Why thaw embryos for PGT?

Preimplantation genetic testing (PGT) encompasses methods used to assess embryos produced through in vitro fertilization (IVF) for genetic abnormalities. These abnormalities may either impact the embryo's ability to successfully implant and result in a healthy pregnancy or can cause inherited genetic conditions in the offspring. There are a few different types of PGT testing:

• ‍PGT-A: It tests for aneuploidy, meaning an atypical number of chromosomes in the embryo.
• ‍PGT-M: It tests for genetic variants in a specific gene that can cause an inherited genetic disorder.
• ‍PGT-SR: It screens for structural re-arrangements in chromosomes.

To obtain genetic material for testing, all types of PGT require a biopsy procedure. This step involves the removal of a small number of cells from the developing embryo, which are then sent to a specialized genetic testing laboratory for analysis. Some IVF clinics may have an in-house genetics lab, but most clinics send out biopsies to a specialized facility for genetic analysis. The biopsy almost always occurs in blastocyst-stage embryos. The cells removed during the biopsy are from the outer part of the embryo, called the trophectoderm.

In most cases, the embryos undergo biopsy during a fresh IVF cycle. This process involves the collection of eggs, immediate fertilization, and subsequent biopsy after several days of culture (usually on Day 5). Since PGT test results can take weeks or longer to return to the patient, the embryos are vitrified (frozen) shortly following biopsy and stored in liquid nitrogen. This freezing period allows for patients to choose which embryos to thaw for transfer based on the PGT results.

Less commonly, patients may choose to biopsy previously frozen embryos for PGT. In this scenario, embryos resulting from a prior egg retrieval have already been cultured and frozen as embryos. To complete PGT testing, these embryos must be thawed for the embryologist to perform a trophectoderm biopsy and then refrozen on the same day. That means the embryos will need to undergo an additional freeze-thaw cycle.

There are two main situations in which patients may choose to complete PGT of their already frozen embryos:

• Re-assessment of previously tested and frozen embryos due to inconclusive PGT results
• PGT for previously untested embryos

Re-assessment of previously tested embryos

Occasionally, embryos biopsied during the initial fresh cycle may yield inconclusive, incomplete, or failed genetic test results. When these issues occur, patients may opt to retest the embryos to obtain definitive results — particularly if no other genetically normal (euploid) embryos are available for transfer and the patients/providers feel that having the PGT result is important. Alternatively, patients may decide to undergo another IVF cycle to produce more embryos or consider a blind transfer of the PGT-inconclusive embryo. Each of these choices carries its own set of advantages and risks.
 
Re-biopsy of embryos with mosaic results (with the aim of confirming chromosomal status) is discouraged by some professional organizations, such as the European Society for Human Reproduction and Embryology (ESHRE), due to a lack of diagnostic value and risk to the embryo.ⁱ Emerging evidence indicates that the transfer of mosaics should be considered (especially low level and certain single chromosome aneuploidies), which negates the need to thaw and retest.ⁱⁱ

Analysis of previously untested embryos

Patients who did not initially opt for PGT during the fresh cycle may later decide to pursue it. Reasons for this decision may include recurrent pregnancy loss (RPL), repeated implantation failure (RIF), advanced maternal age at the time of freezing, gender selection for family balancing (subject to ethical and legal considerations), the use of a gestational carrier, and/or the desire to select the best embryo when many frozen blastocysts are available. Additionally, patients may acquire new medical information, such as being carriers for specific genetic variations or structural rearrangements, prompting them to choose PGT-M or PGT-SR to avoid passing on genetic disorders.

How embryo thawing works

Vitrification is the method currently used for cryopreservation, as it is more efficient than the previous method of slow freezing. During thawing, frozen embryos are recovered to natural physiological temperature, restoring their biological activity. Various protocols and kits are available to IVF labs for this process. All protocols involve extracting embryos from the liquid nitrogen tank, warming them, and replacing the cryoprotectant solution used during freezing with water to rehydrate the cells.ⁱⁱⁱ

Cryoprotectants are crucial for safeguarding the embryos during freezing, as they help draw water out of the cells. Water is removed so that ice crystal formation during freezing is prevented, since ice crystals can cause damage to the embryo.

Embryo thawing is a relatively quick procedure — typically taking less than 15 minutes — and is performed by skilled embryologists within the IVF laboratory. Once successfully thawed, embryos are transferred to a culture medium designed to support their viability. They will then be biopsied by the embryologists.

Success rates when thawing embryos for genetic testing

The success of PGT for previously frozen embryos depends on the ability of the embryo to survive thaw, to avoid additional damage or stress from the additional freeze-thaw cycle, and to achieve an accurate genetic diagnosis.

Thaw survival

Thaw survival rates for frozen embryos are generally high, with most embryos successfully enduring the thawing process. However, high thaw survival rates do not mean that re-biopsy and a second freeze-thaw cycle do not impact embryo quality. In addition, success rates can vary across different laboratories, depending on specific cryopreservation methods, technologies, and skills of the embryologists.^iv

Vitrification, the gold-standard technique for freezing, is associated with notably higher survival rates — typically above 95 percent — in comparison to previously used freezing methods.^v For example, slow-freezing methods tend to yield survival rates of around 70 percent to 90 percent.^vi The key advantage of vitrification lies in its rapid cooling process, minimizing ice crystal formation and potential damage to the cellular structure of the embryo.^vii,viii,ix It is unlikely that any IVF labs still use slow-freezing methods for clinical practice.

Various lab- and embryo-related factors can influence the chances of embryos surviving vitrification:

• ‍Lab-related factors: The cryoprotectants used can be a factor, since certain cryoprotectants can be toxic to the embryos, as well as the speed at which embryos are cooled and warmed.^x
• ^‍Embryo-related factors: Studies have shown that the morphological quality of embryos and their developmental stage before freezing is linked to their ability to survive thawing.^xi,xii,xiii A study of nearly 12,000 vitrified blastocyst-stage embryos found that thaw-survival rates were highest in embryos with lower rates of expansion or a highly scored trophectoderm (e.g., formed of many cells forming a cohesive, uniform layer).^xiv This study also found that embryos frozen on Day 5 had higher rates of survival than those frozen on Day 6.

Genetic testing results

The chance of successfully obtaining a genetic result from previously frozen (then thawed) embryos appears to be comparable to fresh embryos.^xv,xvi Additionally, studies have been highly successful in obtaining genetic results from frozen embryos that previously yielded inconclusive results, and the probability that an embryo is genetically normal does not appear to be altered.^{xvii,xviii,xix,xx}

Promisingly, a multicenter study published in Human Reproduction examined more than 200 embryos that had initially yielded inconclusive PGT-A results. Among the embryos that survived the initial thaw (97 percent), 52 percent were confirmed to be genetically normal. Notably, some of these embryos resulted in successful live births when transferred to patients, underscoring the significant value of re-biopsying undiagnosed blastocysts in cases with initially inconclusive results.^xxi

However, it is important to note that performing a biopsy at the cleavage stage (e.g., Day 3 embryos) is associated with diminished clinical outcomes.^xxii Therefore, it is highly recommended that frozen cleavage-stage embryos thawed for PGT should undergo further culture until the blastocyst stage before biopsy and subsequent refreezing. Using this approach, a study reported equivalent rates of live birth and miscarriage following the PGT analysis of frozen Day 3 embryos that were thawed and cultured to blastocysts compared to embryos that were initially frozen as blastocysts.^xxiii

Risks of an extra freeze-thaw cycle on embryos

There are risks and benefits to subjecting previously tested frozen embryos to additional freeze-thaw cycles and biopsy procedures during preimplantation genetic testing (PGT).  It is essential to explore both the potential impacts of a second freeze-thaw cycle as well as a second freeze-thaw with a double-biopsy. These considerations are crucial for patients and healthcare providers in making informed decisions.

Risks: Analysis of previously untested frozen embryos

Performing PGT on cryopreserved embryos involves subjecting them to a second round of freezing and thawing, which introduces additional risks. Numerous studies have explored the impact of a second freeze-thaw cycle on embryos, with most reporting no significant differences in clinical outcomes (thaw survival, clinical pregnancy, live birth, or miscarriage rates) compared to embryos frozen only once.^{xxiv,xxv,xxvi,xxvii,xxviii}

Conversely, two further studies found that a second freeze-thaw cycle during the PGT analysis of frozen embryos was associated with reduced rates of thaw survival^xxix and live birth.^xxx The negative impact of a second freeze-thaw cycle on live birth rates was also reported in a statistical analysis of the data from several different studies.^xxxi

These factors are especially important to consider because embryos labelled “mosaic” or even "aneuploid” by PGT have resulted in healthy live births in some cases. In other words, abnormal PGT results do not always indicate an abnormal embryo with certainty, and couples should consider this fact when contemplating thawing of untested embryos for PGT biopsy.^xxxii

Risks: Re-biopsy and analysis of previously tested frozen embryos

Embryo biopsies carry inherent risks. Patients considering PGT analysis of frozen embryos that were previously analyzed by PGT in the fresh cycle should consider the potential impact of a second biopsy (re-biopsy) procedure. That said, the effect of the double biopsy, double freeze-thaw approach is not well-documented, and the current evidence is conflicting.

Several studies have investigated the impact of a double biopsy, double freeze-thaw approach on pregnancy rates in comparison to a single biopsy and freeze-thaw cycle, providing conflicting results. Three notable studies reported reduced pregnancy rates associated with the former approach when embryos previously analyzed were thawed for a re-biopsy. One of the largest studies, conducted by Neal et al. (2019), revealed a concerning 25 percent decrease in ongoing pregnancy rates in cases involving a second biopsy and freeze-thaw cycle, compared to embryos biopsied and frozen-thawed once.^xxxiii Similarly, clinical pregnancy rates were found to be reduced in two further studies analyzing the impact of re-biopsy, highlighting the potential adverse impact that re-biopsy of previously tested frozen embryos can have on clinical outcomes.^xxxiv,xxxv

Conversely, two promising studies have reported no detrimental effects on clinical outcomes when embryos undergo double biopsy and double freeze-thaw procedures.^xxxvi,xxxvii Encouragingly, the studies demonstrated a 97 to 100 percent survival rate among embryos subjected to this approach during the second thaw. Furthermore, there were no observed adverse effects on implantation or pregnancy rates compared to embryos undergoing a single biopsy and freeze-thaw cycle. These findings suggest that a double biopsy, double freeze-thaw approach may be a viable option for patients without compromising embryo viability. Notably, the larger study conducted by Cimadomo et al (2018) reported live birth rates similar to those previously reported for embryos biopsied and frozen-thawed only once, reinforcing the potential benefits of this approach.^xxxviii

A 2023 statistical analysis of data from multiple independent studies (a meta-analysis)^xxxix revealed that embryos subjected to re-biopsy and re-vitrification achieved comparable live birth and miscarriage rates to embryos biopsied and vitrified only once. However, it is crucial to note that this study also identified a negative impact of a double freeze-thaw cycle alone on live birth rates, as mentioned above, highlighting the conflicting nature of current evidence on this topic.

These variations in outcomes may arise from differences in laboratory protocols, cryopreservation techniques, and biopsy methods. For example, biopsy and PGT-A procedures lack standardization, and certain methods may be safer and more accurate than others, contributing to the variability in study results.^xl,xli,xlii

While this field remains relatively underexplored, it is important to acknowledge that biopsy and cryopreservation procedures are invasive and can introduce additional risks to embryos.^xliii Therefore, when possible, performing embryo biopsy for PGT during the fresh cycle may be advisable. However, considering the reasonable success rates reported in studies, PGT analysis of frozen embryos can be considered as an alternative to discarding existing embryos or undergoing another cycle of invasive ovarian stimulation.

Risks in women over 40

Females over 40 years old may face unique challenges and additional risks compared to younger patients. It is well-established that as females age, their ovarian reserve decreases, leading to fewer eggs being retrieved during an IVF cycle. Consequently, these individuals may have small numbers of embryos frozen in storage and any damage or loss during the biopsy or freeze-thaw procedures may further reduce the number available for transfer. Embryo quality and competence is also negatively associated with maternal age, and so it is possible that embryos obtained from older patients may have a reduced ability to withstand multiple biopsies and freeze-thaw procedures.^xliv

One reason that embryos may be more subject to the stress of an extra freeze-thaw cycle is due to alterations of mitochondria in oocytes and embryos from females of advanced maternal age. Oocytes from older females often exhibit decreased quality in terms of mitochondrial function, energy (ATP) production, and antioxidant defenses. These factors may make the resulting embryos more susceptible to certain types of damage, such as oxidative stress and DNA damage, which may occur during cryopreservation.^{xlv,xlvi,xlvii,xlviii,xlix} However, research in this area is very limited and unclear.

Another issue to consider is the cost-effectiveness of PGT, especially when it requires an additional thaw. Although there is relatively little evidence related to cost-benefit analysis, one Australian study examined the costs of PGT-A in 2,093 females of advanced maternal age (older than 37) undergoing IVF. They found that the cost was over $28,000 for each additional live birth resulting from PGT.^l

Conclusion

Performing preimplantation genetic testing (PGT) biopsy of embryos during the fresh cycle is advisable. However, some individuals may choose to do PGT on their cryopreserved embryos at a later date — often because their embryos were not previously PGT tested or because certain embryos had inconclusive PGT results.

To complete the procedure, frozen embryos must be thawed to conduct a trophectoderm biopsy.  This process involves subjecting the frozen embryos to a second round of freezing and thawing, which may introduce additional risks. Furthermore, conducting a second biopsy on embryos with inconclusive results imparts additional risks beyond the second freeze-thaw cycle.

However, considering the reasonable success rates reported in studies, PGT analysis of previously frozen and untested embryos is worth considering if the alternative would be discarding existing embryos or undergoing another cycle of invasive ovarian stimulation. Patients should discuss the potential benefits and risks associated with thawing embryos for PGT or re-biopsy with their fertility specialist.