Step 1: The First Thaw (Warming)

Your embryo, which has been stored in liquid nitrogen at -196°C, is carefully warmed using specialized warming media. Modern vitrification (ultra-rapid freezing) techniques have revolutionized cryopreservation—survival rates after thaw exceed 95-98% for high-quality blastocysts.[2]

This step is generally safe. The embryo survives the warming process in the vast majority of cases.

Step 2: Re-expansion and Biopsy

After warming, the blastocyst needs time to recover and re-expand. The cells that collapsed during freezing must reform the fluid-filled cavity (blastocoel). This typically takes 2-6 hours in culture.[3]

Once the blastocyst has re-expanded to a stage suitable for biopsy, an embryologist performs a trophectoderm biopsy—the same procedure done during standard PGT-A. Using laser-assisted techniques, 5-10 cells are removed from the outer layer (trophectoderm, which will become the placenta). The inner cell mass (which becomes the baby) is not touched.[4]

If this is a rebiopsy (meaning the embryo was biopsied once before), the embryologist removes cells from a different section of the trophectoderm to avoid the previously biopsied area.[5]

Step 3: The Refreeze (Re-vitrification)

After the biopsy, the embryo must be frozen again while the biopsied cells are sent to a genetic testing lab for chromosomal analysis. PGT-A results take 1-2 weeks.

This means the embryo undergoes a second freeze-thaw cycle—re-vitrification. It will remain frozen until the results return.

Step 4: The Second Thaw and Transfer

Once the genetic testing results identify an euploid embryo, it is thawed a second time for frozen embryo transfer (FET). The embryo is transferred into the uterus approximately 5-7 days after re-warming.

Total freeze-thaw events: For a rebiopsy scenario, the embryo experiences three freeze-thaw cycles: initial freeze (from fresh cycle), thaw-rebiopsy-refreeze, and final thaw for transfer. For a standard PGT-A cycle, there are only two: biopsy-freeze, thaw for transfer.

The Impact on Embryo Viability

Each procedural step—thawing, biopsying, freezing—carries a very small risk of cellular damage. When you perform these steps multiple times, the risks compound.

What the data shows:

Vitrification survival rates: Modern vitrification has excellent survival rates (>95%) for high-quality blastocysts. However, double vitrification-warming cycles (freeze-thaw-refreeze-thaw) do introduce slightly higher attrition. Studies report survival rates of 90-95% after a second thaw, compared to 95-98% after a first thaw.[2][6]

This means: For every 100 embryos undergoing the full rebiopsy process, 5-10 may not survive to transfer, compared to 2-5 in a standard single-freeze cycle.

Biopsy impact: Trophectoderm biopsy for preimplantation genetic testing is considered safe and does not significantly harm euploid blastocysts when performed once.[7] However, a second biopsy removes additional cells from the trophectoderm, and the cumulative cellular loss may reduce the embryo's capacity to implant or develop a healthy placenta.[8]

Embryo quality matters: High-quality, fully expanded blastocysts with abundant trophectoderm cells tolerate the process better than lower-grade embryos or those with fewer cells.[9] If your embryos were graded as "good" (e.g., 4BB or higher) at the time of initial freezing, they are more likely to withstand the additional manipulation.

Study 1: Cimadomo et al. (2019) - Human Reproduction[1]
This study investigated blastocysts undergoing a second biopsy due to inconclusive results from the first PGT-A attempt. Results:

• Embryos undergoing a second biopsy and double biopsy and double vitrification had lower implantation rates (47% vs 58% for single biopsy)
• Live birth rates were also lower (40% vs 52%)

Conclusion: **A second embryo biopsy may have a detrimental effect on embryo viability and reproductive potential.

Study 2: Escribá et al. (2019) - Reproductive BioMedicine Online[10]
This study looked at blastocysts biopsied and rebiopsied for various reasons (inconclusive, re-expansion for additional testing). Results:

• No significant difference in clinical pregnancy rates between single-biopsied and rebiopsied embryos
• Survival rates after thaw were similar (92% vs 94%)

Conclusion: In selected cases, rebiopsy did not significantly affect pregnancy rates.

Study 3: Capalbo et al. (2016) - Fertility and Sterility[11]
This matched case-control study examined euploid blastocysts that underwent rebiopsy versus controls. Results:

• Rebiopsy was considered a regular strategy in their clinic for inconclusive results
• Pregnancy rates were comparable between groups

Conclusion: With careful case selection, rebiopsy outcomes can be acceptable.

Scenario 1: Testing Previously Frozen, Untested Embryos

Your situation:
You have embryos from a previous IVF cycle that were frozen without genetic testing. Now—due to advanced age (38+), a miscarriage, or repeated implantation failure—you want PGT-A data before embryo transfer.

The reasoning:
Aneuploidy is the leading cause of miscarriage and implantation failure. At age 38, approximately 50% of embryos are aneuploid; at age 42, it's 70-80%.[12] If you transfer an aneuploid embryo, there's a 95%+ chance it will either not implant or end in early miscarriage. PGT-A allows you to identify and prioritize euploid embryos.

The consideration:
You're weighing two risks:

1. Transfer without testing: Risk of miscarriage or failed implantation due to aneuploidy
2. Thaw-biopsy-refreeze: Procedural risk to embryo viability (5-15% reduction in success)

When it makes sense:

• You're 38+ years old (higher baseline aneuploidy risk)
• You have multiple frozen embryos (3+), so losing 1-2 to the procedure is acceptable
• Your embryos were high-quality blastocysts at initial freeze
• You've had a prior miscarriage or multiple failed transfers

When to reconsider:

• You have only 1-2 frozen embryos total
• Your embryos were lower-grade at initial freeze (e.g., 3BC or lower)
• You're under 35 with no miscarriage history (lower baseline aneuploidy risk)

Scenario 2: Rebiopsy After an Inconclusive or "No Result" PGT-A

Your situation:
You did PGT-A on your embryos, but one or more came back with an inconclusive result or "no result." The lab couldn't extract enough DNA or the data was uninterpretable.

The reasoning:
An inconclusive result leaves you in limbo. You don't know if the embryo is euploid (safe to transfer) or aneuploid (high miscarriage risk). Rebiopsy is the only way to get a definitive genetic testing answer for that specific embryo.

The consideration:
This scenario involves the most manipulation: the embryo has already been biopsied once and frozen once. A rebiopsy means a **second biopsy, a second freeze-thaw cycle, and then a third thaw for transfer.

Studies show this is the highest-risk scenario for reduced viability.[1]

When it makes sense:

• You have very few embryos total (e.g., only 1-2), and getting a definitive answer on this one is critical
• The embryo is high-quality and re-expanded well after the first thaw
• Your clinic has significant experience with rebiopsy and can provide their specific success rates

When to reconsider:

• You have other euploid embryos available for transfer
• The embryo quality was borderline at the initial biopsy

You're comfortable transferring "blind" without genetic testing data for this particular embryo

Questions to Ask Your Doctor

Empower yourself with these specific questions at your consultation:

1. "What are your clinic's specific survival rates for embryos after a second thaw?"
   (You want clinic-specific data, not general statistics.)
2. "What are your live birth rates for rebiopsied embryos compared to standard single-biopsy embryos?"
   (This tells you the real-world impact at your clinic.)
3. "Given the quality of my specific embryos (grades), would you recommend this procedure?"
   (High-quality blastocysts tolerate rebiopsy better.)
4. "If we proceed, what is the likelihood of getting another inconclusive result?"
   (For rebiopsy after inconclusive: is there a technical reason the first failed?)
5. "How many embryos do I have, and how many euploid embryos do you expect after PGT-A?"
   (If you have 6 embryos and statistically expect 3 euploid, losing 1 to rebiopsy may be acceptable.)

"What is the alternative if I don't do this procedure?"
(Transfer without testing? New cycle?)