ABB Embryology cheat sheet

Primordial germ cells arise in the yolk sac wall and migrate to the gonadal ridge by week 5-6 of gestation.

Oogenesis arrests in prophase I (diplotene) from before birth until the LH surge; the secondary oocyte arrests at metaphase II until fertilization.

Spermatogenesis: spermatogonia (2n) → primary spermatocyte (2n, after DNA replication) → secondary spermatocyte (n) → spermatid (n) → spermatozoon; meiosis yields 4 haploid cells per primary spermatocyte.

Sertoli cells secrete inhibin and androgen-binding protein and form the blood-testis barrier; Leydig cells produce testosterone under LH stimulation.

FSH drives follicle recruitment and Sertoli cell function; LH triggers ovulation and Leydig cell testosterone synthesis.

The acrosome is a Golgi-derived vesicle over the sperm nucleus containing hyaluronidase and acrosin; the midpiece contains mitochondria for motility.

Fallopian tube ampulla is the usual site of fertilization; the isthmus regulates sperm reservoir release and embryo transport.

Corpus luteum secretes progesterone for luteal support; regression occurs unless rescued by hCG from the implanting blastocyst.

Fertilization sequence: cumulus/oocyte penetration → zona pellucida binding → acrosome reaction → sperm-oocyte membrane fusion → cortical granule release (block to polyspermy) → completion of meiosis II → pronucleus formation.

Two pronuclei (2PN) visible 16-20 h post-insemination define normal fertilization; 1PN, 3PN, or >3PN indicate abnormal fertilization.

Cleavage divisions occur without net growth; human embryos typically reach 2-cell by day 1, 4-8-cell by day 2-3, morula by day 4, blastocyst by day 5-6.

Compaction begins at the morula stage (day 3-4), forming tight junctions and the outer trophectoderm.

Cavitation produces the blastocoel; the inner cell mass (embryoblast) and trophectoderm differentiate by day 5.

Zona hatching occurs around day 6-7; implantation into the endometrium begins about day 7 after fertilization.

Human chorionic gonadotropin is secreted by the syncytiotrophoblast to maintain the corpus luteum.

Gastrulation forms the three germ layers (ectoderm, mesoderm, endoderm) during week 3; neurulation follows in week 3-4.

Standard semen analysis per WHO: volume ≥1.4 mL, concentration ≥16 million/mL, total motility ≥42%, progressive motility ≥30%, normal morphology ≥4% (strict criteria).

Sperm preparation methods: density gradient centrifugation (selects motile, morphologically normal sperm) and swim-up (selects highly motile sperm); both remove seminal plasma and debris.

Intracytoplasmic sperm injection (ICSI) is indicated for severe male factor, prior fertilization failure, or surgically retrieved sperm.

Denuding oocytes with hyaluronidase before ICSI allows assessment of nuclear maturity: germinal vesicle (immature), metaphase I (immature), metaphase II (mature, extruded polar body).

Embryo biopsy for preimplantation genetic testing is typically performed at the blastocyst stage (trophectoderm, 5-10 cells) on day 5-6.

Vitrification uses high cryoprotectant concentrations and ultra-rapid cooling to achieve glass transition, avoiding ice crystal formation.

Intrauterine insemination (IUI) typically uses washed sperm inseminated near ovulation; success depends on tubal patency and motile sperm count.

Gamete and embryo handling should occur in a controlled environment with heated stages (37°C) and pH-buffered media.

Standard incubator conditions: 37°C, 5-6% CO2 (bicarbonate buffering), and 5% O2 in reduced-oxygen (tri-gas) systems; atmospheric O2 is ~20%.

Culture media are bicarbonate-buffered and require CO2 to maintain pH ~7.2-7.4; HEPES-buffered media are used for handling outside the incubator.

Sequential media change composition (e.g., lactate/pyruvate in cleavage, glucose in blastocyst media) to match changing embryo metabolism.

Embryo density and media volume affect paracrine signaling and nutrient availability; group culture can improve outcomes via autocrine/paracrine factors.

Oil overlay reduces evaporation, stabilizes pH and temperature, and limits volatile contaminant exposure.

Embryo metabolism shifts from pyruvate/lactate oxidation in cleavage stages to increased glucose consumption at the blastocyst stage.

Incubator CO2 and temperature must be verified with independent calibrated instruments; drift affects pH and development.

Air quality control includes VOC filtration and monitoring; volatile organic compounds can impair embryo development.

ICSI requires an inverted microscope with micromanipulators, holding pipette, and injection pipette; the sperm is injected into the ooplasm after immobilization.

Before ICSI, sperm are immobilized by crushing the tail with the injection pipette to ensure membrane disruption and DNA delivery.

Assisted hatching (mechanical, laser, or chemical) is performed to breach the zona pellucida and facilitate hatching, often for older patients or prior implantation failure.

Laser zona drilling uses a precise laser to thin or breach the zona; energy and pulse settings must be controlled to avoid embryo damage.

Polar body biopsy samples maternal genetic material; trophectoderm biopsy samples cells fated to become placenta.

Micromanipulation pipettes and instruments must be sterile and handled to avoid cross-contamination between patients.

Intracytoplasmic morphologically selected sperm injection (IMSI) uses high magnification to select sperm with normal nuclear morphology.

Sperm retrieval techniques include testicular sperm extraction (TESE) and microsurgical epididymal sperm aspiration (MESA).

Slow-freezing uses controlled-rate cooling with cryoprotectants (e.g., propanediol, glycerol) and seeding to induce ice formation outside the cell.

Vitrification uses high cryoprotectant concentrations (e.g., ethylene glycol, DMSO, sucrose) and ultra-rapid cooling (>10,000°C/min) to avoid ice crystals.

Cryoprotectants are categorized as permeating (DMSO, ethylene glycol, propanediol) or non-permeating (sucrose); they reduce intracellular ice formation.

Thawing/warming must be rapid to prevent recrystallization; stepwise removal of cryoprotectants prevents osmotic shock.

Oocyte vitrification is more technically demanding than embryo vitrification due to the large volume and meiotic spindle sensitivity.

Post-thaw survival is assessed by morphology and, for blastocysts, re-expansion; fully lysed embryos are not transferred.

Cryopreservation of sperm is standard for donor and fertility preservation; post-thaw motility is a key quality metric.

Storage in liquid nitrogen requires temperature monitoring and inventory tracking to prevent sample loss or mix-ups.

Cleavage-stage grading considers cell number, symmetry, and fragmentation; day 3 embryos are typically 6-8 cells with <10% fragmentation for top quality.

Blastocyst grading uses a numeric expansion score (1-6) plus two letters for inner cell mass (A-C) and trophectoderm (A-C).

Expansion grade 3 indicates a full blastocoel; grade 4 indicates an expanded blastocyst with thinning zona; grade 5 indicates hatching.

Good-quality inner cell mass is compact and numerous; good trophectoderm has many cells forming a cohesive epithelium.

Time-lapse monitoring allows continuous assessment of cleavage timing and morphology without disturbing culture.

Morphokinetic parameters (e.g., time to 2-cell, 4-cell, blastocyst formation) correlate with developmental potential.

Embryo selection may incorporate morphology, morphokinetics, and, where indicated, preimplantation genetic testing for aneuploidy (PGT-A).

Transfer of a single euploid blastocyst reduces multiple gestation risk while maintaining high implantation potential.

Quality control monitors individual tests/materials (e.g., media lot testing, pH and osmolality checks); quality assurance is the broader program ensuring consistent quality.

Media and consumables should be lot-tested (e.g., mouse embryo assay or sperm survival test) before clinical use.

Temperature and CO2 calibration must be documented at defined intervals using calibrated reference instruments.

Laboratory safety includes chemical hazard control, sharps handling, and liquid nitrogen handling precautions (frostbite and asphyxiation risk).

Biological safety involves universal precautions, PPE, and proper disposal of gametes, embryos, and contaminated materials.

Documentation and traceability of gametes/embryos are essential; double-witnessing reduces errors in critical steps.

External proficiency testing and internal audits verify competency and laboratory performance.

Air quality (VOC and particle monitoring) and incubator maintenance are part of routine QC.

Informed consent is required for all assisted reproduction procedures, including disposition of surplus embryos and cryopreserved gametes.

Embryo research and preimplantation genetic testing are subject to national legislation and institutional review; limits vary by jurisdiction.

Donor gamete programs require screening for infectious disease, genetic history, and informed consent regarding anonymity and disclosure.

Confidentiality of patient and donor information is a core professional obligation.

Embryo disposition options include transfer, cryopreservation, donation to research, and disposal, as permitted by law and consent.

Professional standards emphasize competency, continuing education, and adherence to accreditation requirements.

Ethical considerations include avoiding multiple gestation through single embryo transfer and ensuring equitable access.

Record retention and reporting requirements are set by regulatory bodies and accreditation organizations.