Inside the Embryology Lab: A Step-by-Step Look at What Happens to Your Donor Egg Embryos

You've selected your egg donor. The retrieval was successful. Twelve mature eggs are now in the embryology laboratory. But what happens next? For the next 5-7 days, these eggs will undergo a remarkable transformation—from single cells to complex blastocysts ? containing over 100 cells, ready for genetic testing and eventual transfer.

Most patients never see inside the embryology laboratory. It's a controlled, sterile environment where embryologists work with precision measured in micrometers, manipulating cells too small to see without high-powered microscopes. Robust laboratory safeguards continuously monitor critical variables—temperature, pH, and oxygen levels—to maintain optimal embryo development.

This is your behind-the-scenes guide to exactly what happens to your donor egg embryos during their critical first week of development.

Hour 0-6: The ICSI Procedure—Precision Fertilization Under the Microscope

Your eggs have arrived at the laboratory. Time is critical.

Within 4-6 hours of retrieval, eggs must be fertilized—otherwise, they begin aging and losing developmental potential. ICSI is commonly used in donor egg cycles—particularly when sperm quality is suboptimal, with frozen eggs, or to eliminate polyspermy risk. However, some clinics use conventional IVF fertilization when sperm parameters are normal. Your embryologist will determine the optimal approach based on your specific case.

Why ICSI for donor eggs:

Eliminates polyspermy risk (multiple sperm fertilizing one egg)
Allows sperm selection (embryologist chooses highest quality sperm)
Required for frozen eggs (zona pellucida ? hardens during vitrification/warming)

The ICSI workspace: Creating an artificial fallopian tube

The embryologist prepares the ICSI dish inside a specialized workstation:

Laminar flow: Provides sterile air
Heated stage: Maintains precise 37.0°C ± 0.5°C (exact human body temperature)
Micromanipulator: Human-controlled equipment capable to deliver ultimate precision (movements measured in micrometers)
Inverted microscope: 200-400x magnification to visualize subcellular structures

The ICSI procedure step-by-step:

Minute 0-15: Denudation ? (removing cumulus cells ?)

Mature eggs arrive surrounded by protective cumulus cells. The embryologist:

Places eggs in a special solution (37°C, 30-60 seconds)
Gently pipettes eggs to mechanically strip remaining cells
Assesses egg maturity under microscope

Maturity assessment:

Metaphase II (MII): Polar body ? visible—ready for ICSI
Metaphase I (MI): No polar body—maturation may (or may not) happen within several hours
Germinal vesicle (GV): Immature—cannot be fertilized

From the whole amount of retrieved donor eggs, we expect to obtain a major cohort of mature (MII) eggs capable of immediate fertilization using ICSI. A fraction of eggs may be graded “MI”. This part of harvested eggs is not going to be used in patients’ treatment program.

Minute 15-120: Sperm preparation

While eggs equilibrate, the biologist prepares sperm:

Density gradient centrifugation: Separates motile, morphologically normal sperm
Washing: Removes seminal fluid, dead sperm, debris
Selection: Under 400x magnification, specialists select individual sperm based on:

Progressive forward motility
Normal head morphology (oval, smooth outline)
Absence of large cytoplasmic droplets
Straight midpiece and tail

The critical moment: Sperm injection (2-3 minutes per egg)

Using micromanipulators with precision motors:

Step 1: Embryologist stabilizes egg with holding pipette (applying gentle suction)

Step 2: Injection pipette (about 1/10th the width of a human hair) approaches egg at 3 o'clock position

Step 3: Pipette pierces zona pellucida

Step 4: Pipette advances through cytoplasm toward egg center, carefully avoiding polar body and spindle region

Step 5: Small amount of cytoplasm aspirated into pipette (breaks membrane)

Step 6: Single sperm injected with minimal volume

Step 7: Pipette withdrawn; egg released from holding pipette

Immediate post-ICSI:

Injected eggs are placed individually in microdrops of culture medium to:

Prevent evaporation
Maintain stable temperature/pH
Protect from light
Allow gas equilibration

Eggs are returned to the incubator. The laboratory light is dimmed. The eggs begin the most critical process of their existence: fertilization.

Laboratory ICSI standards:

Our senior embryologists perform all donor egg ICSI procedures. We maintain:

Average fertilization rate (across all cycles): 76% (industry standard 70-80%)
Damage rate: <1% (eggs damaged during injection)
Average ICSI time per egg: 1 minute

Hour 16-20 (Day 1): The Fertilization Check—Confirming Success

Approximately 16-18 hours post-ICSI, the first critical checkpoint arrives.

The embryologist removes eggs from the incubator and examines each under an inverted microscope at 200-400x magnification. This evaluation takes seconds but reveals everything about whether fertilization succeeded.

What the embryologist looks for:

Normal fertilization (2PN ?—two pronuclei):

Two distinct pronuclei visible in egg cytoplasm
Two polar bodies in perivitelline space
Both pronuclei approximately equal size
Nucleoli (small dots within pronuclei) clearly visible

This is success. The egg is now officially a "zygote" ? or "fertilized embryo."

Expected fertilization outcomes (donor eggs):

From 12 mature eggs undergoing ICSI using a normozoospermic sperm sample, embryologists typically expect ~85% successful fertilization rate (MII). Our egg donor score system helps track every donor’s individual statistics. Our team recruits only egg donors with consistent fertilization indicators.

The pronuclear scoring system (optional advanced assessment):

Some laboratories grade pronuclear morphology:

Pronuclear position: Central (good) vs. peripheral (concerning)
Pronuclear size symmetry: Equal (optimal) vs. unequal
Nucleolar pattern: Specific arrangements correlate with developmental potential
Cytoplasmic appearance: Clear vs. granular

Research shows pronuclear morphology modestly predicts blastocyst formation, but modern laboratories increasingly rely on later-stage morphology and genetic testing rather than Day 1 scoring.

Day 2-3: Early Cleavage—The First Critical Cell Divisions

The embryos are dividing. But not all divisions are equal.

Until the mid-2010s it was regular to remove embryos from the incubator each day to confirm the developmental stage and overall progress. However, these days embryologists rely on Time Lapse systems.

Day 2 (30-48 hours post-ICSI): The 2-4 cell stage

The zygote undergoes its first cleavage ? division approximately 24-30 hours after ICSI, producing a 2-cell embryo. The second division follows 10-12 hours later, creating a 4-cell embryo by Day 2.

Multinucleation ?:

Normal: Each cell contains one nucleus
Abnormal: Cells with 2+ nuclei (highly associated with chromosomal abnormalities)

Day 3 (60-72 hours post-ICSI): The 6-8 cell stage

By Day 3, embryos should contain 6-8 cells. This is traditionally when embryologists performed detailed grading and embryo transfer (in earlier IVF protocols).

Day 3 grading system:

Modern laboratories use simplified grading:

Grade 1: 7-8 cells, <10% fragmentation ?, symmetric
Grade 2: 6-8 cells, 10-25% fragmentation
Grade 3: 4-6 cells or >25% fragmentation
Grade 4: <4 cells or >50% fragmentation or arrested

The critical question: Continue culture to blastocyst?

Decades ago, embryos were transferred on Day 3. Modern practice extends culture to Day 5-7 (blastocyst stage) because:

Advantages of blastocyst culture:

Natural selection: 40-60% of Day 3 embryos fail to reach blastocyst (eliminates many chromosomally abnormal embryos)
Better synchronization: Blastocyst implants Day 5-6 naturally, matching uterine receptivity
Enables PGT: Trophectoderm biopsy ? only possible at blastocyst stage
Higher implantation rates: 45-60% per blastocyst vs. 20-30% per Day 3 embryo

Our protocol:

For donor egg cycles, we culture all normally developing embryos to blastocyst stage (Day 5-7).

Day 4: Compaction and Morula Formation—The Quiet Before Transformation

Why? Embryos undergo dramatic reorganization called compaction ?. Individual cells become tightly adherent, forming a compact mass called a morula ?. Cell boundaries become invisible under standard microscopy.

What's happening inside (invisible to microscopes):

Cellular changes:

Cell-to-cell contacts strengthen via tight junctions and gap junctions
Cells polarize (develop distinct "inside" vs. "outside" surfaces)
Gene expression shifts dramatically
Cells begin differentiating into two lineages: inner cell mass ? (future baby) vs. trophectoderm ? (future placenta)

Molecular events:

Embryonic genome fully activated: Until Day 4, the embryo relies on maternal proteins stored in the egg. By Day 4, the embryo's own genes take control
Cell fate decisions begin: Complex signaling determines which cells become ICM vs. trophectoderm
Metabolism shifts: Embryo switches from pyruvate/lactate consumption to glucose metabolism

Why this stage is critical:

Embryos that fail to compact properly on Day 4 rarely develop to blastocyst.

Both sequential and single-step media systems can produce excellent results when used by experienced labs with rigorous quality controls. The choice often reflects a lab's validated protocol rather than one approach being universally superior.

By Day 4 evening, morulae should show early signs of cavitation—small fluid-filled spaces forming inside the compact cell mass. Tomorrow, they will transform into blastocysts.

Day 5-7: Blastocyst Formation—The Final Transformation Before Biopsy

Day 5 morning: The moment of truth.

Embryologists remove dishes from the incubator and examine embryos under the microscope. This is the most critical assessment. Embryos that reach blastocyst stage have demonstrated:

Chromosomal competence: (most aneuploid embryos ? arrest before blastocyst)
Metabolic health: (blastocysts have high energy demands)
Developmental synchronization

Our blastocyst culture standards:

All blastocysts graded using Gardner ? system by two independent embryologists
Digital images captured by Embryoscope ?
Blastocysts of grade 3BB or better proceed to biopsy
Lower-grade blastocysts (3BC, 3CC, etc.) assessed individually (may biopsy if patient has limited embryos)

The Biopsy Procedure: Removing 5-10 Cells for Genetic Testing

Only blastocysts suitable for biopsy proceed to the next critical step.

Trophectoderm biopsy for PGT-A requires specialized micromanipulation skills. Unlike ICSI (performed on all donor cycles), biopsy is optional—but recommended for maximizing success rates and minimizing transfer attempts.

Day 4 laser-assisted hatching for all PGT cycles—allows natural herniation and reduces Day 5 handling time.

Trophectoderm biopsy procedure (Day 5, 6, or 7):

Setup:

Blastocyst placed in biopsy dish (microdrop medium on heated stage)
Holding pipette stabilizes embryo (gentle suction)
Biopsy pipette approaches herniating TE cells

Step 1: Cell selection

Embryologist identifies 5-10 herniating TE cells
Avoids cells near ICM
Selects cohesive cell cluster (not individual cells)

Step 2: Aspiration

Biopsy pipette aspirates TE cells into tip
Gentle suction draws cells into pipette
Cell cluster remains attached to blastocyst via narrow connection

Step 3: Laser cutting

Laser pulses sever connection between biopsied cells and embryo
Typically 2-5 laser pulses (millisecond duration each)
Calibrated to cut cell connection without damaging remaining embryo

Step 4: Cell collection

Biopsied cells expelled into PCR tube containing 2-5 microliters buffer
Tube immediately labeled with embryo ID
Cells flash-frozen and shipped to genetic laboratory (NGC has its in-house genetic unit where the biopsy samples are registered and stored)

Step 5: Embryo vitrification ?

Biopsied embryo immediately moved to vitrification protocol
Cannot be left in culture after biopsy (increased risk of collapse/damage)

Entire biopsy: 3-5 minutes per embryo

Critical quality factors:

Cell number: 5-10 cells optimal

Too few (<5 cells): Inadequate DNA, higher amplification failure risk
Too many (>10 cells): Potential embryo damage

Cell viability: Biopsied cells should be intact

Lysed cells release DNA, contaminating sample
Embryologist confirms cells intact under microscope before tubing

Minimal ICM disruption: Biopsy from TE only

ICM cells will form the baby—must not be damaged
Proper technique keeps ICM completely intact

Rapid processing: Minimize embryo time outside incubator

Biopsy standards:

All biopsies performed by senior embryologists (>500 biopsies experience)
Average cell number: 6-8 cells per biopsy
DNA amplification success rate: 96.2%
Embryo survival post-warming: >98%
Biopsy session duration: 3-4 embryos biopsied per 15-minute session

PGT-A Analysis: 10-14 Days in the Genetics Laboratory

Your embryo cells are now in the genetics laboratory. What happens during genetic testing?

(Details covered extensively in previous article "The Hidden Variables: How Embryo Biopsy Timing, Technique, and Lab Quality Affect PGT Accuracy")

Brief overview of PGT-A process:

Days 1-3: DNA extraction and amplification

Cells lysed to release DNA
Whole genome amplification (WGA) creates millions of copies
Quality control: verify amplification success

Days 4-7: Next-generation sequencing (NGS)

DNA fragmented and sequenced
Generates millions of sequence reads
Each chromosome represented by specific number of reads

Days 8-10: Bioinformatics analysis

Software analyzes read counts per chromosome
Compares to reference genome
Calculates copy number for each chromosome (should be 2 copies each)

Days 11-12: Result interpretation and reporting

Geneticist reviews automated calls
Difficult cases reviewed by second analyst
Results categorized: Euploid ? / Aneuploid / Mosaic ? / No result

Day 12-14: Results delivered to patient

In-house laboratory advantage:

Average turnaround: 10-12 days (vs. 14-21 days for external labs)
Direct embryologist-geneticist communication
Same-day result review meetings
Amplification success rate: 96.2%

Typical PGT-A outcomes (from 6 biopsied blastocysts, donor eggs):

65-70% euploid (normal chromosomes—suitable for transfer)
30-35% aneuploid or mosaic (abnormal chromosomes—not recommended for transfer)

Transfer Preparation: Warming and Final Assessment

You've selected which euploid embryo to transfer. The final laboratory steps begin.

Day before transfer:

16:00-18:00: Embryo warming
Embryologist retrieves embryo from liquid nitrogen storage
Warming protocol (20 minutes): Room temp → 37°C dilution steps
Embryo transferred to culture medium
Placed in incubator to recover

Transfer morning (Day of ET):

06:00-07:00: Expansion assessment
Embryologist checks embryo has re-expanded to full blastocyst
Grading reassessed (most embryos maintain or improve grade post-warming)
Blastocoelic ? cavity should be clearly visible
TE and ICM should appear intact

Expected outcomes:

98%+ embryos successfully re-expand
1-2% fail to re-expand (rare but possible)—transfer canceled, next embryo selected

08:00-10:00: Transfer catheter loading

Embryo loaded into soft embryo transfer catheter
Catheter tip contains 20-30 microliters medium + embryo
Air brackets on either side (visible on ultrasound)
Embryologist confirms embryo in catheter under microscope
Physician receives catheter immediately

During transfer (10-15 minutes):

Ultrasound-guided catheter insertion through cervix
Catheter advanced to 1-2cm from uterine fundus
Embryo expelled gently
Catheter withdrawn and checked under microscope (confirms embryo released)

Post-transfer:

Patient rests 10-20 minutes
Embryologist confirms empty catheter
No bed rest required (evidence shows no benefit)

Common transfer protocol:

All transfers performed under abdominal ultrasound guidance
Soft catheters only (reduce endometrial trauma)
Embryologist present in transfer room (immediate catheter verification)

Success rates (NGC 2024 data, euploid donor egg blastocysts):

Clinical pregnancy rate per transfer: 62%
Ongoing pregnancy rate: 58%
Live birth rate per euploid embryo transfer: 56%

What Makes an Excellent Embryology Laboratory?

Not all IVF labs produce equivalent results. Eight critical quality indicators:

Embryologist experience and training

Minimum 2-3 years dedicated embryology training
Ongoing continuing education
Specialization in specific procedures (ICSI, biopsy, vitrification)
Low staff turnover (consistency matters)

Environmental control systems

HEPA-filtered air (removes VOCs, particulates)
Positive pressure rooms (prevents contamination ingress)
Temperature stability ±0.3°C
Humidity control

Incubator technology

Embryoscope > benchtop > large cabinet incubators (Embryoscope / Time-Lapse preferred—no need to remove embryos for assessment purposes)
Tri-gas system (CO₂, O₂, N₂ independently controlled)
Temperature recovery <30 seconds after door opening
Regular calibration (monthly minimum)

Culture medium quality

Pharmaceutical-grade components
Testing before clinical use
Mouse embryo assay (MEA) verification
Expiration date tracking

Quality control protocols

Daily incubator checks (temperature, CO₂, O₂)
Weekly quality audits
Monthly embryologist competency assessments
Annual proficiency testing

Volume and outcomes tracking

Minimum 200-300 cycles/year for consistent expertise
KPI monitoring: fertilization rates, blastocyst rates, pregnancy rates
Outcome correlation: identify which variables affect success

Laboratory accreditation

National licensing requirements
ISO for medical laboratories

Equipment redundancy

Backup incubators
Generator power backup
Liquid nitrogen supply redundancy
Emergency protocols

NGC Laboratory infrastructure:

Class 10,000 cleanroom environment (HEPA filtration)
2 Embryoscopes and 8 benchtop incubators
RI Witness system for biomaterial tracking safety
Tri-gas system (6% CO₂, 5% O₂, balance N₂)
24/7 environmental monitoring with alerts
Backup power systems (dual generators)
ISO 15189 accredited
8,000 PGT cycles annually

Faqs

Can I visit the embryology laboratory to see where my embryos are cultured?

For contamination control reasons, embryology laboratories typically do not allow patient visits while embryos are in culture. However, many clinics offer virtual lab tours via video or provide viewing windows where you can observe embryologists working. In our case the embryology lab is designed in glass allowing visitors to view the insides.

How long can embryos remain frozen—does storage duration affect success rates?

Embryos can remain vitrified indefinitely without quality degradation. The oldest successfully transferred embryo was frozen for 27 years. Studies comparing embryos frozen 1 year vs. 10+ years show no difference in pregnancy rates or neonatal outcomes. What matters is the embryo's quality at the time of vitrification, not how long it remains frozen. Your embryos are equally viable whether transferred next month or in a decade.

What happens if my embryos arrest (stop developing) before reaching the blastocyst stage?

Embryo arrest is a natural selection process—it occurs when the embryo has genetic abnormalities preventing further development. From 8 fertilized donor eggs, 1-3 typically arrest before blastocyst stage (this is normal and expected). The embryologist monitors daily and identifies arrested embryos (they stop dividing or fragment severely). These embryos are removed from culture and discarded. You'll be informed of any arrested embryos in your Day 5-7 blastocyst update.

Why does my clinic use different culture media brands than other clinics—does it matter?

Multiple reputable culture media manufacturers exist (Vitrolife, Irvine Scientific, Cooper Surgical, Fujifilm). Well-designed media from any manufacturer can produce excellent outcomes when used correctly. What matters more than brand is: (1) proper media handling and storage, (2) lot testing before clinical use, (3) using sequential media systems (switching from cleavage to blastocyst medium), and (4) maintaining optimal incubator conditions. Ask your clinic about their media validation process, not just which brand they use.

Should I pay extra for time-lapse imaging systems (Embryoscope) to monitor my embryos?

Time-lapse incubators capture images every 10-20 minutes without removing embryos from the incubator, creating a developmental "movie." Benefits: (1) Avoids environmental disturbance from daily assessments, (2) Provides morphokinetic data (timing of cell divisions), (3) May slightly improve embryo selection. However, multiple studies show time-lapse does not significantly increase pregnancy rates compared to standard incubation when embryos are also undergoing PGT-A (genetic testing already provides superior selection information). Time-lapse is most valuable when NOT using PGT-A or when you have many morphologically similar embryos to choose between. Ask your clinic if the added cost justifies the modest benefit for your specific situation.

Scientific References

Practice Committee of the American Society for Reproductive Medicine. Intracytoplasmic sperm injection (ICSI) for non-male factor indications: a committee opinion. Fertil Steril. 2020;114(2):239-245.

Alpha Scientists in Reproductive Medicine and ESHRE Special Interest Group of Embryology. The Istanbul consensus workshop on embryo assessment: proceedings of an expert meeting. Hum Reprod. 2011;26(6):1270-1283.

Doyle N, Gainty M, Eubanks A, et al. Donor oocyte recipients do not benefit from preimplantation genetic testing for aneuploidy to improve pregnancy outcomes. Hum Reprod. 2020;35(11):2548-2555.

Gardner DK, Schoolcraft WB. Culture and transfer of human blastocysts. Curr Opin Obstet Gynecol. 1999;11(3):307-311.

Edgar DH, Gook DA. A critical appraisal of cryopreservation (slow cooling versus vitrification) of human oocytes and embryos. Hum Reprod Update. 2012;18(5):536-554.

Rienzi L, Gracia C, Maggiulli R, et al. Oocyte, embryo and blastocyst cryopreservation in ART: systematic review and meta-analysis comparing slow-freezing versus vitrification to produce evidence for the development of global guidance. Hum Reprod Update. 2017;23(2):139-155.

ESHRE Guideline Group on Good Practice in IVF Labs, De Los Santos MJ, Apter S, et al. Revised guidelines for good practice in IVF laboratories (2015). Hum Reprod. 2016;31(4):685-686.

Swain JE. Optimal human embryo culture. Semin Reprod Med. 2015;33(2):103-117.

Kovacic B, Vlaisavljevic V. Influence of atmospheric versus reduced oxygen concentration on development of human blastocysts in vitro: a prospective study on sibling oocytes. Reprod Biomed Online. 2008;17(2):229-236.

Kaser DJ, Racowsky C. Clinical outcomes following selection of human preimplantation embryos with time-lapse monitoring: a systematic review. Hum Reprod Update. 2014;20(5):617-631.