Protocol - Male Fertility Status

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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.

Specific Instructions

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:

  • Urinate
  • 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.

  1. Name
  2. Birth date
  3. ID code
  4. Period of abstinence from ejaculation
  5. Date and time of collection
  6. Completeness of sample
  7. Difficulties collecting the sample

Lab staff should record the following:

  1. 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:

pH _______________

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)

1.5 (1.4-1.7)

Total sperm number (106 per ejaculate)

39 (33-46)

Sperm concentration (106 per ml)

15 (12-16)

Total motility (PR + NP, %)

40 (38-42)

Progressive motility (PR, %)

32 (31-34)

Vitality (live spermatozoa, %)

58 (55-63)

Sperm morphology (normal forms, %)

4 (3.0-4.0)

Other consensus threshold values


> 7.2

Peroxidase-positive leukocytes (106 per ml)

< 1.0

MAR test (motile spermatozoa with bound particles, %)

< 50

Immunobead test (motile spermatozoa with bound beads, %)

< 50

Seminal zinc (_mol/ejaculate)

≥ 2.4

Seminal fructose (_mol/ejaculate)

≥ 13

Seminal neutral glucosidase (mU/ejaculate)

≥ 20

Personnel and Training Required

The processing and analyses procedures should be performed by a certified laboratory technician.

Equipment Needs

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.

Requirement CategoryRequired
Major equipment Yes
Specialized training Yes
Specialized requirements for biospecimen collection No
Average time of greater than 15 minutes in an unaffected individual No
Mode of Administration



Adolescent, Adult


Adult men and boys 14 and older

Selection Rationale

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.



Human Phenotype Ontology Decreased fertility in males HP:0012041 HPO
Human Phenotype Ontology Male infertility HP:0003251 HPO
caDSR Form PhenX PX101601 - Male Fertility Status 6872587 caDSR Form
Derived Variables


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

Protocol Name from Source

WHO Laboratory Manual for the Examination and Processing of Human Semen, Fifth Edition, 2010


World Health Organization (WHO). (2010). WHO laboratory manual for the examination and processing of human semen (5th ed.). Geneva, Switzerland: WHO.

General References

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.

For infertility:

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.

Protocol ID


Export Variables
Variable Name Variable IDVariable DescriptiondbGaP Mapping
PX101601110000 Immunobead test (motile spermatozoa with more
bound beads show less
PX101601100000 MAR test (motile spermatozoa with bound particles) N/A
PX101601090000 Peroxidase-positive leukocytes N/A
PX101601010000 Semen volume N/A
PX101601130000 Seminal fructose N/A
PX101601140000 Seminal neutral glucosidase N/A
PX101601120000 Seminal zinc N/A
PX101601080000 pH N/A
PX101601030000 Sperm concentration 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
Reproductive Health
Measure Name

Male Fertility Status

Release Date

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

Measure Protocols
Protocol ID Protocol Name
101601 Male Fertility Status

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