Embryo Biopsy For Genetic Testing
EMBRYO BIOPSY
EMBRYO BIOPSY
- Biopsy procedures
Opening the zona pellucida
Removal of the cellular material - Developmental stage to perform the biopsy
ZONA OPENING
1- MECHANICAL OPENING
- Direct Puncture
- Partial Zona Dissection
2- CHEMICAL OPENING (Ac. Tyrode’s ph=2.3)
3- PHOTOTHERMOLYSIS (Laser)
ZONA OPENING
MECHANICAL OPENING
Direct Puncture: Performed with the use of a beveled pipette. Not clinically applied in the human for blastomere biopsy but used for polar body biopsy Verlinsky and Cieslak, 1993
Partial Zona Dissection: Described for human oocytes to facilitate sperm penetration Also useful for Assisted Hatching. Involves making a slit in the ZP by a sharp closed microneedle.
ZONA OPENING
CHEMICAL OPENING: Acid Tyrode Solution
The most widely used approach (cheap option)
Human ZP is more resistant to AT than mouse ZP
Larger, rounder hole than with PZD. Size of the hole not always easy to control
Two separate pipettes are usually used (double holder). Drilling pipette with an inner ø of 5 -7 µm plus the aspiration pipette. Zona opening: Chemical opening
Limiting the extent and duration of AT exposure is necessary to avoid acidification of medium and cell lysis
Target site: between two blastomeres
Embryo wash after AT exposure are recommended
Useful for early cleavage stages but inappropriate for oocytes
Photothermolysis: laser
Non contact laser: easily adapted to the microscope. Laser is transmitted through a 45X objective
Laser technique reduces time of biopsy procedure
Quick, simple, safe and efficient procedure with no need for micropipette changes
A direct relationship between the hole diameter (µm) and the exposure time (ms)
Effective and focalised without dispersion. Minimal absorption by the culture dish and the medium
Safe, with no mechanical, thermal or mutagenic effects
BIOPSY
POLAR BODY BIOPSY (day 0/day1)
EARLY CLEAVAGE EMBRYO BIOPSY (day 3)
BLASTOCYST BIOPSY (day 5)
POLAR BODY BIOPSY
1st polar body:2-3 hours after oocyte pick up (<6h). Degeneration or fragmentation of the 1st pb
Small hole of 18-25 µm. (not less than 15 µm)
Ac. Tyrode’s is not recommended. It could be harmful and compromise the viability of the oocyte. Mechanical zona opening and laser technology are the best options.
Pipettes for pb biopsy: bevelled biopsy pipette
Zona opening and laser technology
Sperm Microinjection(ICSI). Not IVF.
LIMITATIONS
Only genetic maternal contribution can be evaluated
Premature division of chromatids could also lead to a difficult interpretation of the results
No ethical objections or legal restrictions to polar body biopsy exist
BENEFITS
Useful for maternal structural and numerical chromosome aberrations and in certain monogenetic diseases
Polar bodies often degenerate. They are expected to have no biological role in the embryo development
No embryonic cells are removed
No reduction of the cellular mass
BLASTOMERE BIOPSY
Place the embryo with the chosen cell to biopsy at the 3 o’clock position. The cell should contain a single, clearly visible nucleus
Zona opening: mechanical, Acid Tyrode’s, Laser. Small hole of approx. 40 µm.
Special pipettes for blastomere aspiration.
The cell should be partially aspirated and pulled out rather than completely aspirated
Cell removal: Aspiration is the most widely used
Place the biopsied cell far from the embryo
Keep the embryo immediately in the incubator
BLASTOMERE BIOPSY
BLASTOMERE BIOPSY
It allows the detection of maternal, paternal and early post-fertilisation defects
1 or 2 blastomeres; Preferable to remove only one cell. If it is necessary to remove two, the same hole has to be used
The removal of too many blastomeres can be detrimental
Fragmented embryos(>35%) or embryos with a low development rate should not be biopsied.
Mosaicism of the cells
TROPHOECTODERM BIOPSY
Blastocyst biopsy is an emerging technique
Provides more cells to analyse
Interesting in monogenic diseases (more DNA available)
Lower degree of mosaicism
ICM remains fully intact
Requires a high blastocyst rate, an optimized culture system and specific laboratory expertise
Genetic results should be obtained in <24 hours in order to avoid cryopreservation
Blastocyst biopsy on day 5 and transfer on day 6
TROPHOECTODERM BIOPSY
Zona Pellucida drilling. A small gap 25-30 µm directly opposite the ICM (morning of day 5/ day 3-4)
Incubation 4 h to allow blastocoele expansion and spontaneous herniation of trophectoderm cells trophectoderm cells
Dissection of 3-10 trophectoderm cells using laser pulses
Blastocyst incubation and transfer on late day 5 or morning day 6 (hatched blastocysts).
First cases reported needed cryopreservation
TROPHOECTODERM BIOPSY