Class 12 Lab Tech ART

Semen Analysis and Andrology Standards (WHO-2021)

  • Sample Collection Protocol:

    • Requires 2−72-7 days of abstinence prior to collection.

    • Must be performed twice for accuracy.

    • Complete liquefaction should occur within 60 min60\,\text{min}.

  • WHO-2021 Normal Criteria (Lower Reference Limits - LRL):

    • pH: ≥7.2\ge 7.2

    • Volume: ≥1.4 mL\ge 1.4\,mL

    • Concentration: ≥16×106/mL\ge 16 \times 10^6/mL

    • Total Sperm Count: ≥39×106\ge 39 \times 10^6

    • Total Motility: ≥42%\ge 42\%

    • Progressive Motility: ≥30%\ge 30\%

    • Vitality (Live Sperm): ≥54%\ge 54\%

    • Normal Morphology: ≥4%\ge 4\%

  • Velocity Categories (WHO-2021):

    • Rapid: ≥25 μm/sec\ge 25\,\mu m/sec

    • Slow: 5−25 μm/sec5-25\,\mu m/sec

    • Non-progressive: ≤5 μm/sec\le 5\,\mu m/sec

    • Immotile: No active movement detected.

  • Diagnostic Classifications:

    • Normospermia: All parameters meet or exceed the LRL. Note that volume, concentration, and morphology are independent in normospermia but correlate in pathologies (e.g., testicular atrophy or androgen deficiency).

    • Aspermia: Complete lack of semen (often due to retrograde ejaculation).

    • Azoospermia: Absence of sperm in the ejaculate.

    • Cryptozoospermia: Sperm absent from fresh preparations but found in a centrifuged pellet.

    • Necrozoospermia: Absence of living sperm in the ejaculate.

    • Leukospermia: Presence of leukocytes above threshold (typically <1×106/mL< 1 \times 10^6/mL in health; higher indicates inflammation).

    • Haemospermia: Presence of erythrocytes (can be a marker for prostate cancer).

    • Hypospermia: Semen volume below the LRL.

    • Oligozoospermia: Total sperm count below the LRL.

    • Teratozoospermia: Percentage of normal morphology forms below the LRL.

    • Asthenozoospermia: Sperm motility below the LRL.

    • OTA (Oligo-Terato-Asthenozoospermia): Approximately 40%40\% of cases are idiopathic.

Advanced Andrology Testing and Morphology

  • Vitality Testing for Immotile Sperm: Used to distinguish live immotile sperm from dead sperm.

    • Hypo-osmotic Swelling (HOS) Test: Uses the membrane capability of live cells to swell in hypo-osmotic solutions.

    • Eosin Test: A dye exclusion test where dead cells take up the stain.

    • Sperm Tail Flexibility Test (STFT).

    • Laser Assisted Immotile Sperm Selection (LSISS).

  • Non-Sperm Constituents in Semen:

    • Squamous epithelial cells.

    • Bacilli (bacteria).

    • Monocytes and Polymorphonuclear leukocytes.

    • Sperm precursors: Spermatids.

  • Sperm DNA Fragmentation (SDF): Assesses the integrity of the paternal genetic contribution.

  • Sperm Preparation for ART (Assisted Reproductive Technology):

    • Swim Up Technique: Relies on motile sperm swimming out of seminal plasma into an over-layered medium.

    • Density Gradient Centrifugation (DGC): Uses layers of silica particles (typically 40%40\% and 80%80\%) to separate mature sperm from debris, epithelial cells, immature sperm, and white blood cells (WBC). Typical protocol: 20 min20\,\text{min} centrifugal force at 700 RCF700\,RCF.

    • Surgical Sperm Extraction (for Azoospermic Patients):

      • PESA: Percutaneous epididymal sperm aspiration.

      • TESE / Micro-TESE: Testicular sperm extraction.

Cryopreservation of Gametes and Embryos

  • Physics and Biology of Freezing:

    • Water makes up 50%50\% of cell volume and can form lethal crystalline ice (a volume increase of approximately 9%9\%).

    • Prevention Strategies:

      1. Dehydration: Removing water using solutes like Sucrose or PVP.

      2. Cryoprotection: Replacing water with Cryoprotective Agents (CPAs) such as PROH, Ethylene Glycol (EG), DMSO, or Glycerol (first described by Polge, Smith, and Parkes in Nature, 1949).

  • Freezing Methods:

    • Slow Freezing: Cooling from 37∘C37^{\circ}C to −196∘C-196^{\circ}C at a rate of −1∘C/min-1^{\circ}C/min. This promotes controlled crystallization.

    • Vitrification: Ultra-rapid cooling (0.2 sec0.2\,\text{sec}) at speeds up to 60,000∘C/min60,000^{\circ}C/min to reach a glass-transition status (∼−130∘C\sim -130^{\circ}C) without ice formation. Requires high concentrations of viscous CPAs (e.g., 15% v/v EG+15% v/v DMSO+0.5M Sucrose15\%\,v/v\,EG + 15\%\,v/v\,DMSO + 0.5M\,Sucrose).

    • Warming Protocol: Extremely fast (96,000∘C/min96,000^{\circ}C/min) to avoid the "danger zone" of devitrification/ice formation (0∘C0^{\circ}C to −146∘C-146^{\circ}C).

IVF Laboratory: Oocyte and Embryo Management

  • Environment: Controlled using an ISO Class 7 air system.

  • Ovum Pick-up (OPU): The surgical retrieval of oocytes.

  • Oocyte Maturation Stages:

    • GV (Germinal Vesicle) Stage: Prophase I.

    • MI Stage: Metaphase I.

    • MII Stage: Metaphase II (Mature oocyte ready for fertilization).

  • AI-Based Oocyte Evaluation (AERIS - Automatic Egg Ranking Intelligent System):

    • Uses Bayesian neural networks to predict blastocyst formation probability.

    • Parameters Assessed:

      • Zona Pellucida (ZP): Thickness, birefringence, shape.

      • Perivitelline Space (PVS): Size (large/fragmented is sub-optimal), granulation.

      • Polar Body (PB): Shape, fragmentation (fragmented = low quality), size.

      • Meiotic Spindle (MS): Visualization, length, birefringence, retardance.

      • Cytoplasm: Presence of vacuoles, refractile bodies, CLCG, or SER clusters.

      • Whole Oocyte Size: Giant oocytes are typically excluded.

  • In Vitro Maturation (IVM):

    • Used for anovulatory patients (high risk of OHSS), patients with thrombophilia/estrogen-sensitive tumors, or previous non-responders to FSH/LH.

    • Protocol involves OPU of immature follicles followed by 24−48 h24-48\,\text{h} incubation in media enriched with FSH+LH before denudation and ICSI.

Fertilization and Embryo Transfer (ET)

  • Selection Techniques:

    • Conventional IVF: Oocytes and sperm are co-incubated.

    • ICSI (Intracytoplasmic Sperm Injection): Direct injection for poor sperm quality/quantity, IVF failures, or IVM/PGT cases.

    • IMSI (Intracytoplasmic Morphologically Selected Sperm Injection): High-magnification (×1500\times 1500) selection compared to standard ICSI (×300\times 300).

    • PICSI: Sperm selection based on hyaluronic acid binding.

  • Embryo Evaluation:

    • Morphokinetics: Brightfield time-lapse microscopy monitoring PB extrusions, Pronuclei (PN) formation, and Gardner metrics for blastocyst morphology.

    • Cleavage: Assessment of direct/reverse cleavage (DC/RC), symmetry, and multinucleation (MNB).

  • Embryo Transfer Guidelines:

    • Window of Implantation (WOI): Critical timing for successful transfer.

    • Embryo Number (IFA Recommendations):

      • Single Embryo Transfer (SET): Mandatory/95% in females <35y<35y (first 3 cycles), egg donation (ED), or high risk.

      • 2 Embryos: For patients aged 35−41y35-41y.

      • 3 Embryos: For patients aged ≥42y\ge 42y.

    • Timing: Day 2-3 (low zygote counts/logistics) or Day 5-7 (standard default for higher pregnancy rates and PGT).