WHO laboratory manual for the examination and processing of human semen

Computer assisted semen analyzers in andrology research and veterinary practice

Baseline DNA damage in spermatozoa from fertile and infertile men was compared using a modified alkali single cell gel electrophoresis (comet) assay. Semen from normozoospermic fertile, normozoospermic infertile and asthenozoospermic infertile (World Health Organization criteria, 1992) samples were studied. No significant difference was observed in levels of baseline damage between the three groups. A median value for baseline damage of approximately 20% (80% head DNA) was obtained in all samples. Irradiation with X-rays (5-30 Gy) produced no additional damage in fertile samples when median values were examined. However, irradiation with 30 Gy X-rays produced significant damage in both infertile groups. Hydrogen peroxide (40 microM) treatment induced significant damage in the asthenozoospermic group, whereas 100 microM H2O2 was required to cause significant damage in the normozoospermic fertile and infertile samples. Within the fertile population a subgroup in which percentage head DNA was greater than 80% was observed in both treated and untreated specimens. This subgroup significantly decreased with treatment in both infertile groups. We conclude that the asthenozoospermic infertile group is more susceptible to damage than the normozoospermic infertile group, which in turn is more susceptible than the fertile group. The fertile group contains a resistant subpopulation of spermatozoa with relatively intact DNA.

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A comparison of baseline and induced DNA damage in human spermatozoa from fertile and infertile men, using a modified comet assay.

Two fertility trials were undertaken to evaluate the relationship between boar semen quality and fertility (conception rate and litter size) after on-farm artificial insemination (AI). Trial 1 included 98 ejaculates from 27 boars, and trial 2 included 72 ejaculates from 26 boars. The semen quality was measured by computer-assisted semen analysis (CASA) using the Hobson Sperm Tracker. Boar semen was diluted in a standard extender (Beltsville Thawing Solution, BTS), dispensed into 75 ml allquots each containing 1.5 x 10(9) spermatozoa and dispatched to farms by overnight mail for use by their normal AI procedures. Randomly selected 75 ml aliquots of semen from each boar were also sent to the institute of Zoology for CASA measurement. Prior to CASA analysis, the spermatozoa were recovered from the BTS using Percoll gradients, resuspended in trisbuffered saline media containing 40 mM Ca++, and incubated at 39 degrees C. Parameters of sperm motion were measured after 0, 2, 4, and 6 hours of incubation. Various multiple regression models based on measured motion parameters could account for up to 24% of the variation in litter size. Using logistic regression, highly significant (P < 0.0001) models explaining conception rate in terms of sperm motion were derived for trial 2 only. The change in sperm velocity during the first 2 hours of incubation and the magnitude of the velocity parameters after 2 hours were identified as the most consistent indicators of fertility. Other attributes of sperm quality, i.e., frequency of spontaneous acrosome reactions (AR) and ARs induced by ionophore A23187 or solubilized pig zona pellucida, were also examined. When the "within-trial" median litter size was used as a way of allocating ejaculates to "high" or "low" litter-size groups, higher litter size was associated with lower frequency of both spontaneous and induced AR. These results demonstrate that fertility information can be derived from the CASA analysis of boar semen provided it is combined with a period of incubation in capacitating conditions.

Objectively measured boar sperm motility parameters correlate with the outcomes of on-farm inseminations: results of two fertility trials.

Male factor infertility: Evaluation and management

To determine which sperm movement characteristics are related to in vitro fertilization rates, semen and swim-up preparations used for in vitro fertilization in 108 patients were assessed using the Hamilton-Thorn HTM-2030 Motility Analyzer (HTMA) and other sperm tests. There were highly significant correlations between manual and HTMA results for sperm concentration (Spearman r = 0.881; P less than 0.001) and the percentage of motile spermatozoa (Spearman r = 0.580; P less than 0.001). The percentage of motile spermatozoa with average path velocities greater than 10 microns/s and greater than 20 microns/s, straight line and curvilinear velocity, linearity (straight line velocity vs curvilinear velocity), amplitude of lateral head displacement, and beat-cross frequency were significantly higher in the insemination medium after selection of motile spermatozoa by the swim-up technique than in the semen. Linearity (P less than 0.01), the percentage of morphologically normal spermatozoa (P less than 0.05) and straight line velocity (P less than 0.05) in semen, and the percentage of motile spermatozoa with average path velocities greater than 10 microns/s in both semen (P less than 0.05) and insemination medium (P less than 0.05) were significantly correlated with in vitro fertilization rate when examined by a nonparametric (Spearman) test. With logistic regression analysis of all data, only the diagnoses of male infertility and tubal disease, linearity in semen, and the percentage of motile spermatozoa with average path velocities between 10 and 20 microns/s in insemination medium were significantly related to in vitro fertilization rates.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship between sperm motility assessed with the Hamilton-Thorn motility analyzer and fertilization rates in vitro.

The morphology of spermatozoa bound to the zona pellucida (ZP) of 264 oocytes that had failed to fertilize in 58 in vitro fertilization procedures was studied by light microscopy. The percentage of spermatozoa with normal morphology bound to the ZP (mean 77, range 8-100) was significantly higher than in the insemination medium (mean 42, range 2-80). The abnormal spermatozoa bound to the ZP had small oval (47%), pyriform (46%), amorphous (5%) and tapering (2%) heads. Other abnormalities of morphology were not observed in ZP-bound spermatozoa. The mean rates of binding to the ZP of spermatozoa with normal morphology (44, 95% confidence limits 42-46, number bound/ZP/10(5)/ml inseminated) were much higher than for abnormal spermatozoa with small oval 6.5 (5.5-7.6), pyriform 5.9 (4.8-7.3), amorphous 1.0 (0.5-2.0) and tapering 2.5 (1.2-5.3) heads. The morphologically abnormal forms not found on the ZP were infrequent in the insemination medium (tail defects, large oval, round, pin and duplicate heads and cytoplasmic droplets) but upper 95% confidence limits for standardized binding ratios of less than 60% indicated that these were unlikely to bind to the zona with rates approaching those of normal spermatozoa (standardized binding ratio 180%). A large number of uniformly normal spermatozoa bound to the ZP when the percentage normal morphology in the insemination medium was greater than 40%. The proportions of abnormal spermatozoa in the insemination medium. Spermatozoon head dimensions were measured with a micrometer in samples from 14 patients. While there were no consistent changes in all samples, the means for head width and area were significantly larger and the ratio of the length to width was smaller for spermatozoa on the ZP than for those in the insemination medium. The head length and ratio of length to width of spermatozoa in samples with good morphology were significantly less than in samples with poor morphology. The percentage of spermatozoa with normal morphology, motility and the ratio of head length to width in the insemination medium were positively correlated with the percentage of spermatozoa with normal morphology bound to the ZP. Logistic regression analysis showed that the diagnosis of tubal infertility and the rate of binding of spermatozoa with normal morphology to the ZP were positively related to fertilization rates in vitro while the rate of binding of spermatozoa with pyriform heads was negatively related. In conclusion. human ZP are highly selective for spermatozoa with normal morphology. The frequency of binding of abnormal spermatozoa to the ZP was mainly dependent on semen quality.(ABSTRACT TRUNCATED AT 400 WORDS)

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Morphology of spermatozoa bound to the zona pellucida of human oocytes that failed to fertilize in vitro.

The proportion of human sperm with poor morphology but normal intact acrosomes detected with pisum sativum agglutinin correlates with fertilization in vitro

The human acrosome reaction was induced with the calcium ionophore A23187 and the proportion of reacted spermatozoa was determined with fluorescein-labelled Pisum sativum agglutinin. Human oocytes that failed to fertilize in vitro were used to test binding of spermatozoa to the zona pellucida (ZP) and oolemma. Differential labelling of spermatozoa with fluorescein and rhodamine was used to control for variability in the oocytes. Spermatozoa labelled with one fluorochrome were treated with A23187 and mixed with equal numbers of motile untreated control spermatozoa labelled with the other fluorochrome. The mixture was incubated with zona-intact and zona-free oocytes for 4 h. The sperm-ZP binding ratio of test to control spermatozoa was significantly decreased with increasing proportions of acrosome reacted spermatozoa. In contrast, the sperm-oolemma binding ratio was significantly increased with A23187 treatment. This suggests that acrosome-reacted spermatozoa do not bind to the human ZP.

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Inducing the human acrosome reaction with a calcium ionophore A23187 decreases sperm-zona pellucida binding with oocytes that failed to fertilize in vitro.

D. Y. Liu

Papers

Relationship between sperm motility assessed with the Hamilton-Thorn motility analyzer and fertilization rates in vitro.

Morphology of spermatozoa bound to the zona pellucida of human oocytes that failed to fertilize in vitro.

The proportion of human sperm with poor morphology but normal intact acrosomes detected with pisum sativum agglutinin correlates with fertilization in vitro

Inducing the human acrosome reaction with a calcium ionophore A23187 decreases sperm-zona pellucida binding with oocytes that failed to fertilize in vitro.

Inhibition of human sperm-zona pellucida and sperm-oolemma binding by antisperm antibodies