ABB Andrology cheat sheet

WHO 5th-edition lower reference limits (5th centile): volume 1.4 mL, total sperm number 39 million, concentration 15 million/mL, total motility 40%, progressive motility 32%, normal morphology 4%, vitality 58%, leukocytes <1 million/mL.

Volume is measured by weight (1 mg = 1 µL) or in a graduated cylinder; low volume with absent sperm suggests retrograde ejaculation or incomplete collection, not necessarily low production.

Concentration is assessed in a Neubauer hemocytometer; count at least 200 sperm in two dilutions, and report the mean. Duplicate counts should agree within ~95% Poisson limits.

Motility is graded a (rapid progressive), b (slow progressive), c (non-progressive), d (immotile); total motility = a+b+c, progressive = a+b.

Vitality uses eosin-nigrosin or eosin Y: dead sperm take up the dye (membrane damaged); a live-but-immotile fraction >5% suggests flagellar or metabolic defect rather than death.

Morphology by strict (Tygerberg) criteria: normal sperm has smooth oval head 4-5 µm long, 2.5-3.5 µm wide, acrosome 40-70% of head, midpiece 1.5x head length, single straight tail.

Abstinence interval of 2-7 days is required before collection; longer abstinence increases volume and may reduce motility and morphology.

Semen liquefies within 60 min at room temperature; failure to liquefy suggests prostatic enzyme deficiency and complicates analysis.

Density-gradient centrifugation (e.g., 40%/80% isotonic colloid) selects motile, morphologically normal sperm and removes debris, leukocytes, and seminal plasma; superior recovery of functional sperm vs. simple wash.

Swim-up relies on active migration into overlaid medium; yields high-motility sperm but lower total recovery than gradient, so it is preferred when concentration is adequate.

Sperm washing removes seminal plasma, which contains prostaglandins and decapacitation factors; needed before intrauterine insemination and IVF.

Capacitation occurs in the female tract or in vitro with bicarbonate, calcium, and cholesterol acceptors (albumin); required before acrosome reaction and fertilization.

Acrosome reaction can be assessed with PSA-FITC or calcium ionophore challenge; the acrosome must be intact before capacitation and reacted afterward.

Sperm DNA fragmentation (SCSA, TUNEL, Comet) measures chromatin damage; elevated fragmentation is associated with poor ART outcomes and may be reduced by density gradient or testicular sperm.

Hyaluronan binding assay (HBA) estimates sperm maturity and zona-binding competence; low binding correlates with abnormal morphology.

Cryopreservation uses glycerol as cryoprotectant with controlled slow cooling; post-thaw motility typically drops 30-50% from pre-freeze values.

GnRH from the hypothalamus stimulates pituitary gonadotrophs to release LH and FSH in pulses; LH drives Leydig-cell testosterone production, FSH supports Sertoli-cell function and spermatogenesis.

Testosterone exerts negative feedback on the hypothalamus and pituitary; estradiol (from aromatization) also suppresses GnRH and LH.

Primary hypogonadism: low testosterone with elevated LH/FSH (testicular failure). Secondary: low testosterone with low or inappropriately normal LH/FSH (central defect).

Total testosterone should be drawn in the morning (07:00-10:00) because concentrations peak then; repeat confirmatory testing before diagnosing hypogonadism.

Free testosterone is calculated from total testosterone, SHBG, and albumin (e.g., Vermeulen equation); low SHBG states (obesity, insulin resistance) can mask low free testosterone.

Hyperprolactinemia suppresses GnRH and causes hypogonadotropic hypogonadism; prolactin should be measured in men with low testosterone and low/normal gonadotropins.

Azoospermia with normal FSH and normal testicular size suggests obstruction; very high FSH (>2x upper limit) with small testes suggests non-obstructive (primary) failure.

hCG stimulates Leydig cells (used to treat hypogonadotropic hypogonadism and to test Leydig-cell reserve); FSH is added when spermatogenesis does not recover.

Internal QC: run control materials (fixed sperm suspensions or video recordings) with each batch; monitor concentration, motility, and morphology against established means and Levey-Jennings charts.

External quality assessment (EQA/proficiency testing) compares the laboratory's results with peer laboratories; participation is expected for accreditation (CAP, CLIA, ISO 15189).

Duplicate hemocytometer counts should differ by no more than the 95% Poisson sampling error for the number of cells counted; larger discrepancies trigger a recount.

Motility QC uses recorded video or standardized slides; inter-technician variability is assessed by replicate reads and by kappa statistics for grading agreement.

Morphology QC requires periodic re-standardization against reference slides; inter-observer agreement is monitored and technicians re-trained when drift is detected.

Equipment: hemocytometers and pipettes are calibrated and verified; incubators, water baths, and refrigerators are temperature-monitored with logs.

Documentation: SOPs, reagent lot numbers, expiration dates, and corrective actions are recorded; records are retained per regulatory requirements (often ≥2 years, longer for some states).

Proficiency-testing failures require root-cause analysis and documented corrective action before results are reported again.

IVF: oocytes are inseminated with washed motile sperm (typically 50,000-100,000 motile sperm per oocyte) or by ICSI when severe male factor is present.

ICSI injects a single sperm into the oocyte cytoplasm; indications include severe oligozoospermia, prior fertilization failure, and surgically retrieved sperm.

Surgical sperm retrieval: TESA/PESA for obstructive azoospermia; micro-TESE for non-obstructive azoospermia, where focal spermatogenesis may be found.

Sperm selection for ICSI may use hyaluronan binding, MSOME (high-magnification morphology), or PICSI; these aim to reduce DNA-damaged sperm.

Embryo culture uses sequential or single-step media with controlled pH (bicarbonate/CO2) and osmolality; temperature and gas (5-6% CO2, low O2) are tightly controlled.

Vitrification (ultra-rapid cooling with cryoprotectants) is the dominant method for embryo and oocyte cryopreservation; survival rates exceed those of slow freezing for oocytes.

Fertilization check occurs 16-18 h post-insemination; presence of two pronuclei confirms normal fertilization, while three pronuclei indicate polyspermy.

Time-lapse incubation allows continuous embryo monitoring without disturbing culture conditions; selection algorithms remain adjunctive to standard morphological grading.

CLIA regulates clinical laboratory testing in the U.S.; andrology/semen analysis is generally classified as moderate complexity, requiring appropriate personnel qualifications and proficiency testing.

CAP accreditation requires documented SOPs, QC, competency assessment of personnel, and participation in proficiency testing for regulated analytes.

OSHA Bloodborne Pathogens Standard (29 CFR 1910.1030) requires exposure control plans, PPE, hepatitis B vaccination offers, and safe handling of semen and blood specimens.

Semen and reproductive tissues are handled under universal precautions; all specimens are treated as potentially infectious regardless of patient history.

HIPAA governs privacy of patient health information; results, donor records, and identifying data must be protected and disclosed only as permitted.

Informed consent is required for semen donation, cryopreservation, and use of sperm in ART; donors are screened for genetic and infectious diseases per FDA tissue regulations (21 CFR 1271).

FDA donor eligibility rules require testing for HIV, HBV, HCV, syphilis, gonorrhea, chlamydia, and HTLV, plus genetic and family history screening.

Ethical standards prohibit reproductive cloning and heritable genome editing; gamete and embryo disposition must follow documented patient directives.