Semen Analysis

Specimen Requirements and Procedure

The semen sample is collected by the man through self-masturbation either at home or near the laboratory. The semen should be collected after a minimum of 3 days and a maximum of 7 days of abstinence.[6] Clear instructions for collecting the semen sample should be provided beforehand to ensure that all fractions of the ejaculate are collected completely. Any loss of the sample must be reported to the laboratory during submission. Human male semen can vary significantly in composition at various times; therefore, collecting 2 or 3 samples for more accurate data is advisable.[7]

The sample should be collected in a clean, wide-mouthed container that is nontoxic to spermatozoa. The sperm container should be kept at ambient temperature between 20 °C and 37 °C. If the patient is unable to produce a suitable sample through masturbation, semen can be collected in nontoxic condoms during sexual intercourse. Latex condoms should not be used in these situations as these condoms have chemical agents that can affect sperm viability and motility.[8]

The semen should be delivered to the laboratory within 1 hour of its collection so that dehydration and temperature changes do not affect the sperm quality. Safety guidelines must be strictly followed in the laboratory while handling semen samples. The sample should be analyzed using standardized methods described in the World Health Organization (WHO) laboratory manual to examine and process human semen.[9] The laboratory should implement internal and external quality control measures to ensure accurate and reliable analysis of the semen samples.[10]

Diagnostic Tests

According to the WHO laboratory manual for the examination and processing of human semen published in 2010, semen analysis involves the following steps:

Specimen Liquefaction

In the first 5 minutes, liquefaction of the specimen is performed by placing the sample on an incubator at 37 °C or on the bench. Allowing time for liquefaction helps semen to become more homogenous and watery, with only some areas of coagulation. Spermatozoa that are immobilized in the coagulum gain the ability to move. Liquefaction can take up to 60 minutes. If liquefaction does not occur naturally, reagents may be used to facilitate the process; however, this can also affect the composition of the seminal plasma and sperm motility and morphology, which needs to be noted.[9]

Volume Measurement

The semen volume is measured first, followed by semen pH. Wet preparations are made to assess the appearance, motility, and dilution required for optimal spermatozoa counting. Attention should be given to the agglutination of spermatozoa, where motile sperm are stuck to one another. Testing for anti-sperm antibodies must be considered in such situations. Agglutination can affect sperm motility and concentration. Sperm motility is best tested within an hour of collection.

Vitality Assessment

Sperm vitality is assessed, especially if the sperm number is low. The integrity of the cell membranes helps identify if the immotile sperms are dead or alive. The semen is diluted to assess the sperm number. Tests for sperm antibodies, such as the mixed antiglobulin reaction test, immunobead test, check for peroxidase-positive cells, and similar biochemical tests, are performed as needed. The semen needs to be sent to the microbiology laboratory within 3 hours of collection if required. Finally, smears are prepared to study sperm morphology after 4 hours. Properly conducted semen analyses are relatively labor-intensive and complex, so the best results are obtained in laboratories that perform these tests regularly.[9]

Results, Reporting, and Critical Findings

The WHO has provided normal reference limits for semen analysis. The following values represent the accepted 5th percentile for the measured parameters:

• Volume: >1.5 mL
• pH: >7.2
• Total sperm number: 39 million sperm per ejaculate or more
• Morphology: >4% normal forms using the Tygerberg method 
• Vitality: >58% live sperm
• Progressive motility: >32%
• Total (progressive motility and nonprogressive 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 per ejaculate
  • Seminal zinc: >2.4 mcmol per ejaculate
  • Seminal neutral glucosidase: <20 mU per ejaculate [11]

Clinical Significance

Semen analysis is performed as part of the assessment of male factor infertility. Obtaining a detailed medical and sexual history, performing a thorough physical examination, and conducting semen analysis are crucial to ensure a complete diagnostic assessment.[11][12] If the semen analysis is normal according to the WHO criteria, a single semen specimen may be sufficient, but some experts still recommend 2 separate specimens. If the semen analyses are abnormal, the analysis can be repeated 3 months after completing another complete spermatogenesis cycle. Analysis can be repeated earlier if the sperm count is low or absent. A more comprehensive and detailed review of male infertility diagnosis and treatment can be found in our review article on the subject.[5]

Low Semen Volume 

• When the semen volume is low (<1.5 mL), and especially when it is less than 1 mL, a urine specimen should be collected immediately after ejaculation. The urine specimen thus collected is centrifuged and microscopically examined for spermatozoa. The presence of substantial sperm in the post-ejaculatory urine specimen confirms retrograde ejaculation.[11] Retrograde ejaculation may result from spinal cord injury, transurethral prostatectomy, retroperitoneal lymph node dissection, diabetes mellitus, transverse myelitis, multiple sclerosis, or psychogenic causes.[13]

• Incomplete semen collection or loss of part of the sample during collection could lead to low semen volume. 

• Low semen volume and low sperm count can occur in men with testosterone deficiency. 

• Low semen volume with no sperm or severely low sperm count could be secondary to ejaculatory duct obstruction or congenital bilateral absence of the vas deferens where the seminal vesicles have not developed.[14]

Low Sperm Count

Men with a sperm count <10 million/mL with sexual dysfunction or with clinical findings suggestive of endocrine pathology should have their hormone levels checked. Initial labs should include a serum total testosterone (8 AM to 10 AM) and a serum follicle-stimulating hormone (FSH). If the total testosterone is low (<300 ng/mL), a second measurement of the total and free testosterone, serum luteinizing hormone (LH), and prolactin should be performed.[11] Patients with borderline test results should have their tests repeated. Initial hormonal screening is recommended for all male infertility patients.[5]

• Low sperm count, low testosterone, high FSH, and high LH: This pattern suggests primary hypogonadotropic hypogonadism, affecting both spermatogenesis and Leydig cell function. Chromosomal anomalies, such as Klinefelter’s syndrome and Y chromosome microdeletion (sperm count is <5 million/mL), should be ruled out through karyotyping.[15] Other causes include survivors of cancer treatments, such as radiation or chemotherapy; testicular trauma; torsion of the testes; or infections such as mumps.[16]

• Low sperm count, normal testosterone, normal LH, and high FSH: This pattern suspects seminiferous tubule damage without Leydig cell dysfunction and abnormal spermatogenesis.

• Low sperm count, low testosterone, normal or low FSH, and normal or low LH or hypogonadotropic hypogonadism: In such cases, it is crucial to check other pituitary hormone deficiencies, including thyroid function (free T4), cortisol at 8 AM, and prolactin levels. High prolactin levels suggest a prolactin-secreting tumor.

• Low sperm count, high testosterone, high LH, and normal FSH: This pattern could indicate partial androgen insensitivity syndrome in men. Men with this condition may have gynecomastia and display a range of genital presentations, from hypospadias to normal male genitalia, due to improper androgen receptor function.[17]

• Low sperm count, normal testosterone, normal LH, normal FSH, and normal testis size: Evaluate for genital tract obstruction:
• Congenital absence of the vas deferens, identified through a physical examination and low semen pH, and confirmed on scrotal or transrectal ultrasound, is often associated with cystic fibrosis mutations confirmed by genetic testing or an absent kidney.[18][19]
• Ejaculatory duct obstruction can also be secondary to infections such as chlamydia, gonorrhea, tuberculosis, or vasectomy. In such cases, scrotal or transrectal ultrasound reveals dilated seminal vesicles.[20] Sperm, in such cases, has poor progressive motility.[11]

• Low sperm count and low LH in muscular man: Inquire about androgen abuse.[21]

• Low sperm motility: Low motility does not significantly impact the likelihood of natural conception unless a very high proportion of sperm is immotile.[22]  In such cases, artificial reproductive techniques, such as intracytoplasmic sperm injection, can be used to help treat male infertility. 

Sperm Vitality

A high number of immotile and nonviable sperms may be due to epididymal pathology.[23] Many immotile and viable sperms may be due to structural defects in the flagellum.[24] 

Sperm Morphology

Abnormal sperm morphology is suggestive of a spermatogenesis problem. Assisted reproduction, such as intracytoplasmic sperm injection, may be considered. However, the impact of sperm morphology assessment on pregnancy rates after in vitro fertilization (IVF) remains controversial.[25] 

Cells in the Ejaculate

The presence of round cells in the ejaculate must be assessed using peroxidase activity and leukocyte markers. Men with >1 million leukocytes/mL (pyospermia) must be evaluated to rule out genital tract inflammation or infection.[26]

Enhancing Healthcare Team Outcomes

Lifestyle changes such as quitting smoking, reducing alcohol intake, maintaining a healthy diet, exercising regularly, losing weight if obese, avoiding toxic lubricants during intercourse, and maintaining a normal scrotal temperature can improve male factor fertility.[27][28] A low or absent sperm count in the semen may be due to hypothalamic-pituitary failure, primary testicular failure, or obstruction of the genital tract. Gonadotropin drugs can improve fertility in patients with hypogonadotropic hypogonadism.[28][29] 

Genetic counseling should be offered to individuals with karyotypic abnormalities, including Klinefelter's syndrome, Y chromosome deletions, congenital bilateral absence of the vas deferens, and CFTR gene mutation.[30] Surgical treatment of ejaculatory duct obstruction can help improve fertility and is an alternative treatment to assisted reproductive procedures such as intracytoplasmic sperm injection and IVF.[20]

Semen analysis, when combined with a detailed medical and sexual history and a thorough physical examination, provides a comprehensive evaluation of a male's fertility potential. This approach guides the treatment of both the individual and the couple seeking relief from their infertility.