Improvement of post-thaw sperm motility in poor quality human semen.

OBJECTIVE: To find alternative cryopreservation methods to improve the post-thaw fertilizing capacity of poor quality human sperm. DESIGN: Controlled clinical study. SETTING: Fertility clinic of a teaching hospital. PATIENTS: Men with poor quality semen samples, i.e., asthenozoospermia (< 40% motile sperm) and/or oligozoospermia (< 20 x 10(6) sperm/mL). Fertile sperm donors were used for comparison. INTERVENTIONS: Semen samples were divided into four aliquots and slowly diluted 1:1 with: [1] n-tris (hydroxymethyl) methyl-2-amino ethane sulfonic acid (TES) and tris (hydroxymethyl) aminomethane (Tris)-citric acid-egg yolk buffer with 12% glycerol (TEST), [2] TEST+CryoSeeds (Cell Systems, Ltd., Cambridge, UK), [3] TEST + 10 mM dithiothreitol (DTT), or [4] TEST+CryoSeeds + 10 mM DTT. Cryovials were frozen using slow staged cooling and static vapor freeze and stored at -196 degrees C. MAIN OUTCOME MEASURE: The frozen aliquots were randomly thawed and, after 15 minutes at 37 degrees C, motion analysis was performed. RESULTS: The percent motility after freeze-thaw in TEST was significantly decreased to 42 +/- 5% of prefreeze motility (P < 0.001). Addition of CryoSeeds with holding at -5 degrees C for 10 minutes resulted in 47 +/- 6% of prefreeze motility, which was not different than TEST alone. Addition of DTT to TEST significantly improved post-thaw motility over TEST alone to 71 +/- 7% of initial motility (P < 0.01). The combination of CryoSeeds and DTT further improved post-thaw motility to 80 +/- 10% of initial motility, which was not different than the neat semen. CONCLUSION: The present results suggest that DTT, a reducing agent that prevents oxidation of sulfhydryl groups, protects poor quality spermatozoa from excessive cryodamage. Thus, DTT along with seeding may be a useful addition when long-term storage of poor quality semen is crucial for maintaining reproductive potential.

Comparison of motility and flow cytometric assessments of seminal quality in fresh, 24-hour extended and cryopreserved human spermatozoa.

Functional differences among fresh 24-hour extended and cryopreserved human spermatozoa were assessed using both computer-assisted semen analysis (CASA) and flow cytometry. The objective was to determine if there were interrelationships among various qualitative parameters of the fresh and treated samples when assessed by these two automated methods. Fertile donor specimens (n = 15) were split and examined for sperm motility and curvilinear velocity using CASA within 1 hour postejaculation, after 24 hours in TEST-yolk buffer at 5 degrees C and after cryopreservation in TEST-yolk-glycerol medium. Flow cytometric analyses were performed on 24-hour extended and cryopreserved (CP) samples after fluorescent staining with rhodamine 123 to quantify mitochondrial function and carboxydimethyl fluorescein diacetate and propidium iodide to assess plasma membrane integrity. The percentages of spermatozoa with functional mitochondria and intact membranes along with the proportion of dead cells were identified and quantified by flow cytometry. Quadrant analyses of these data were used to determine the relative red and green fluorescent intensities. The initial sperm motility was correlated to the motility observed for the 24-hour stored and the CP samples. The sperm velocity of both the initial and the 24-hour extended samples was correlated to the velocity of CP samples. As for the comparison of the two automated methods for assessing seminal quality, the only sperm motion parameter that was correlated with a sperm population identified by flow cytometry (quadrant 4) was the curvilinear velocity of the sperm after 24 hours storage (r = 0.69) and after cryopreservation (r = 0.74). The present findings indicate that additional research is needed to determine if prefreeze analyses of donor sperm could be useful in predicting the post-thaw integrity of CP samples and, thereby, be useful in screening potential semen donors.