Sperm Analysis & Sperm Function Test: Lecture Notes

Historical Background

• Anton van Leeuwenhoek (1677) – first visualized sperm with his handmade single–lens microscope; mistook the sperm head for a miniature baby.

• John Hunter (18ᵗʰ century) – performed the first recorded insemination with donor semen (AID), proving pregnancy is possible without coitus.

• Evolution from primitive lenses to today’s high-resolution inverted microscopes (e.g. for ICSI & micro-TESE) illustrates the parallel growth of our knowledge of male infertility.

Core Definitions & Semen Parameters

• Male infertility = any condition reducing a man’s probability of achieving pregnancy.

• Semen report focuses on three primary domains:

  • Concentration

  • Motility

  • Morphology

• Classical terminology (now being phased out):

  • Oligozoospermia – low concentration

  • Asthenozoospermia – poor motility

  • Teratozoospermia – abnormal morphology

• Current WHO 6ᵗʰ Edition discourages these labels in favour of decision limits.

Etiological Framework

1. Defects in Sperm Production

• Hypothalamo–pituitary failure (↓ FSH/LH)

• Hyper-stimulation with testicular non-response (testicular failure)

• Normal stimulation but intrinsic tubular failure

2. Defects in Sperm Delivery

• Congenital absence of vas/epididymis (e.g. CFTR mutations)

• Acquired obstruction (infections: TB, gonorrhoea; post-surgery)

3. Functional/Structural Sperm Defects

• Immotile-cilia syndromes (Kartagener/PCD)

• Acrosome, zona-binding, oolemma-fusion or pronuclear formation defects – increasingly diagnosed via ART failures (e.g. ICSI fertilisation failure).

4. Accessory-Gland Pathology (35 % of cases)

• Prostatitis, seminal-vesicle or Cowper’s infection can alter volume, pH, ROS load, liquefaction.

5. Varicocele (≈ 35 %)

• Grades II–III most likely to impair semen; mechanism via scrotal hyper-thermia & oxidative stress.

Treatability Spectrum (approximate distribution)

• Untreatable sub-fertility (oligo/astheno/terato) – 75 %

• Treatable infertility (hCG deficiency, obstructive azoospermia, antisperm antibodies, sexual-dysfunction, drug toxicity) – 12 %

• Irreversible sterility (Sertoli-cell-only, XY azoospermia factor deletion) – 13 %

WHO Reference Values – Evolution

• 2010 dataset criticised for selection bias (1 953 recent fathers only from Nordic & Western nations). 2020 expands to 3 733 men, adding Egypt, Iran, China, Italy, yet Asian Indian data still lacking.

WHO 6ᵗʰ Edition – New Structure

A. Test Categories

1. Basic (routine)

   • Volume, pH, appearance, viscosity, odour

   • Rapid vs slow progressive motility (A vs B)

   • Systematic strict morphology

   • Vitality screening if total motility < $40\%$

2. Extended

   • Peroxidase leukocyte count, antisperm antibodies (MAR/IBT)

   • Sperm DNA fragmentation (TUNEL, SCSA, SCD/Halo, Comet)

3. Research/Advanced

   • Oxidative stress assays (MiOXSYS, ORP)

   • Sperm–oocyte binding/penetration tests, CASA, reactive oxygen species quantification

B. Decision Limits (replace “normal/abnormal”)

➡ Counselling: “Normal” – expectant management; “Borderline” – optimise lifestyle, short medical therapy, timed‐coitus; “Pathological” – prompt advanced evaluation ± ART.

Clinical Workflow Using Decision Limits

1. Detailed history: duration of subfertility, coital frequency, occupational heat, anabolic steroids, drugs, co-morbidities.

2. Physical exam: body habitus (metabolic syndrome), testis volume, varicocele, secondary sexual characters.

3. Baseline semen ×2 (2–7 days abstinence)

4. Categorise via decision limits → choose next step:

   • Borderline only morphology ⇒ DNA fragmentation, antioxidant/lifestyle, re-test

   • Severe motility deficit ⇒ infection work-up, ROS, move toward ICSI

   • Very low count (<$5\times10^{6}$) ⇒ genetics (karyotype, Y micro-deletion), endocrine panel (FSH/LH/T/E2/prolactin).

Sperm DNA Fragmentation (SDF)

• Intact chromatin vital for fertilisation, embryogenesis & miscarriage avoidance.

• Lesion types

  • Single-strand breaks (SSB) – potentially repairable by oocyte.

  • Double-strand breaks (DSB) – mostly irreparable, high risk of failed implantation & miscarriage.

Causes

Pre-testicular

• Age > 40, smoking, obesity, diabetes, chemotherapy, radiotherapy.

Testicular

• Varicocele, high scrotal temperature, orchitis, ROS from immature germ cells, leucocytospermia.

Post-testicular

• Epididymal/vas obstruction, prolonged abstinence, cryo-damage.

➡ Leads to the entity MOSI – Male Oxidative Stress Infertility (>50 % of “unexplained” cases).

Testing Modalities

• TUNEL (fluorescent break labelling)

• SCSA (flow cytometry – gold standard)

• SCD/Halo (chromatin dispersion) – clinic-friendly

• Comet assay (single-cell electrophoresis)

Interventions & Typical Reduction in SDF

Endocrine Phenotypes & Medical Therapy

Hypogonadotropic Hypogonadism (HH)

• Small testes, low T & gonadotropins.

• Treatment: $hCG$ 1 500–2 000 IU 2×/week ± recombinant FSH after 3–6 months.

Obesity / “PCO-Male” (Metabolic Syndrome)

• ↑ aromatisation → high E₂, low T.

• Aromatase inhibitors (Anastrozole 1 mg/day; Letrozole 2.5 mg alt-day) for ≥ 8 weeks.

• Monitor $T/E_2$ ratio (goal >10).

Empirical Agents (use selectively)

• SERMs (Clomiphene 25–50 mg/d) – boosts endogenous Gn release.

• Antioxidant cocktails (CoQ10 200–400 mg, carnitine 2 g, NAC 600 mg).

• Avoid indiscriminate poly-pharmacy; always have an objective endpoint (repeat semen after one spermatogenic cycle ≈ 70 days).

Genetics in Male Infertility (Indications & Tests)

Surgical Sperm Retrieval

• PESA/TESA – percutaneous epididymal / testicular aspiration

• TESE – open biopsy extraction

• Micro-TESE – operating microscope (×25–40) to target dilated tubules; success in non-obstructive azoospermia (45 → 60 %).

  • Pros: highest retrieval, minimal tissue loss

  • Cons: cost, operative time, learning curve

ART Utilisation & Outcomes

• European registry 2009: IVF 28.9 %, ICSI 28.7 % cycles.

• US data (JAMA 2015): ICSI use without male factor rose from 36 % (1996) to 76 % (2012).

• Yet pregnancy per cycle in ESHRE 2016: IVF 27.1 %, ICSI 24 %. ⇒ Over-reliance on ICSI without addressing underlying pathology yields limited gains.

Practical Clinic Algorithm

1. Two semen analyses → categorize via decision limits.

2. If pathological:

   • Start parallel female evaluation.

   • Varicocele Doppler; scrotal temperature management.

   • Endocrine + genetics ± scrotal US.

3. Correctable lesion ⇒ surgery/medical first (varicocelectomy, hCG, AI etc.)

4. Persisting severe factor ⇒ choose retrieval + ICSI; consider sperm selection (MACS/PICSI) if ↑ SDF.

5. Prior counselling on realistic success, miscarriage risk, costs, donor options.

Ethical, Philosophical & Public-Health Points

• Label “infertile” can be stigmatising; decision‐limit terminology shifts focus to actionable next steps.

• Need for region-specific reference ranges (e.g. large Indian multi-centre project still lacking).

• Overuse of ICSI inflates cost & medicalises reproduction; comprehensive male work-up is ethical and cost–efficient.

• Lifestyle change (weight loss, smoking cessation) remains cheapest, safest “treatment”.

Key Numerical & Formulae Recap

• Normal semen volume ≥ $1.4\,\text{mL}$

• Normal concentration ≥ $20\times10^{6}/\text{mL}$

• Borderline motility $35!–!49\%$; Pathological < $35\%$

• Strict morphology pathological when < $4\%$

• Spermatogenesis cycle ≈ $64\text{ days}$ → repeat semen study after ≥ 70 days of any intervention.

High-Yield Take-Home Messages

• Always examine & investigate the male; semen analysis alone is insufficient.

• Use WHO 6ᵗʰ decision limits for clearer counselling.

• DNA fragmentation is common even with "normal" semen; test when history suggests ROS or ART failure.

• Varicocele and accessory-gland infection together account for ~70 % of identifiable causes – both are treatable.

• Medical therapy is effective in specific endocrine phenotypes; avoid blanket antioxidant or hormonal cocktails.

• ICSI is powerful but NOT a substitute for proper diagnosis; treat what is treatable first.