Protocol - Male Fertility Status
The World Health Organization’s (WHO’s) 2010 WHO Laboratory Manual for the Examination and Processing of Human Semen, Fifth Edition, provides a standardized protocol for collection, analyses, and interpretation of sperm parameters such as sperm numbers, sperm motility, sperm morphology, and azoospermia.
Motility values may not be as accurate if samples are shipped overnight, but morphology should be unaffected. Please note that the WHO Laboratory Manual for the Examination and Processing of Human Semen undergoes periodic updates, and the Expert Review Panel encourages researchers to check with World Health Organization (WHO) for updates.
Note that the following information is a summary, the full protocol is in the WHO laboratory manual available here.
Collection of Semen Sample
- Provide the man with clear written and verbal instructions how to properly collect the sample.
- Sperm should be collected after a minimum of 2 days of abstinence or a maximum of 7 days of abstinence from ejaculation.
Instruct him to do the following:
- Wash hands and penis with soap. Rinse. Dry off with fresh disposable towel.
- Masturbate and ejaculate into the sterile specimen container. Cap the container. If some of the sperm is not ejaculated directly into the specimen container it should be recorded how much (% of sample) was not captured. This fraction of the sample should never be collected and processed.
- Ensure that a specimen label with subject’s name and identification number is affixed to the side of the container.
He should record the following information at the time of sample collection.
- Birth date
- ID code
- Period of abstinence from ejaculation
- Date and time of collection
- Completeness of sample
- Difficulties collecting the sample
Lab staff should record the following:
- Time between collection and analyses
Keep the sample at 20° to 37° C. The time between the time of collection and delivery to the laboratory should be approximately 1 hour. The laboratory should begin analysis within 3 hours of collection. Ideally the sample is collected at or near the laboratory.
Processing and Analyses of Semen Sample
Normally, semen samples will liquefy within 30 minutes, and once this occurs laboratory staff can proceed with semen analysis.
Follow the detailed instructions in the lab manual to measure and record the following characteristics of the sample.
Semen volume (ml) __________________
Total sperm number (106 per ejaculate) __________________
Sperm concentration (106 per ml) ___________________
Total motility (PR + NP, %) _______________________
Progressive motility (PR, %) _________________________
Vitality (live spermatozoa, %) ________________________
Sperm morphology (normal forms, strict criteria %) __________________
Other consensus threshold values that may be reported:
Peroxidase-positive leukocytes (106 per ml) _______________
MAR test (motile spermatozoa with bound particles, %) ______________
Immunobead test (motile spermatozoa with bound beads, %) __________
Seminal zinc (_mol/ejaculate) _________________
Seminal fructose (_mol/ejaculate) _________________
Seminal neutral glucosidase (mU/ejaculate) ______________
Interpreting the Results
The following values represent lower reference limits for the semen parameters (5 centiles and their 95% confidence intervals)
Lower reference limit
Semen volume (ml)
Total sperm number (106 per ejaculate)
Sperm concentration (106 per ml)
Total motility (PR + NP, %)
Progressive motility (PR, %)
Vitality (live spermatozoa, %)
Sperm morphology (normal forms, %)
Other consensus threshold values
Peroxidase-positive leukocytes (106 per ml)
MAR test (motile spermatozoa with bound particles, %)
Immunobead test (motile spermatozoa with bound beads, %)
Seminal zinc (_mol/ejaculate)
Seminal fructose (_mol/ejaculate)
Seminal neutral glucosidase (mU/ejaculate)
Protocol Name from Source:
WHO Laboratory Manual for the Examination and Processing of Human Semen, Fifth Edition
Personnel and Training Required
The processing and analyses procedures should be performed by a certified laboratory technician.
Highly specialized laboratory equipment is necessary to perform accurate semen analyses. Many groups rely on a Computer-Assisted Sperm Analysis (CASA) machine, particularly for motility assessments.
|Specialized requirements for biospecimen collection||No|
|Average time of greater than 15 minutes in an unaffected individual||No|
Mode of Administration
Adult men and boys 14 and older
The WHO Laboratory Manual for the Examination and Processing of Human Semen is the most comprehensive manual available for standardized protocols of semen collection and analyses.
|Common Data Elements (CDE)||World Health Organization Human Semen Examination Laboratory Sample Manual Assessment Description Text||5746939||CDE Browser|
|Human Phenotype Ontology||Decreased fertility in males||HP:0012041||HPO|
|Human Phenotype Ontology||Male infertility||HP:0003251||HPO|
Process and Review
The Expert Review Panel #5 (ERP 5) reviewed the measures in the Reproductive Health domain.
Guidance from ERP 5 includes:
Added a new measure
New Data Dictionary
World Health Organization (WHO). (2010). WHO laboratory manual for the examination and processing of human semen (5th ed.). Geneva, Switzerland: WHO.
For environmental exposures:
Serrano, M., Gonzalvo, M. C., Sánchez-Pozo, M. C., Clavero, A., Fernández, M. F., López-Regalado, M. L., Mozas, J., Martínez, L., & Castilla, J. A.(2014). Adherence to reporting guidelines in observational studies concerning exposure to persistent organic pollutants and effects on semen parameters. Human Reproduction, 29(6), 1122-1133.
Guzick, D. S., Overstreet, J. W., Factor-Litvak. P., Brazil, C. K., Nakajima, S. T., Coutifaris, C., . . . Vogel, D. L.; National Cooperative Reproductive Medicine Network. (2001). Sperm morphology, motility, and concentration in fertile and infertile men. New England Journal of Medicine, 345(19), 1388-1393.
Murray, K. S., James, A., McGeady, J. B., Reed, M. L., Kuang, W. W., & Nangia, A. K. (2012). The effect of the new 2010 World Health Organization criteria for semen analyses on male infertility. Fertility and Sterility, 98(6), 1428-1431.
Rohayem, J., Fricke, R., Czeloth, K., Mallidid, C., Wistuba, J., Krallmann, C., Zitzmann, M., & Kliesch, S. (2015). Age and markers of Leydig cell function, but not of Sertoli cell function predict the success of sperm retrieval in adolescents and adults with Klinefelter’s syndrome. Andrology, 3(5), 868-875.
|Variable Name||Variable ID||Variable Description||dbGaP Mapping|
|PX101601110000||Immunobead test (motile spermatozoa with more||N/A|
|PX101601100000||MAR test (motile spermatozoa with bound particles)||N/A|
|PX101601140000||Seminal neutral glucosidase||N/A|
|PX101601070000||Sperm morphology (normal forms, strict criteria)||N/A|
|PX101601050000||Progressive motility (PR)||N/A|
|PX101601040000||Total motility (PR + NP)||N/A|
|PX101601060000||Vitality (live spermatozoa)||N/A|
|PX101601020000||Total sperm number||N/A|
Male Fertility Status
February 26, 2010
This measure describes how to properly collect, analyze, and interpret the characteristics of a semen sample for the assessment of male fertility status.
Sperm and semen characteristics, such as sperm count, motility and morphology, as well as semen volume and semen fructose, can influence a man’s fertility status. By measuring these parameters, researchers and clinicians may be able to identify a potential cause of the couple’s infertility related to the male partner. Additionally, abnormalities in sperm and semen may be related to environmental exposures, illicit drugs, or medications.
Reproductive health, semen, sperm, motility, morphology, fertility, World Health Organization, WHO