The Procedure for Semen Analysis as described in the World Health Organization's Laboratory Manual for the Examination and Processing of Human Semen (2021)

By: Logan Hunt

In 2021, the World Health Organization, or WHO, published the sixth edition of their Laboratory Manual for the Examination and Processing of Human Semen, which provides standardized guidelines to physicians and other medical professionals performing semen analysis. Semen analysis is a technique that medical professionals use to analyze the characteristics of a male’s semen and sperm cells to identify possible causes of male infertility. A sperm cell is the male sex cell that fertilizes a female’s egg cell, while semen is the fluid containing sperm cells that men release during ejaculation. The WHO created the first laboratory manual for semen analysis in 1980, a time when there was no standardized way to analyze a male’s semen sample. The WHO laboratory manual was one of the first documents to standardize a procedure for evaluating human semen to look for sperm abnormalities, and it remains one of the most widespread methods of evaluating a male’s fertility potential and determining the causes of male infertility.

  1. Background
  2. History of Semen Analysis
  3. WHO Manual’s Semen Analysis Protocol
  4. Impacts


The WHO manual provides physicians with a method to identify the causes of male infertility, which is the inability of a male to impregnate a fertile female after at least one year of unprotected intercourse. In typical cases, when a male and female engage in unprotected intercourse, the male releases semen that carries sperm cells into the female. Semen is the fluid that carries and nourishes the sperm cells. Sperm cells join with a female’s egg cell in the process of fertilization, which forms a zygote. That zygote travels to the uterus, where it will develop into an embryo and eventually a fetus throughout the duration of pregnancy. When a male or female is infertile, they are unable to have a child through intercourse alone. Researchers define a couple as infertile, which the manual characterizes as subfertility, when they have had unprotected sex for twelve or more months without a resulting pregnancy.

The semen analysis procedure in the WHO manual is one of the most common ways of diagnosing male infertility. The WHO is an international organization of government officials from across the world that routinely sets standards and guidelines for global health, which is why many organizations use their world-wide manual for semen analysis. The first step of conducting a semen analysis includes collecting a semen sample from the male patient, usually through them ejaculating in a private room at a clinic. The medical practitioner then uses a microscope and various tools to assess several characteristics of sperm that contribute to male fertility potential. While the WHO’s semen analysis technique looks at many characteristics of the sperm and semen, only three of those characteristics highly correlate with fertility: sperm count, morphology, and motility. Sperm count is the amount of sperm cells in semen. Sperm morphology is the shape of sperm, which consists of a head, a tail, and a midpiece that connects the two. Motility refers to a sperm cell’s ability to move efficiently.

History of Semen Analysis

Prior to the WHO manual’s publication, physicians William Cary and Robert Hotchkiss, both of whom studied the male reproductive tract, developed one of the first early semen analysis procedures and published their results in 1934. Their semen analysis followed three steps, including collection of the sperm and semen, macroscopic examination, and microscopic examination, which is similar to the WHO’s process. First, Cary and Hotchkiss recommended that the male either collects their semen in a quiet room at the clinic or collects their sample outside of the clinic in a glass bottle then brings it into the clinic at their appointment. If a participant cannot produce a semen sample without having intercourse and does need to use a condom, the authors advised that the condom should be washed and dried prior to collection of a specimen to prevent any foreign bodies from the condom. Second, for the macroscopic examinations, the physicians analyzed the amount of semen present, its color and appearance, thickness, and pH. Third, their microscopic examinations included looking at the samples under a microscope and reviewing the density, motility, and morphology of the sperm.

From 1934 until the manual’s publication in 1980, various other researchers developed their own methods to test semen and sperm, but the techniques remained unstandardized. In 1934, Samuel Raynor Meaker, a physician who studied male infertility, published his book A Practical Manual and Clinical Procedure, which described his semen analysis technique for male fertility. In that book, he emphasized sperm endurance, in addition to sperm count, motility, morphology. Researchers worldwide developed their own analysis techniques, which analyzed similar sperm characteristics, but those did not follow identical procedural steps. According to Fernando Tadeu Andrade-Rocha in an article analyzing the history of semen analysis, semen analysis became a laboratory practice in the mid-1930s, but there were not defined, universal methods for the technique until 1980. Without a standardized protocol, the semen analysis technique varied amongst medical practitioners and researchers and led to inconsistency of results within the medical community.

In 1980, the WHO published their first laboratory manual for the examination and processing of human semen, which standardized the semen analysis technique and allowed its use to become widespread. The manual contains specific protocols to maintain the quality of semen analysis across labs throughout the world as well as to allow the comparison of results between said labs. Prior to the WHO manual, there were very few well defined ranges for the normal variations of sperm characteristics, and none with international agreement. The manual includes a step-by-step breakdown of performing the semen analysis and the internal and external conditions of the semen sample that the physicians should control for a proper analysis.

Since their initial 1980 publication, the WHO has continually updated the manual to incorporate advances in reproductive medicine and feedback from researchers. As of 2024, they have updated their manual five times, with the Department of Sexual and Reproductive Health and Research of the WHO publishing the most recent sixth edition in 2021. The manual has been translated into several languages including Italian and Korean. In response to feedback from individuals using the methods they developed to conduct semen analysis, the WHO devised an editorial committee to review the methods and change or update the standards as needed. The sixth edition defines the most recent findings on how professionals should conduct a semen analysis and recommends parameters for assessing the quality of semen and sperm.

WHO Manual’s Semen Analysis Protocol

The process of semen analysis as set by the WHO begins with the collection procedure. For semen sample collection, the WHO recommends that a male ejaculate in a private room at a clinic into a clean, wide-mouthed container made of plastic. Within five minutes of collection, the manual advises that the healthcare professional place the sample in an incubator maintained at thirty-seven degrees Celsius. Although the analysis of one ejaculate sample is enough for the investigation of infertility in the male, the manual explains that examining two or three ejaculates can give physicians a better baseline of a male’s fertility potential. That is typically done because there can be variation in semen samples from the same person depending on their spermatogenic cycle, which is the time it takes for the body to create new sperm and for those sperm cells to mature. Using semen from different spermatogenic cycles means that a completely new batch of sperm cells would be present in the semen sample each time, allowing the clinician to understand more about the overall sperm characteristics of the male rather than the particular characteristics of one ejaculation.

After obtaining a semen sample, the WHO instructs the medical professional to evaluate the sperm motility after the sample has liquified, which usually happens about thirty to sixty minutes after collection. Sperm liquefaction is a process where the ejaculate, which is gel-like after ejaculation, becomes watery. During intercourse, that liquification allows the sperm to move through the female reproductive tract. Sperm motility is related to pregnancy rates as it helps determine a sperm’s ability to move through the female reproductive system to the egg, which is in the fallopian tube, for fertilization. The WHO details that the healthcare professional should mix the liquified semen sample with either a small, specialized mixer, known as an orbital mixer, or manually mix the sample for fifteen to thirty seconds to ensure even distribution of the sperm across the sample. The manual then advises the medical professional to place ten microliters of the fully mixed sample on a clean microscope slide warmed to thirty-seven degrees Celsius. After the sample on the slide stabilizes, the WHO states that the professional must assess the sperm motility under a light microscope at 200 to 400 times magnification.

Following sample preparation, the manual states that the healthcare worker must measure sperm motility through the eyepiece of the microscope. The manual suggests using an eyepiece that contains a grid image because it makes it easier to assess one small area at a time. The WHO explains that when the sperm concentration is high, professionals should only evaluate the top row of the grid, but if the sperm concentration is low, the healthcare worker should evaluate the entire grid. Physicians often use CASA to better detect and analyze the motility of the sperm cells. The physicians count sperm with different motility levels in two rounds of counting. There are four categories of sperm according to motility level denoted as A through D, with rapidly progressive sperm, or grade A, being the most motile and grade D being the most immotile sperm. In the first round of counting, the physician counts the number of grade A and B sperm, while in the second round of counting they count the number of grade C and D sperm. The manual reports that the healthcare worker should analyze 200 sperm on each microscope slide and compare the amounts of each grade of sperm motility between the samples to ensure the values are within acceptable range of each other. The WHO explains that the goal of analyzing the sperm motility is to report the average motility grade, with grade A indicating higher male fertility potential.

The WHO manual then states that determining the sperm count of the sample is a predictor of a male’s ability to fertilize the egg cell of the female. A low amount of sperm makes it less likely that a sperm cell will travel to and enter the egg, meaning that a low sperm count directly correlates with infertility. Various forms of abnormal sperm concentration are linked to fertility issues. For example, azoospermia is the lack of sperm in the semen while oligozoospermia is lower than normal sperm counts. When the male has no sperm present in his semen, he has a condition known as aspermia.

To begin the process of counting the amount of sperm present, the manual explains that a medical professional must prepare a microscope slide after thoroughly mixing and preparing the sample. The professional then must dilute the sample to the appropriate degree based on the amount of sperm the physician sees when using the microscope at 200- and 400-times magnification. The manual states that when there is more sperm in the sample, the healthcare workers must add more diluting liquid to ensure all samples have the same density of sperm. Next, the WHO details that the professional must load the sample into the counting chamber. The manual recommends that the professional count the sperm present only in the central grid, but if the central grid contains fewer than 200 sperm, the WHO advises that the worker also analyze the other eight grids. The manual also explains that the professional can only count sperm in which both the head and tail is present inside the grid. The WHO details that the healthcare worker should count sperm on a few different microscope slides, and if those values are acceptably close, the professional can then estimate the number of sperm per ejaculate. Finally, the manual states that the medical workers must determine whether the sample has a high or low male sperm count by multiplying the average concentration of sperm by ejaculate volume.

The WHO manual also includes the specific protocols to assess the semen morphology. In order to penetrate through the outer layer of the egg and fertilize it, the sperm must be a certain shape, which means that males with abnormal sperm morphology have a lower fertility potential. As with other analyses, the WHO manual states that the whole semen sample should be mixed well so that the medical professional can extricate a small amount and put it onto the surface of a plastic slide, which the professional will later place under the microscope. Next, the manual explains that the healthcare worker must drag another slide at a forty-five-degree angle across the first slide to thin out and separate the individual sperm. After the sample has air dried, the WHO states that the healthcare worker should stain the sample to make the details of the sperm morphology easier to see. The manual states that the professional then magnifies the sample 1,000 times its original size and uses oil immersion, which is a technique where the lab professional adds oil on top of the slide cover. That allows the sperm cells to light up and become more magnified, making it easier for examiners to analyze sperm morphology. After, the manual explains that the medical worker examines the three main components of sperm including their head, tail, and the midpiece that connects the two. The WHO advises that professionals study around 200 sperm cells to make claims about the sperm morphology of the male participant.

The WHO manual has guidelines to score the sperm morphology, which includes categories of sperm abnormalities specific to each component of the sperm cell, such as the head, tail, and midpiece. The WHO states that common defects in the sperm’s head include a head size less than forty percent or greater than seventy percent of the normal head area and an asymmetrical or non-oval shape, as well as a double head, which occurs when the sperm has two heads, rather than one. Next, the manual details that common midpiece defects in sperm include having an irregular shape, being too thick or thin, and presenting in a bent or coiled manner. In addition, the WHO manual notes that the tail of some sperm can be short, bent, coiled, broken, or even a double tail. The tail helps the sperm cell efficiently swim through the female reproductive tract to fertilize the egg, so an abnormal tail shape could prevent the sperm from reaching the egg. According to an article by S. S. Vasan, a physician who studies male health, research demonstrates that pregnancy is possible with semen samples with low morphology scores since fertilization only requires one normal sperm cell to fertilize the egg given the other parameters are efficient.


The WHO manual for semen analysis is an internationally standardized guideline for  proper analysis of sperm and the fertility potential of males. According to the WHO manual, clinical and research laboratories use the procedure set forth by the manual to understand the reproductive function in males, diagnose males with subfertility, plan treatment for subfertile couples, and conduct large-scale population studies and research activities, such as looking at the effects of viral, environmental, and other toxins on male reproductive function. In an article reviewing the WHO’s semen analysis procedure titled “The Sixth Edition of the WHO Manual for Human Semen Analysis: A Critical Review and SWOT Analysis,” the researchers, who were from various institutions worldwide, state that the WHO’s sixth edition laboratory manual provides beneficial insights into the management of male infertility around the globe. They also note that the sixth edition added more data from males of Southern Europe, Asia, and Africa that researchers previously considered to be underrepresented in the data in earlier editions of the laboratory manual.

The semen analysis procedure the WHO manual details helps physicians potentially determine causes of male infertility. If the physician finds no abnormal results through semen analysis, they can further investigate other issues that may cause infertility, such as examining the patient’s reproductive system for an infection or obstruction of the genital tract that prevents ejaculation or defects in hormone secretions that impact sperm production. If a physician does find abnormal results through a semen analysis, they can then recommend solutions for the male’s infertility, including using assisted reproductive technologies. Assisted reproductive technologies, or ART, are fertility treatments that involve a physician surgically removing the egg cells from a female and sperm cells from a male. Then, the professional artificially fertilizes the egg cell with the sperm in a laboratory, then implants the fertilized egg back into the female’s uterus. One common type of ART is intracytoplasmic sperm injection, or ICSI, during which a medical professional directly injects a sperm cell into the egg cell, bypassing the need for sperm to be motile for fertilization. Those treatments allow many couples that would otherwise not be able to conceive to do so. However, without knowing that they are infertile, a couple may not seek those options.

As of 2024, semen analysis as described by the sixth edition of the WHO manual remains a standardized tool that allows physicians to determine causes of male infertility and provide solutions for those individuals with male infertility to conceive.


  1. Anamthathmakula, Prashanth and Wipawee Winuthayanon. “Mechanism of Semen Liquefaction and Its Potential for a Novel Non-Hormonal Contraception.” Biology of Reproduction 103 (2020): 411–426.,the%20causes%20of%20male%20infertility. (Accessed April 2, 2024).
  2. Andrade-Rocha, Fernando Tadeu. “On the Origins of the Semen Analysis: A Close Relationship with the History of the Reproductive Medicine.” Journal of Human Reproductive Sciences 10 (2017): 242–255. (Accessed April 2, 2024).
  3. Boitrelle, Florence, Rupin Shah, Ramadan Saleh, Ralf Henkel, Hussein Kandil, Eric Chung, Paraskevi Vogiatzi, Mohamed Arafa, Armand Zini, and Ashok Agarwal. “The Sixth Edition of the WHO Manual for Human Semen Analysis: A Critical Review and SWOT Analysis.” Life 11 (2021): 1368. (Accessed April 2, 2024).
  4. Cary, William H. and Robert S. Hotchkiss. “A Differential Stain That Advances the Study of Cell Morphology.” JAMA 102 (1934): 587–590. (Accessed April 2, 2024).
  5. Centers for Disease Control and Prevention. “What Is Assisted Reproductive Technology?” CDC. (Accessed April 2, 2024).
  6. Cleveland Clinic. “Conception.” Cleveland Clinic. (Accessed April 2, 2024).
  7. Cleveland Clinic. “Semen Analysis.” Diagnostics and Testing. (Accessed April 2, 2024).
  8. Male Contraceptive Initiative. “Semen vs. Sperm: Role and Responsibilities.” Male Contraceptive Initiative. (Accessed April 2, 2024).
  9. Mayo Clinic. “Abnormal Sperm Morphology: What Does It Mean?” Mayo Clinic.,reach%20and%20penetrate%20an%20egg. (Accessed April 2, 2024).
  10. Mayo Clinic. “ICSI.” Mayo Clinic. (Accessed April 2, 2024).
  11. Milne Library. “Differential Staining Techniques.” Microbiology: A Laboratory Experience.,the%20surface%20of%20the%20slide. (Accessed April 2, 2024).
  12. Taylor, Alison. “Extent of the Problem.” The BMJ 327 (2003): 434–436.,appropriate%20to%20start%20investigations%20sooner. (Accessed April 2, 2024).
  13. University of Wyoming. “Spermatogenesis.” University of Wyoming.,is%20called%20the%20spermatogenic%20wave. (Accessed April 2, 2024).
  14. Vasan, S. S. “Semen Analysis and Sperm Function Tests: How Much to Test?” Indian Journal of Urology 27 (2011): 41–48. (Accessed April 2, 2024).
  15. Wang, Christina,  Michael Mbizvo, Mario P. Festin, Lars Björndahl, Igor Toskin, and other Editorial Board Members of the WHO Laboratory Manual for the Examination and Processing of Human Semen. “Evolution of the WHO “Semen” Processing Manual from the First (1980) to the Sixth Edition (2021). Fertility and Sterility 117 (2022): 237–245. (Accessed April 2, 2024).
  16. Wang, Christina, & Ronald S. Swerdloff. “Limitations of Semen Analysis as a Test of Male Fertility and Anticipated Needs from Newer Tests.” Fertility and Sterility 102 (2014): 1502–1507. (Accessed April 2, 2024).
  17. World Health Organization. WHO Laboratory Manual for the Examination and Processing of Human Semen Sixth Edition. World Health Organization, 2021. (Accessed April 2, 2024).



Aubrey Pinteric


Arizona State University. School of Life Sciences. Center for Biology and Society. Embryo Project Encyclopedia.


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Monday, July 8, 2024 - 13:14

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