies on their ability to capacitate as they travel through the female reproductive tract. During this process, cholesterol is released from the plasma membrane, altering its architecture and dynamics. Using ISolate gradients, we obtained high (L90)- and low (L45)-quality spermatozoa from asthenozoospermic human semen samples. We tested the hypothesis that the lower fertilizing ability of asthenozoospermic L90 cells could be related to a lower ability to increase their membrane fluidity during capacitation. We assessed two sets of fluorescent probes: (i) DPH, TMA-DPH and PA-DPH which senses the hydrophobic core, cytosolic and exofacial leaflets of the bilayer, respectively and (ii) Laurdan, sensitive to the amount of water molecules intercalated between lipid moieties of the membrane (membrane hydration). Before capacitation, membrane fluidity of asthenozoospermic sperm populations was similar to the corresponding fractions of normozoospermic cells when evaluated with DPH, TMA-DPH or PA-DPH. Asthenozoospermic whole samples displayed lower plasma membrane hydration than normozoospermic cells as evidenced with Laurdan. After capacitation, asthenozoospermic L45 and L90 cells failed to increase their membrane fluidity in opposition to normozoospermic cells. Interestingly, membrane hydration significantly correlated with the main sperm motion parameters analysed, being a low membrane hydration associated with poor sperm movement. These results show that low-motility spermatozoa are unable to respond to capacitation with the necessary changes in membrane fluidity. This defect in sperm plasma membrane rheology may be responsible for their poor functional quality and low fertilizing ability.

Superoxide dismutase content in sperm correlates with motility recovery after thawing of cryopreserved human spermatozoa

OBJECTIVE To investigate the correlation between sperm superoxide dismutase (SOD) content and motility recovery after thawing of cryopreserved human sperm, based on the rationale that this antioxidant enzyme provides protection against reactive oxygen species-induced damage during cryopreservation. DESIGN Prospective study. SETTING Private infertility institute and university-based research laboratory. PATIENT(S) Forty-two consenting normozoospermic patients consulting for infertility. INTERVENTION(S) The SOD content was measured in sperm from unfractionated samples and in sperm recovered from the pellet fraction obtained after discontinuous density gradient centrifugation. MAIN OUTCOME MEASURE(S) Sperm motility was evaluated post-thaw in the two sets of samples and motility recovery was plotted against the sperm SOD content to determine their correlation. RESULT(S) There was a significant positive correlation between motility recovery after thawing and SOD content in sperm from the 90% gradient pellet containing highly purified mature sperm. There was also a significant negative correlation between motility after thawing and SOD content in the unfractionated sample. CONCLUSION(S) The positive correlation between post-thaw motility recovery and SOD content in mature spermatozoa provides a good predictor of post-thaw motility recovery after cryopreservation.

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

Summary. 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.