What constitutes morphological normal and abnormal human sperm heads

Abstract

The percentage morphologically normal sperm appears to be of predictive value in the in vitro fertilization laboratory. However, the methodology used in this context is technically inaccurate and imprecise (subjective) and needs to be improved. Nevertheless, most laboratories continue to use such techniques. It is therefore not surprising to find that three different sperm morphology classification systems are in use. Some of the published methods include, the World Health Organization system (WHO), the Tygerberg strict criteria (TSC) and the Dusseldorf criteria (DC) to define morphologically normal sperm. Each of these methods use different criteria and different cut-off values to define sperm morphology normality in patients. For WHO it is >30%, for TSC it is >14% and for DC it is >30%. Consequently, there is no objective morphological criteria for defining normal spermatozoa in human semen at present. Such criteria can be established only on the basis of extensive studies that assess the morphometric characteristics of spermatozoa. Therefore, there is a great need to standardize methodology in this context. The second problem lies with the training of technicians. Unfortunately implementation of visual semen analysis often differs between laboratories. Moreover, few laboratories systematically train their technicians by one standard method, and monitor within and between technician variability. Accurate and precise visual semen analysis will only be achieved by implementing a program of international standardization and technician training and proficiency testing. The third problem is that specimen handling and preparation for evaluation of sperm morphology are not standardized and this needs to be done to the highest degree possible. ln this investigation four microscopic techniques were used to study sperm morphology. The purpose of this part of the investigation was to test whether Papanicolaou stained (PAP) sperm smears studied by means of bright field microscopy represents a reliable method to study sperm morphology when compared to more sophisticated microscopic techniques. Consequently Bright field microscopy (PAP staining), Normaski differential interference microscopy (NDIM), Scanning electron microscopy (SEM) and Confocal microscopy of normal and abnormal sperm types were compared. However, a new technique had to be developed for preparing sperm for confocal microscopy. For this purpose both unfixed and fixed sperm were embedded in agarose to avoid motion artifacts during confocal imaging. Both groups of unfixed and fixed sperm were placed in PBS buffer containing O.874mM dihexaoxacarbocyanine iodide (D!OC6(3)) at room temperature. Sperm were pre-loaded for 30 minutes with Tetramethyl rhodamine methylester (TMRM). DIOC6(3) is a lipophilic fluorescent dye, and it has been found that the fluorescent intensity of sperm increased over time. For Laser Scanning Confocal Microscopy 3D sperm morphology was reconstructed from a series of 20 - 30 optical sections taken at 0.2:m intervals through each sperm. ln order to examine 3D morphology a series of projected (rotated) views were reconstructed to allow visualization of a 3D animation set. All forms of abnormal sperm could be clearly identified by all four microscopic techniques. Despite the fact that human sperm structure can be visualized and studied more comprehensively by means of both NDIM and confocal microscopy than with bright field microscopy, the latter technique of PAP stained smears is adequate to identify abnormal sperm morphology on a routine basis in the clinical laboratory. However, confocal microscopy of human sperm reveals that sperm may be scored normal/abnormal on the basis of its orientation. Only slight rotation of a 3D constructed image of a spermatozoon by means of confocal microscopy can change the classification of a sperm from normal to abnormal. ln the second part of the investigation bright field microscopy of PAP stained smears were used to test the reliability/repeatability of scoring among three technicians from three different laboratories using the Tygerberg Strict Criteria. All three technicians scored the same 77 patients. While two technicians scored within a relatively close range, the third technician varied more when TSC was employed. One technician classified 43"/o of the samples in a different TSC category when compared to a second technician. A fourth technician scored the same sperm smears but used the WHO criteria (1992). The scores for TSC and WHO were consequently compared. It was found that a 3-5% TSC range of abnormal sperm corresponded to a 6-30"/" WHO range.