Semen Analysis

Semen analysis is the cornerstone laboratory test of male fertility, assessing the volume and quality of ejaculate, sperm number, motility, morphology and vitality. It does not measure fertilising capacity directly and results overlap between fertile and infertile men, so interpretation requires clinical context.

Biology:

• Semen is made of sperm (about 5% of volume) from the testes and epididymis, and seminal plasma from the seminal vesicles (about 65 to 70%, fructose-rich, coagulating factors), prostate (about 20 to 30%, citric acid, zinc, PSA, acid phosphatase), bulbourethral glands and epididymis.
• Liquefaction occurs in 15 to 60 minutes through PSA-mediated proteolysis of semenogelin.
• Spermatogenesis takes about 74 days, and epididymal transit 10 to 14 days, so a change in the semen picture reflects events 2 to 3 months earlier.

WHO 6th edition (2021) lower reference limits (5th centile, with 95% confidence interval) for fertile men:

• Semen volume: 1.4 mL (1.3 to 1.5).
• Total sperm number: 39 million per ejaculate (35 to 40).
• Sperm concentration: 16 million/mL (15 to 18).
• Total motility (progressive plus non-progressive): 42% (40 to 43).
• Progressive motility: 30% (29 to 31).
• Vitality (live spermatozoa): 54% (50 to 56).
• Normal forms: 4% (3.9 to 4.0, strict criteria).
• Other: pH 7.2 or higher, round cells below 1 million/mL (leukocytes), anti-sperm antibody (MAR test) below 50% coated.
• The lower limits represent the 5th centile and are not thresholds of infertility, and about 5% of fertile men fall below them.

Terminology:

• Normozoospermia: parameters above limits. Oligozoospermia: low concentration. Asthenozoospermia: low motility. Teratozoospermia: abnormal morphology. Oligoasthenoteratozoospermia (OAT): all three. Azoospermia: no sperm in the pellet. Cryptozoospermia: sperm only in centrifuged pellet. Aspermia: no ejaculate. Leukocytospermia: leukocytes above 1 million/mL. Necrozoospermia: all sperm dead.

Indications: infertility evaluation (first-line male test), vasectomy follow-up, fertility preservation, before assisted reproduction, evaluation after chemotherapy or testosterone use, and medicolegal cases.

Pre-analytic factors that determine the validity of the sample (and therefore the clinical interpretation):

• Abstinence: 2 to 7 days (ideally 2 to 3 days). Longer abstinence raises volume and count but lowers motility and increases DNA damage.
• Collection: masturbation into a sterile, non-toxic container, in a private room at the laboratory or at home if delivered within 30 to 60 minutes, kept at body temperature (20 to 37 degrees Celsius). No lubricants, saliva, or standard condoms (spermicidal). Special non-toxic condoms for religious or psychological reasons.
• Report any loss of ejaculate, fever or illness within the last 3 months, drugs and supplements, and time of collection.
• Because of the high biological variation (up to 50% for counts), two or more samples 2 to 4 weeks apart (7 days to 3 months) are needed to establish a diagnosis, and a third sample if the first two differ markedly.

Clinical significance of abnormal results:

• Low volume (hypospermia, below 1.4 mL): incomplete collection, retrograde ejaculation, ejaculatory duct obstruction, congenital bilateral absence of vas deferens (acidic pH below 7.2, absent fructose), hypogonadism, or partial seminal vesicle disease.
• Azoospermia: obstructive (normal FSH, normal testis volume, absent vasa, low volume) versus non-obstructive (high FSH, small testes).
• Oligozoospermia: varicocele, hormonal disturbance, genetic causes, infection and toxins.
• Asthenozoospermia: varicocele, infection, antisperm antibodies, genital tract inflammation, epididymal dysfunction, ciliary disorders (primary ciliary dyskinesia, Kartagener syndrome, immotile sperm with normal vitality).
• Teratozoospermia: globozoospermia (round-headed, absent acrosome), macrocephalic head defects (AURKC), and pinhead sperm. Morphology alone is a weak predictor of natural fertility.
• Agglutination or antibodies: immunological infertility after vasectomy, trauma or infection.

Procedure and interpretation:

• Macroscopic: liquefaction, viscosity, appearance, volume, pH.
• Microscopy: sperm concentration by haemocytometer (improved Neubauer chamber), motility classified as progressive, non-progressive and immotile (within 60 minutes), vitality (eosin-nigrosin, hypo-osmotic swelling) when motility is under 40%, and morphology (Papanicolaou stain, strict Tygerberg criteria). Assess round cells and peroxidase-positive leukocytes (leukocytospermia above 1 million/mL), agglutination and aggregation.
• Automated systems (CASA) provide kinematics but need quality control. Quality assurance with internal and external controls is essential.

Interpretation steps:

• Compare with reference limits. Remember that results should be integrated with history, examination, hormones and partner factors.
• Azoospermia confirmation: examine the centrifuged pellet (3000 g for 15 minutes) on at least two occasions. Perform fructose and pH tests, post-ejaculatory urine for retrograde ejaculation (sperm in urine), FSH, LH, testosterone, karyotype, Y chromosome microdeletions, CFTR testing, and testicular or transrectal ultrasound.
• Post-vasectomy: examine at 12 weeks (or after 20 ejaculations). Clearance is azoospermia (or at most rare non-motile sperm, under 100,000/mL).
• Additional tests when indicated:
• Anti-sperm antibodies (mixed antiglobulin reaction or immunobead test).
• Sperm DNA fragmentation (TUNEL, SCSA, SCD, comet) in recurrent pregnancy loss, unexplained infertility, or failed assisted reproduction (fragmentation index above 25 to 30% is abnormal).
• Leukocyte detection and semen culture or PCR for chlamydia and other pathogens.
• Reactive oxygen species testing (specialist).
• Genetic and hormonal evaluation.
• Bacteriological tests (urethral swab, midstream urine) when leukocytospermia, pyospermia or symptoms are present.

Report: include abstinence time, collection location, time of production and analysis, and any problems with collection.

Management follows the cause (see infertility article). Specific actions related to semen analysis:

Counselling and lifestyle

• Explain that an abnormal result needs confirmation, that values fluctuate, and that spermatogenesis cycles take 3 months.
• Stop smoking, reduce alcohol, avoid anabolic steroids and exogenous testosterone (stop for at least 3 to 6 months, with hCG and FSH to restore), lose weight, avoid prolonged heat exposure (hot tubs, laptops on the lap, tight underwear), and review medications and occupational exposures.
• Antioxidants (vitamin C, E, coenzyme Q10 200 mg daily, L-carnitine 2 g daily, zinc 25 to 50 mg, selenium 100 micrograms) may modestly improve parameters, with weak evidence of live birth benefit. Repeat the analysis after 3 months.

By finding

• Hypogonadotropic hypogonadism: hCG with recombinant FSH. Clomiphene citrate 25 to 50 mg daily or enclomiphene, or anastrozole 1 mg daily, in selected men with low testosterone and normal or low-normal gonadotropins.
• Varicocele: microsurgical varicocelectomy or percutaneous embolisation for clinical varicocele with abnormal semen and infertility. Benefit in azoospermia may allow sperm to appear in some.
• Infection or leukocytospermia: culture-directed antibiotics (doxycycline 100 mg twice daily for 14 days, levofloxacin 500 mg daily for 14 days, or azithromycin 1 g single dose for chlamydia), anti-inflammatory therapy, treatment of partner.
• Retrograde ejaculation: pseudoephedrine 60 to 120 mg or imipramine 25 mg twice daily, alkalinised urine (sodium bicarbonate) for sperm retrieval.
• Ejaculatory duct obstruction: transurethral resection of the ejaculatory ducts.
• Obstructive azoospermia: reconstructive surgery (vasovasostomy, vasoepididymostomy, transurethral resection) or sperm retrieval (PESA, MESA, TESE) with ICSI.
• Non-obstructive azoospermia: hormone optimisation (clomiphene, hCG, FSH) and micro-TESE, with genetic counselling for karyotype and AZF deletions (AZFa and b complete deletions predict failure of sperm retrieval, AZFc allows retrieval in about 50%).
• Severe oligo-, astheno- and teratozoospermia: intrauterine insemination for mild male factor (total motile count after wash above 5 million), IVF with ICSI for severe disease (total motile count under 5 million).
• Antisperm antibodies: ICSI preferred, corticosteroids have been used with limited evidence.
• Globozoospermia and ciliary defects: ICSI with assisted oocyte activation.
• High DNA fragmentation: lifestyle, antioxidants, varicocele repair, shorter abstinence, testicular sperm for ICSI.

Other uses

• Post-vasectomy: clear at 12 weeks with azoospermia or non-motile rare sperm before stopping contraception.
• Fertility preservation: cryopreserve at least three samples before gonadotoxic therapy or testosterone replacement, preferably before treatment starts. Sperm banking in adolescents (from Tanner 3 or when spermarche occurs).

Follow-up

• Repeat analysis 3 months after intervention, with ongoing couple-based counselling. Use of genetic counselling for inherited disorders.