This chapter reviews the current limitations of CASA in routine clinical andrology and describes a path by which it could become a robust analytical technology that can be employed in accredited expert clinical andrology laboratories. Particular focus is on the need for premarketing validation by manufacturers for CASA-based semen analysis applications, as well as the impact that software differences have on the absolute values reported for sperm kinematics. Without close comparability between CASA systems it will continue to be extremely difficult to establish either decision limits or reference ranges that can be applied clinically for either diagnostic or prognostic purposes.

Discusses the development, application and limitations of computer-aided sperm analysis (CASA) methods, including the deriation of kinematic measures of human sperm motility. Explains the technical and biological factors that limit CASA's functionality for human semen analysis and summarizes expert recommendations on the use of CASA for human semen analysis and sperm kinematics analysis (including sperm-mucus penetration and sperm hyperactivation). Issues related to the non-comparability of different CASA systems are considered, along with quality control for CASA. A strategy for validating a CASA system for human semen analysis, based on expectations of accuracy and precision, is also provided. Finally the use of CASA for analyzing sperm function tests, and new and future CASA technology (including the application of artificial intelligence technqiues) are surveyed.

The physiology and basic principles of sperm washing and other preparative techniques are described, including the limitations and even dangers posed by some methods. Optimized methods for sperm preparation using direct swim-up from semen and density gradient centrifugation are provided as standard operating procedures (SOPs) for easy use at the bench. Methods are focussed on standardization and robustness, and minimizing the risk iatrogenic damage to the spermatozoa and avoiding errors. There are sections on selecting which method to use, dealing with atypical semen specimens, processing multiple specimens, and potential new technology for preparing human spermatozoa for use in assisted conception treatment.

Provides methods for a range of common sperm function tests that are structured as standard operating procedures (SOPs) for easy use at the bench. Methods are focussed on objectivity, robustness, standardized reporting, controlling the risk of errors, and minimizing measurement uncertainty. Includes sperm hyperactivation, acrosome reaction testing, and sperm-zone pellucida binding tests (hemi-zona and competititve binding formats). A protocol for using the sperm survival test in also provided. Limitations of the hyaluronan bibding assay, and of sperm fertilizing ability testing using zona pellucida-free hanster oocytes, are summarized.

Discusses the origins and detection of sperm DNA fragmentation, including cliniCal indications for such testing. Provides methods for a range of common techniques for assessimg sperm DNA fragmentation that are structured as standard operating procedures (SOPs) for easy use at the bench, inclding the TUNEL assay, Comet assay, and the sperm chromatin dispersion test (Halosperm test).

Sampling of cervical mucus, its assessment, and the investigation of sperm-mucus interaction are described in detail. Methods are provided for a range of common tests that are structured as standard operating procedures (SOPs) for easy use at the bench. Methods are focussed on objectivity, robustness, standardized reporting, controlling the risk of errors, and minimizing measurement uncertainty. Includes the post-coital test, Kurzrok-Miller slide test, the Kremer capillary tube sperm penetration test, and the sperm-cervical mucus contact sldie test. An explanation of crosed hostility testing is also included, along with a discussion of complementary tests and the use of cervical mucus surrogates.

This chapter provides an overview of 20 primary recommendations for safe working in the andrology laboratory, along with sections on accident prevention, appropriate clothing and the proper use of personal protective equipment (PPE), fire safety, dealing with spills, use and disposal of biological materials, chemical hazards, compressed gases, and cryogenics.

The basic princples of cryobiology are described for both slow freezing and vitrification of spermatozoa. Specific aspects of cryopreserving human spermatozoa are discussed in detail, incluidng the formulation of cryopreservation media and their proper use. Alternative packaging devices are discussed in relation to the achievement of correct cooling and warming curves as well as effective biocontainment. High security straws are recommended as the best method to use from both perspectives, and a standard operating procedure (SOP) for easy use at the bench is provided. SOPs for human sperm vitrification techniques are also gven. Quality control and risk management aspects of sperm freezing and for cryobank organzation are described. Finally, there is a section on sperm donation.

Standard operating procedures (SOPs) for processing both epididymal and testicular spermatozoa are provided.

Provides methods for a range of common additional assessments on semen that are structured as standard operating procedures (SOPs) for easy use at the bench. Methods are focussed on objectivity, robustness, standardized reporting, controlling the risk of errors, and minimizing measurement uncertainty. Includes anti-sperm antobodies (direct and indirect MAR test), immunocytochemical staining of leukocytes, meaaurement of reactive oxygen species and seminal plasma redox state, and biochemical tests on the seminal plasma to evaluate acessory gland function (zinc for the prostate, frutose for the seminal vesicles and alpha-glusidase for the epidiymis) and investigate abnormalities of the sequence of ejaculation. There is also a discussion on the microbiological examination of semen,.