Introduction

Semen analysis is a laboratory test that is performed to assess male fertility. Infertility is defined as the inability to conceive after 1 year of unprotected sexual intercourse. About 15% of all couples in reproductive-aged couples experience infertility. Infertility in a male is assessed by taking a detailed medical and sexual history, a complete physical examination, and semen analyses. The male factor significantly contributes to 30% of the infertility cases and is a contributing factor in about half.[1][2][3]

Semen is released in boluses during ejaculation. It contains a mixture of spermatozoa that are produced in the testes, processed in the epididymides, and mixed with secretions from the male accessory sex organs, including the prostate, seminal vesicles, bulbourethral glands, and the epididymides.[4] Semen can be assessed by various attributes: the total number of spermatozoa, the fluid volume, sperm concentration, and the nature of the spermatozoa; their viability, motility, and shape as well as the composition of the secretions. Detailed analysis of these factors can help identify the reason for the male factor infertility.[5]

Specimen Requirements and Procedure

The semen sample is collected by the man by self-masturbation near the laboratory or at home. It is recommended that semen is collected after a minimum of 3 days and a maximum of 7 days of abstinence.[6] Clear instructions regarding the collection of the semen sample must be provided before collection to ensure all fractions of the ejaculate are collected and complete. Any loss of the sample must be reported to the laboratory during submission. Human male semen can vary significantly in its composition at various times, and hence it is best to collect 2 or 3 samples of semen for more accurate data.[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 deg C and 37 deg C. If the patient is unable to produce a suitable sample by 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 adhered to at the laboratory while handling semen samples. The sample should be analyzed using standardized methods described in the WHO laboratory manual for the examination and processing of human semen.[9] The laboratory should employ both internal and external quality control measures to ensure the semen samples are analyzed accurately and reliably.[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:

In the first 5 minutes, liquefaction of the specimen is performed by placing the sample on an incubator at 37 degrees Celsius 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 happen on its own, reagents can be used to facilitate the process, but this can also affect the composition of the seminal plasma, sperm motility, and morphology and needs to be noted.[9]

The semen volume needs to be measured. Following this, semen pH is measured, and wet preparations are made. Wet preparations help assess the appearance, motility, and dilution required to optimally assess the number of spermatozoa. Attention should be paid to the presence of agglutination of spermatozoa, where motile sperms 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.

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, checking 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 needed. Finally, smears are prepared to study sperm morphology after 4 hours. Properly done semen analyses are relatively labor-intensive and can be 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 limits of reference for semen analysis. The values mentioned in the chart below represent the accepted 5th percentile for the parameters measured.[11]

• 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

Semen analyses are performed as part of the assessment of male factor infertility. It is imperative to take a complete medical and sexual history as well as perform a thorough physical examination in addition to the semen analysis to provide a complete diagnostic assessment.[11][12] If the semen analysis is normal per the WHO criteria, a single semen specimen may be sufficient, but two separate specimens are still recommended by some experts. If the semen analyses are abnormal, then the analysis can be repeated 3 months after completion of another complete spermatogenesis cycle. It 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 (<1.5 ml)

• When the semen volume is low, especially 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), then a second measurement of the total and free testosterone, serum luteinizing hormone (LH), and prolactin (PRL) should be performed.[11] Those patients who have borderline values will need to have their tests repeated. Some experts recommend initial hormonal screening in all-male infertility patients.[5]

• Low sperm count, low testosterone, high FSH, and high LH: suspect primary hypergonadotropic hypogonadism (that affects both the spermatogenesis and Leydig cell function). Rule out chromosomal anomalies - such as Klinefelter’s syndrome, Y chromosome microdeletion (sperm count is <5 million/ml) by karyotyping these individuals.[15] Other causes include survivors of cancer treatment, including radiation, chemotherapy, testicular trauma, torsion of the testes, or an infection like mumps.[16]
• Low sperm count, normal testosterone and normal LH, high FSH: suspect 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: check for other pituitary hormone deficiencies including thyroid function (free T4), cortisol at 8 am, check prolactin levels.  High prolactin levels are suggestive of a prolactin-secreting tumor.
• Low sperm count, high testosterone and high LH, normal FSH: suspect partial androgen resistance syndrome in men. The androgen receptor is not functioning optimally in such men. They are phenotypically male with gynecomastia and with a variable presentation from hypospadias to normal male genitalia.[17]
• Low sperm count, normal testosterone, normal LH and normal FSH, normal testis size: evaluate for genital tract obstruction:
• congenital absence of the vas deferens (physical examination, low semen pH, confirmed on scrotal /transrectal ultrasound) 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 like chlamydia, gonorrhea, tuberculosis, or vasectomy. Scrotal or transrectal ultrasound in such cases shows dilated seminal vesicles.[20] Sperms have poor progressive motility in such cases.[11]
• Low sperm count and low LH in muscular man - inquire about androgen abuse.[21]
• Low sperm motility by itself does not have a significant impact on the likelihood of natural conception unless a very high proportion of sperms are immotile.[22]  In such cases, artificial reproductive techniques (ART) like intracytoplasmic sperm injection (ICSI) can be used to help treat male infertility. 

Sperm Vitality

A high number of immotile and non-viable sperms may be due to epididymal pathology.[23] A high number of 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 (ICSI). Whether the morphologic assessment of sperm has a significant impact on pregnancy rates after IVF or not is controversial.[25] 

Cells in the Ejaculate

The presence of round cells in the ejaculate must be assessed with 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 measures including smoking cessation, reducing alcohol intake, healthy eating, exercise, weight loss if obese, avoiding toxic lubricants during intercourse, and ensuring the scrotal temperature is not increased can improve male factor fertility.[27][28] 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 deletion, CBAVD, and CFTR gene mutation.[30] Surgical treatment of ejaculatory duct obstruction can help improve fertility.[20] 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.