Semen Analysis

Last updated date: 18-Aug-2023

Originally Written in English

Semen Analysis


Despite debate about the therapeutic use of sperm analysis, male fertility research continues to rely on a standardized examination of sperm properties. This is especially true for infertility clinics in underdeveloped and rich countries alike.

A sperm and sperm analysis is a test of a man's sperm and sperm. Its findings, often known as a sperm count or male fertility test, indicate how many sperm are discharged, as well as their form and movement. Semen is the thick fluid that emerges from men's penis during sexual intercourse.

Other optional tests or advanced technology have not been extensively used. The current study discusses significant developments in sperm analysis as reported in the new World Health Organization (WHO) handbook for sperm analysis. The most significant modification in the handbook is the inclusion of evidence-based publications as references to derive normalcy threshold values.


Semen analysis definition 

Semen analysis definition 

Semen analysis is a laboratory test used to determine male fertility. After one year of unprotected sexual intercourse, infertility is defined as the inability to conceive. Infertility affects around 15% of all reproductive-aged couples. A full medical and sexual history, a thorough physical examination, and sperm analysis are used to determine male infertility. The male factor significantly contributes to 30% of infertility cases and is a contributing factor in almost half of all cases. 

During ejaculation, sperm is discharged as boluses. It is made up of spermatozoa generated in the testes, processed in the epididymides, and combined with secretions from male accessory sex organs such as the prostate, seminal vesicles, bulbourethral glands, and the epididymides.

The total quantity of spermatozoa, fluid volume, sperm concentration, and the type of the spermatozoa; their viability, motility, and shape, as well as the composition of the secretions, are all factors to consider when evaluating sperm. A thorough examination of these factors can aid in determining the cause of male factor infertility.


Who needs a semen analysis?

semen analysis

Reasons you may need a semen analysis include:

Male infertility: If a couple has been having problems conceiving, there might be a sperm anomaly. In certain circumstances, this is related to a problem with a man's sperm. A semen analysis determines the possibility that a guy will be able to create a pregnancy.

Vasectomy follow-up: A vasectomy's success is determined by a semen analysis. This treatment closes the sperm-depositing tubes in the sperm. If there are no sperm in the sperm, the vasectomy was successful, and a man cannot conceive a woman.


Specimen Requirements and Procedure

sperm sample

The male collects the sperm sample through self-masturbation near the laboratory or at home. It is suggested that sperm be collected after a minimum of three days and a maximum of seven days of abstinence. To guarantee that all fractions of the ejaculate are collected and complete, clear instructions on the collection of the semen sample must be provided prior to collection.

Any sample loss must be notified to the laboratory throughout the submission process. Because the content of human male sperm can fluctuate greatly over time, it is advisable to collect two or three samples of sperm for more reliable results.

The sample should be collected in a clean, wide-mouthed, spermatozoa-safe container. The sperm container should be stored at a temperature between 20 and 37 degrees Celsius. If the patient is unable to generate a satisfactory sample through masturbation, semen might be collected during sexual intercourse using nontoxic condoms. Latex condoms should not be used in these circumstances since they contain chemical compounds that might impair sperm viability and motility.

So that dehydration and temperature fluctuations do not impact sperm quality, the sperm should be sent to the laboratory within 1 hour of being collected. When handling sperm samples in the laboratory, certain safety rules must be followed.

The sample should be tested according to the WHO laboratory protocol for the testing and processing of human sperm. To guarantee that the sperm samples are correctly and reliably evaluated, the laboratory should use both internal and external quality control procedures.


Diagnostic Tests

Diagnostic Tests

Semen analysis, according to the WHO laboratory handbook for the inspection and processing of human sperm issued in 2010, entails the following steps:

During the first 5 minutes, the specimen is liquefied by placing it on an incubator at 37 degrees Celsius or on the bench. Allowing time for liquefaction aids in the formation of more homogeneous and watery semen with just minor regions of coagulation. Spermatozoa trapped in the coagulum develop the capacity to migrate.

It might take up to 60 minutes for liquefaction to occur. If liquefaction does not occur on its own, chemicals can be employed to aid the process; however, this can have an impact on the composition of the seminal plasma, sperm motility, and morphology, which must be considered.

The volume of the sperm must be measured. Following that, the pH of the sperm is determined and moist preparations are created. Wet preparations aid in determining the appearance, motility, and dilution necessary to determine the amount of spermatozoa. Attention should be made to the existence of spermatozoa agglutination, which occurs when motile sperms become hooked to one another. In such cases, anti-sperm antibody testing should be explored. Agglutination can have an impact on sperm motility and concentration. It is recommended to measure sperm motility within an hour of collection.

Sperm vitality is evaluated, particularly if the sperm count is low. The integrity of the cell membranes aids in determining whether immotile sperms are alive or dead. The sperm count is determined by diluting the sperm. As needed, testing for sperm antibodies are done, such as the mixed antiglobulin reaction test, immunobead test, looking for peroxidase positive cells, and similar biochemical procedures.

If necessary, the sperm must be transported to the microbiological laboratory within 3 hours of collection. After 4 hours, smears are generated to investigate sperm morphology. Because proper semen analyses are labor-intensive and complicated, the best findings are achieved in laboratories that perform these tests on a regular basis. 


Results, Reporting, Critical Findings

Semen Analysis Findings

The WHO has established standard standards of reference for sperm analysis. The numbers in the chart below are the acceptable 5th percentile for the parameters assessed. 

  • Volume = >1.5 ml
  • pH = >7.2
  • Total sperm number = 39 million sperm per ejaculate or more
  • Morphology = >4 percent normal forms using the tygerberg method 
  • Vitality = >58% live sperm
  • Progressive motility = >32%
  • Total (progressive motility and non-progressive motility) = >40% 
  • No sperm agglutination
  • Viscosity = <2 cm after liquefaction 
  • Optional investigations 
    • Mixed antiglobulin reaction test with <50% motile spermatozoa with bound particles 
    • Immunobead test with <50% motile spermatozoa with bound beads
    • Seminal fructose =>13 mcmol/ejaculate
    • Seminal zinc = >2.4 mcmol/ejaculate
    • Seminal neutral glucosidase = <20 milliunits /ejaculate


Clinical Significance

Clinical Significance

Semen studies are carried out as part of the evaluation of male factor infertility. In order to offer a comprehensive diagnostic assessment, a complete medical and sexual history, as well as a thorough physical examination, must be obtained in addition to the semen analysis. If the semen analysis is normal according to WHO guidelines, a single semen specimen may be adequate, however, some experts still urge two different specimens.

If the sperm analyses are abnormal, the assay might be redone three months later after another full spermatogenesis cycle. If the sperm count is poor or missing, the procedure might be repeated early. 


Low Semen Volume (<1.5 ml)

  • When the volume of ejaculated sperm is little, less than 1 ml, a urine specimen should be taken soon after ejaculation. The urine sample is next centrifuged and microscopically inspected for spermatozoa. The presence of a significant amount of sperm in the post-ejaculatory urine samples demonstrates retrograde ejaculation. Spinal cord damage, transurethral prostatectomy, retroperitoneal lymph node dissection, diabetes mellitus, transverse myelitis, multiple sclerosis, or psychogenic reasons can all produce retrograde ejaculation.
  • Incomplete sperm collection or the loss of a portion of the sample during collection might result in a low sperm volume.
  • Men with testosterone insufficiency may have reduced sperm volume and count.
  • Low sperm count or volume with no sperm might be caused by ejaculatory duct blockage or congenital bilateral lack of the vas deferens where the seminal vesicles have not matured.


Low Sperm Count

Men with sperm counts of less than 10 million/ml, sexual dysfunction, or clinical signs indicative of endocrine illness should have their hormone levels examined. Initial labs should include a serum total testosterone (8 a.m. to 10 a.m.) and a serum follicle-stimulating hormone (8 a.m. to 10 a.m.) (FSH). If the total testosterone level is below 300 ng/ml, a second measurement of total and free testosterone, serum luteinizing hormone (LH), and prolactin (PRL) should be taken.

Patients with questionable results will need to have their testing redone. Some specialists advise all-male infertility patients to have an initial hormonal test.

  • Low sperm count, low testosterone, high FSH, and high LH: primary hypergonadotropic hypogonadism is suspected (that affects both the spermatogenesis and Leydig cell function). By karyotyping these people, you can rule out chromosomal defects such as Klinefelter's syndrome and Y chromosome microdeletion (sperm count is 5 million/ml). Other causes include survivors of cancer treatment, such as radiation and chemotherapy, testicular trauma, testicular torsion, or an infection such as mumps. 
  • Low sperm count, normal testosterone and LH, and high FSH: seminiferous tubule injury without Leydig cell failure and aberrant spermatogenesis are suspected.
  • Low sperm count, low testosterone, normal or low FSH, normal or low LH, or hypogonadotropic hypogonadism: look for additional pituitary hormone deficits, such as thyroid function (free T4), cortisol at 8 a.m., and prolactin levels. A prolactin-secreting tumor is indicated by elevated prolactin levels.
  • Men with low sperm count, high testosterone and LH, and normal FSH should be suspected of having partial androgen resistance syndrome. In such males, the androgen receptor is not working adequately. They have a masculine phenotype with gynecomastia and a varied appearance ranging from hypospadias to normal male genitalia.


  • Low sperm count, normal testosterone, normal LH and normal FSH, normal testis size: evaluate for genital tract obstruction:
  1. Congenital vas deferens absence (physical examination, low sperm pH, verified on scrotal/transrectal ultrasonography) is frequently accompanied with cystic fibrosis mutations proven by genetic testing or a missing kidney.
  2. Infections such as chlamydia, gonorrhea, TB, or vasectomy can potentially cause ejaculatory duct blockage. In such circumstances, scrotal or transrectal ultrasonography reveals dilated seminal vesicles. In such circumstances, sperms exhibit low progressive motility.


  • Low sperm count and low LH in muscular man - inquire about androgen abuse. 
  • Low sperm motility by itself has little effect on the chances of natural conception unless a large fraction of sperms are immotile. Artificial reproductive treatments (ART) such as intracytoplasmic sperm injection (ICSI) can be used to assist treat male infertility in such circumstances.


Sperm Vitality

Epididymal disease may be the cause of a significant quantity of immotile and non-viable sperms. The presence of a large number of immotile and viable sperms may be related to structural flaws in the flagellum. 


Sperm Morphology

A spermatogenesis disorder is indicated by abnormal sperm morphology. Intracytoplasmic sperm injection, for example, is a type of assisted reproduction (ICSI). It is debatable if morphologic evaluation of sperm has a substantial influence on pregnancy rates following IVF. 


Cells in the Ejaculate

Peroxidase activity and leukocyte markers must be used to determine the presence of round cells in the ejaculate. Men with more than one million leukocytes per milliliter (pyospermia) should be investigated to rule out genital tract inflammation or infection.


How to improve semen analysis results?

Smoking cessation, alcohol reduction, proper diet, exercise, weight loss if obese, avoidance of toxic lubricants during intercourse, and prevention of scrotal temperature rise can all enhance male factor fertility. Low or missing sperm count in the sperm can be caused by hypothalamic-pituitary failure, primary testicular failure, or genital tract blockage. In people with hypogonadotropic hypogonadism, gonadotropins can increase fertility.

Genetic counseling should be offered to individuals with karyotypic abnormalities, including Klinefelter’s syndrome, Y chromosome deletion, CBAVD, and CFTR gene mutation. Surgical treatment of ejaculatory duct obstruction can help improve fertility. This is an alternative treatment to assisted reproductive procedures like intracytoplasmic sperm injection (ICSI) and in-vitro fertilization (IVF).

Semen analysis, when performed along with a detailed medical and sexual history together with a thorough physical examination, can help provide an accurate picture of a male's fertility potential and help guide the treatment of both the individual and the couple seeking relief from their infertility.


Sperm function testing

Sperm functional testing is best conducted in a specialist academic institution, as clinics typically lack the requisite equipment and competence. The sole possible exception is the hemizona test, in which the oocytes may be divided into two equal halves using a stereo microscope and a microsurgical blade.

Although significant improvements in this discipline have been made in the last 20 years, our understanding of the cellular and molecular foundation for this illness remains restricted. Indeed, we know very little about the physiology of a normal human spermatozoon, let alone a defective one.


The ideal sperm function test or battery of tests should permit the following clinical steps:

  1. Diagnosis of a specific sperm dysfunction;
  2. Prediction of fertilisation or pregnancy rates; and 
  3. Indication of an appropriate therapy to alleviate the identified sperm dysfunction.


Due to a lack of material, proper equipment, and experience in certain centers, the andrology inquiry should concentrate on a practical diagnostic approach.

A normal routine sperm analysis conducted by a competent individual should aid the consulting physician in the therapy of choice, but couples referred to tertiary care centers specializing in andrology testing can be further reviewed with sperm function tests. The European Society of Human Reproduction and Embryology and the World Health Organization have chosen induced acrosome response and sperm-binding assays as research tests for clinical use. A meta-analysis of sperm function tests found that the sperm–zona pellucida binding and triggered acrosome response assays had a strong predictive potential for fertilisation result.

A prior study revealed that sperm morphology is strongly connected to certain sperm functions. Furthermore, evaluating sperm morphology, particularly the structure of the acrosome, is an excellent way to evaluate sperm fertilizing capacity. This link is understandably strongest when the physiological inducer, zona pellucida, is utilized to generate acrosome response for diagnostic purposes.

Similarly, sperm morphology is strongly related to the capacity of the sperm cell to adhere to the zona pellucida. Small acrosomes in sperm make it more vulnerable to cell death and non-physiological acrosomal loss. The size of an acrosome represents the physiological capability of sperm activity and, as a result, male reproductive potential.


What do abnormal results mean for my ability to help a woman conceive?

Abnormal findings indicate that you have a lower-than-average likelihood of conceiving a woman. However, a sperm analysis is not the only aspect to consider when assessing male infertility. Extra testing is frequently required to learn more. These tests can either confirm or rule out:

  • Blockages that prevent the body from releasing sperm into semen.
  • Infection.
  • Low sperm count.
  • Low testosterone or hormone abnormalities.
  • Side effects of medications or other medical issues.



A thorough semen analysis determines the amount and quality of fluid discharged during ejaculation. It assesses both the liquid component, known as semen or seminal fluid and the tiny, moving cells known as sperm. It is frequently used to assess male infertility. A simplified variant of this test looks just for the presence of sperm in sperm a few months after a guy has undergone a vasectomy to see if the operation was effective.

The addition of sperm cryopreservation procedures, the extension of the sperm preparation section, and the inclusion of additional appendices have all contributed to the creation of a user-friendly laboratory handbook. The most significant modification in the handbook is the inclusion of evidence-based publications as references to derive normalcy threshold values.

Aside from these revisions, the first assessment and handling techniques, namely sample collection, initial macroscopic inspection, and early microscopic inquiry remain mostly unchanged from the fourth edition of the manual.