Characterization of spermatozoa by planar morphometry.

OBJECTIVE

To define the morphometric characteristics of normal sperm heads, and compare them to sperm head measurements used to define normal morphology using strict criteria.

DESIGN

Computerized image analysis of selected normal and abnormal seminal fluid specimens collected for routine male fertility studies.

SETTING

Research laboratory at the Department of Medical Technology, Bowling Green State University, Bowling Green, OH.

PATIENTS

Sixty adult male patients who submitted semen samples for routine analysis. Fifty percent had normal seminal fluid analysis results. The remaining 50% demonstrated abnormal sperm morphology. CRITERION STANDARD: Microscopic evaluation of sperm head morphology. Sperm fitting the criteria of normal as defined by WHO (1987 and 1992) and Kruger (1988) were classified as normal. Sperm with a post nuclear area of less than 40% were classified as acrosomal deficient.

MAIN OUTCOME MEASURES

Measurements made from stained seminal fluid smears included sperm head area, perimeter, acrosomal area, percent acrosome, Ferret's diameters, aspect ratio, shape factor, and specific length. Normal sperm heads (NL group) were compared to sperm heads demonstrating an acrosomal deficiency (AD group) for statistically significant differences using multivariate analysis of variance (MANOVA) and analysis of variance (ANOVA). Stepwise discriminant analysis was used to remove duplicating variables. Discriminant analysis was used to classify the sperm heads into NL and AD groups. Receiver-operating characteristic (ROC) curves were applied to the 2 most influential variables in order to identify a cutoff that best distinguishes normal from acrosomal deficient sperm heads.

RESULTS

MANOVA and ANOVA showed all 10 variables to be statistically significant (p < .002). Discriminant analysis correctly assigned 98.7% of the normal sperm heads to the NL group and 99.0% of sperm with acrosomal deficiency to the AD group. The percent acrosome and acrosomal area were determined to be the 2 most influential variables. ROC analysis identified a cutoff of 3.6 mu2 for acrosomal area as having the highest sensitivity and specificity (99.7% and 88.0%, respectively). Similarly, a cutoff of 44% for percent acrosome gave a sensitivity of 92.3% and a specificity of 88.7%. The coefficient of variation (CV) for each of the 10 variables determined from 20 day-to-day replicates of a normal semen smear ranged from 2.6% to 8.4%.

CONCLUSION

Computerized image analysis is able to define a reference range for sperm head area, percent acrosome, and acrosomal area that may be used to differentiate normal form abnormal sperm heads. Maximum and minimum Ferret's diameters measure sperm head length and width, respectively. Mean maximum Ferret's diameter, minimum Ferret's diameter, and maximum-minimum Ferret's diameter ratio correspond closely to the WHO (1987) midpoint for normal sperm head length, width, and length-width ratio. The average percent acrosome of normal sperm heads determined by morphometry closely correlate to the WHO (1992) and Kruger (1988) midpoints for percent acrosome.