Explaining the counter-intuitive effectiveness of trophectoderm biopsy for PGT-A using computational modelling

Reviewer #1 (Public Review):

Summary:

The manuscript presents a compelling model to explain the impact of mosaicism in preimplantation genetic testing for aneuploidies.

Strengths:

A new view of mosaicism is presented with a computational model, that brings new insights into an "old" debate in our field. It is a very well-written manuscript.

Weaknesses:

Although the manuscript is very well written, this is in a way that assumes that the reader has existing knowledge about specific terms and topics. This was apparent through a lack of definitions and minimal background/context to the aims and conclusions for some of the author's findings.

There is a need for some examples to connect real evidence and scenarios from clinical reports with the model.

Reviewer #2 (Public Review):

Summary:

Although an oversimplification of the biological complexities, this modeling work does add, in a limited way, to the current knowledge on the theoretical difficulties of detecting mosaicism in human blastocysts from a single trophectoderm biopsy in PGT. However, many of the premises that the modeling was built on are theoretical and based on unproven biological and clinical assumptions that could yet lead to be untrue. Therefore, the work should be considered only as a simplified model that could assist in further understanding of the complexities of preimplantation embryo mosaicism, but assumptions of real-world application are, at this stage, premature and should not be considered as evidence in favour of any clinical strategies.

Strengths:

The work has presented an intriguing theoretical model for elaborating on the interpretation of complex and still unclear biological phenomena such as chromosomal mosaicism in preimplantation embryos.

Weaknesses:

Lines 134-138: The spatial modeling of mitotic errors in the embryo was oversimplified in this manuscript. There is only limited (and non-comprehensive) evidence that meiotic errors leading to chromosome mosaicism arise from chromosome loss or gain only (e.g. anaphase lag). This work did not take into account the (more recognised) possibility of mitotic nondisjunction where following the event there would be clones of cells with either one more or one less of the same chromosome. Although addressed in the discussion (lines 572-574), not including this in the most basic of modeling is a significant oversight that, based on the simple likelihood, could significantly affect results.

General comment: the premise of the manuscript is that an embryologist (embryology laboratory) is aware of and can accurately quantify the number of cells in a blastocyst or TE biopsy. The reality is that it is not possible to accurately do this without the destruction of the sample which is obviously not clinically applicable. Based on many assumptions the findings show that taking small biopsies poorly classifies mosaic embryos, which is not disputed. However, extrapolating this to the clinic and making suggestions to biopsy a certain amount of cells (lines 539-540) is careless and potentially harmful by suggesting the introduction of potential change in clinical practice without validation. Additionally, no embryologist in the field can tell how many cells are present in a clinical TE biopsy, making this suggestion even more impractical.

On a more general clinical consideration, the authors should acknowledge that when reporting findings of unproven clinical utility and unknown predictive values this inevitably results in negative consequences for infertile couples undergoing IVF. It is proven and established that when couples face the decision on how to manage a putative mosaicism finding, the vast majority decide on embryo disposal. It was recently reported in an ESHRE survey that about 75% of practitioners in the field consider discarding or donating to research embryos with reported mosaicism. A prospective clinical trial showed that about 30% live birth rate reduction can be expected if mosaic embryos are not considered (Capalbo et al., AJHG 2021). The real-world experience is that when mosaicism is reported, embryos with almost normal reproductive potential are discarded. The authors should be more careful with the clinical interpretation and translation of these theoretical findings.

There is a robust consensus within the field of clinical genetics and genomics regarding the necessity to exclusively report findings that possess well-established clinical validity and utility. This consensus is grounded in the imperative to mitigate misinterpretation and ineffective actions in patient care. However, the clinical framework delineated in this manuscript diverges from the prevailing consensus in clinical genetics. Clinical genetics and genomics prioritize the dissemination of findings that have undergone rigorous validation processes and have demonstrated clear clinical relevance and utility. This emphasis is crucial for ensuring accurate diagnosis, prognosis, and therapeutic decision-making in patient care. By adhering to established standards of evidence and clinical utility, healthcare providers can minimize the potential for misinterpretation and inappropriate interventions. The framework proposed in this manuscript appears to deviate from the established principles guiding clinical genetics practice. It is imperative for clinical frameworks to align closely with the consensus guidelines and recommendations set forth by professional organizations and regulatory bodies in the field. This alignment not only upholds the integrity and reliability of genetic testing and interpretation but also safeguards patient well-being and clinical outcomes.

References:
ACMG Board of Directors. (2015). Clinical utility of genetic and genomic services: a position statement of the American College of Medical Genetics and Genomics. Genetics in Medicine, 17(6), 505-507. https://doi.org/10.1038/gim.2014.194.
Richards, S., Aziz, N., Bale, S., Bick, D., Das, S., Gastier-Foster, J., ... ACMG Laboratory Quality Assurance Committee. (2015). Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genetics in Medicine, 17(5), 405-424. https://doi.org/10.1038/gim.2015.30

Line 61: "Self correction" - This terminology is unfortunately indiscriminately used in the field for PGT when referring to mosaicism and implies that the embryo can actively correct itself from a state of inherent abnormality. Apart from there being no evidence to suggest that there is an active process by which the embryo itself can correct chromosomal errors, most presumed euploid/aneuploid mosaic embryos will have been euploid zygotes and therefore "self-harm" may be a better explanation. True self-correction in the form of meiotic trisomy/monosomy rescue is of course theoretically possible but not at all clinically significant. The concept being conveyed in this part of the manuscript is not disputed but it is strongly suggested that the term "self correction" is not used in this context, nor in the rest of the manuscript, to prevent the perpetuation of misinformation in the field and instead use a better description.

Lines 69-73: The ability to quantify aneuploidy in known admixtures of aneuploid cells is indeed well established. However, the authors claim that the translation of this to embryo biopsy samples is inferred with some confidence and that if a biopsy shows an intermediate chromosome copy number (ICN), that the biopsy and the embryo are mosaic. There are no references provided here and indeed the only evidence in the literature relating to this is to the contrary. Multifocal biopsy studies have shown that an ICN result in a single biopsy is often not seen in other biopsies from the same embryo (Capalbo et al 2021; Kim et al., 2022; Girardi et al., 2023; Marin, Xu, and Treff 2021). Multifocal biopsies showing reciprocal gain and loss which would provide stronger validation for the presence of true mosaicism are also rare. In this work, the entire manuscript is based on the accuracy of ICN in a biopsy being reflective of mosaicism in the embryo. The evidence however points to a large proportion of ICN detected in embryo biopsy potentially being technical artifacts (misdiagnosing both constitutionally normal and abnormal (meiotic aneuploid) embryos as mosaic. Therefore, although results from the modelling provide insight into theoretical results, these can not be used to inform clinical decision-making at all.

Lines 87-89: The authors make the claim that emerging evidence is suggestive that the majority of embryos are mosaic to some degree. If in fact, mosaicism is the norm, the clinical importance may be limited.

Line 102-103: The statement that data shows that the live birth rate per ET is generally lower in mosaic embryos than euploid embryos is from retrospective cohort studies that suffer from significant selection bias. The authors have ignored non-selection study results (Capalbo et al, ajhg 2021) that suggest that putative mosaicism has limited predictive value when assessed prospectively and blinded.

Lines 94-98: The authors have misrepresented the works they have presented as evidence for biopsy result accuracy (Kim et al., 2023; Victor et al 2019; Capalbo et al., 2021; Girardi et al., 2023, and any others). These studies show that a mosaic biopsy is not representative of the whole embryo and can actually be from embryos where the remainder of the embryo shows no evidence of mosaicism. There is also a missing key reference of Capalbo et al, AJHG 2021, and Girardi et al., HR 2023 where multifocal biopsies were taken.

Lines 371-372: "Selecting the embryo with the lowest number of aneuploid cells in the biopsy for transfer is still the most sensible decision". Where is the evidence for this other than the modeling which is affected by oversimplification and unproven assumptions? Although the statement seems logical at face value, there is no concrete evidence that the proportion of aneuploid cells within a biopsy is valuable for clinical outcomes, especially when co-evaluated with other more relevant clinical information.

Lines 431-463: In this section, the authors discuss clinical outcome data from the transfer of putative mosaic embryos and make conclusions about the relationship between ICN level in biopsy and successful pregnancy outcomes. The retrospective and selective nature of the data used in forming the results has the potential to lead to incorrect conclusions when applied to prospective unselected data.

Reviewer #3 (Public Review):

Unfortunately, this study fails to incorporate the most important variable impacting the ability to predict mosaicism, the accuracy of the test. The fact is that most embryos diagnosed as mosaic are not mosaic. There may be 4 cases out of thousands and thousands of transfers where a confirmation was made. Mosaicism has become a category of diagnosis in which embryos with noisy NGS profiles are placed. With VeriSeq NGS it is not possible to routinely distinguish true mosaicism from noise. An analysis of NGS noise levels (MAPD) versus the rate of mosaics by clinic using the registry will likely demonstrate this is the case. Without accounting for the considerable inaccuracy of the method of testing the proposed modeling is meaningless.

Recent data using more accurate methods of identifying mosaicism indicate that the prevalence of true preimplantation embryonic mosaicism is only 2%, which is also consistent with findings made post-implantation. This model fails to account for the possibility that, because so few embryos are actually mosaic, there is actually no relevance to clinical care whatsoever. In fact, differences in clinical outcomes of embryos designated as mosaic could be entirely attributed to poor embryo quality resulting in noise levels that make NGS results fall into the "mosaic" category.

Additional comments:

Indeed, as more data emerges, it appears that the majority of embryos from both healthy and infertile couples are mosaic to some degree (Coticchio et al., 2021; Griffin et al., 2022).

This statement should be softened as all embryos will be considered mosaic when a method with a 10% false positive rate is applied to 10 more parts of the same embryo. The distinction between artifact and true mosaicism cannot be made with nearly all current methods of testing. When virtually no embryos display uniform aneuploidy in a rebiopsy study, there should be great concern over the accuracy of the testing used. The vast majority of aneuploidy is meiotic in origin.

Experimental data provides strong evidence that, for the most part, the biopsy result obtained accurately represents the chromosome constitution of the rest of the embryo (Kim 96 et al., 2022; Navratil et al., 2020; Victor et al., 2019).

This statement is incorrect given published systematic review of the literature indicates a 10% false positive rate based on rebiopsy results.

This shows that accurately classifying a mosaic embryo based on a single biopsy is not robust.

This is exactly why the practice of designating embryo mosaics with intermediate copy numbers should not exist.

Author response:

Reviewer #1 (Public Review):

Summary:

The manuscript presents a compelling model to explain the impact of mosaicism in preimplantation genetic testing for aneuploidies.

Strengths:

A new view of mosaicism is presented with a computational model, that brings new insights into an "old" debate in our field. It is a very well-written manuscript.

Weaknesses:

Although the manuscript is very well written, this is in a way that assumes that the reader has existing knowledge about specific terms and topics. This was apparent through a lack of definitions and minimal background/context to the aims and conclusions for some of the author's findings.

There is a need for some examples to connect real evidence and scenarios from clinical reports with the model.

We thank the reviewer for their assessment. Some background was condensed for space, and we wrote the manuscript to be understood by readers with existing reproductive genetics background. We will add more detail and explain terminology more clearly. There are a number of published case studies that can link real-life clinical data with the model’s findings. We will include a summary of them in the text.

Reviewer #2 (Public Review):

Summary:

Although an oversimplification of the biological complexities, this modeling work does add, in a limited way, to the current knowledge on the theoretical difficulties of detecting mosaicism in human blastocysts from a single trophectoderm biopsy in PGT. However, many of the premises that the modeling was built on are theoretical and based on unproven biological and clinical assumptions that could yet lead to be untrue. Therefore, the work should be considered only as a simplified model that could assist in further understanding of the complexities of preimplantation embryo mosaicism, but assumptions of real-world application are, at this stage, premature and should not be considered as evidence in favour of any clinical strategies.

Strengths:

The work has presented an intriguing theoretical model for elaborating on the interpretation of complex and still unclear biological phenomena such as chromosomal mosaicism in preimplantation embryos.

We thank the reviewer for this detailed review, and that they see the value of theoretical modelling. We agree that this model makes simplifications; we took this simplified approach to focus on the core contradiction between clinical experience and previous modelling. Expanding the model to consider additional aspects of balanced mitotic nondisjunctions and technical accuracy is something we want to address; we are discussing whether this is something that can be practically added to this manuscript, or will involve enough work that should be developed as a further study.

Weaknesses:

Lines 134-138: The spatial modeling of mitotic errors in the embryo was oversimplified in this manuscript. There is only limited (and non-comprehensive) evidence that meiotic errors leading to chromosome mosaicism arise from chromosome loss or gain only (e.g. anaphase lag). This work did not take into account the (more recognised) possibility of mitotic nondisjunction where following the event there would be clones of cells with either one more or one less of the same chromosome. Although addressed in the discussion (lines 572-574), not including this in the most basic of modeling is a significant oversight that, based on the simple likelihood, could significantly affect results.

As above, we certainly plan to address this in future modelling; developing the model to account for this while also incorporating the issue of technical uncertainty in the state of each cell in the biopsy from sequencing.

General comment: the premise of the manuscript is that an embryologist (embryology laboratory) is aware of and can accurately quantify the number of cells in a blastocyst or TE biopsy. The reality is that it is not possible to accurately do this without the destruction of the sample which is obviously not clinically applicable. Based on many assumptions the findings show that taking small biopsies poorly classifies mosaic embryos, which is not disputed. However, extrapolating this to the clinic and making suggestions to biopsy a certain amount of cells (lines 539-540) is careless and potentially harmful by suggesting the introduction of potential change in clinical practice without validation. Additionally, no embryologist in the field can tell how many cells are present in a clinical TE biopsy, making this suggestion even more impractical.

We will revise this to make the technical limitations of clinical TE biopsies clearer.

On a more general clinical consideration, the authors should acknowledge that when reporting findings of unproven clinical utility and unknown predictive values this inevitably results in negative consequences for infertile couples undergoing IVF. It is proven and established that when couples face the decision on how to manage a putative mosaicism finding, the vast majority decide on embryo disposal. It was recently reported in an ESHRE survey that about 75% of practitioners in the field consider discarding or donating to research embryos with reported mosaicism. A prospective clinical trial showed that about 30% live birth rate reduction can be expected if mosaic embryos are not considered (Capalbo et al., AJHG 2021). The real-world experience is that when mosaicism is reported, embryos with almost normal reproductive potential are discarded. The authors should be more careful with the clinical interpretation and translation of these theoretical findings.

The clinical potential of mosaic embryos is much more nuanced than a simple ‘they should be discarded’ or ‘they should be treated like euploid embryos’. While the study mentioned by the reviewer (Capalbo et al., AJHG 2021) does indeed suggest that embryos with putative low level mosaicism have good potential, it also suggests that embryos with putative high level mosaicism are largely to be considered aneuploid and should therefore be discarded. Therefore, even the mentioned study supports a ‘ranking’ of embryos by their mosaic result. Furthermore, large controlled retrospective studies have indicated that even high level mosaic embryos have reproductive potential (Viotti Fertility & Sterility 2021 and Viotti F&S 2023). Recent case reports have shown that mosaicism can occasionally persist from embryo to late gestation and even birth, at times associating with negative medical findings. Therefore, while the true clinical potential of embryos classified as mosaic is still being defined, here we are merely suggesting that from a modelling standpoint, the features of mosaicism detected with PGT-A can help guide clinical decisions (complementing the observations reported in the clinical studies).

There is a robust consensus within the field of clinical genetics and genomics regarding the necessity to exclusively report findings that possess well-established clinical validity and utility. This consensus is grounded in the imperative to mitigate misinterpretation and ineffective actions in patient care. However, the clinical framework delineated in this manuscript diverges from the prevailing consensus in clinical genetics. Clinical genetics and genomics prioritize the dissemination of findings that have undergone rigorous validation processes and have demonstrated clear clinical relevance and utility. This emphasis is crucial for ensuring accurate diagnosis, prognosis, and therapeutic decision-making in patient care. By adhering to established standards of evidence and clinical utility, healthcare providers can minimize the potential for misinterpretation and inappropriate interventions. The framework proposed in this manuscript appears to deviate from the established principles guiding clinical genetics practice. It is imperative for clinical frameworks to align closely with the consensus guidelines and recommendations set forth by professional organizations and regulatory bodies in the field. This alignment not only upholds the integrity and reliability of genetic testing and interpretation but also safeguards patient well-being and clinical outcomes.

References:

ACMG Board of Directors. (2015). Clinical utility of genetic and genomic services: a position statement of the American College of Medical Genetics and Genomics. Genetics in Medicine, 17(6), 505-507. https://doi.org/10.1038/gim.2014.194.

Richards, S., Aziz, N., Bale, S., Bick, D., Das, S., Gastier-Foster, J., ... ACMG Laboratory Quality Assurance Committee. (2015). Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genetics in Medicine, 17(5), 405-424. https://doi.org/10.1038/gim.2015.30

We will update where necessary to match these references.

Line 61: "Self correction" - This terminology is unfortunately indiscriminately used in the field for PGT when referring to mosaicism and implies that the embryo can actively correct itself from a state of inherent abnormality. Apart from there being no evidence to suggest that there is an active process by which the embryo itself can correct chromosomal errors, most presumed euploid/aneuploid mosaic embryos will have been euploid zygotes and therefore "self-harm" may be a better explanation. True self-correction in the form of meiotic trisomy/monosomy rescue is of course theoretically possible but not at all clinically significant. The concept being conveyed in this part of the manuscript is not disputed but it is strongly suggested that the term "self correction" is not used in this context, nor in the rest of the manuscript, to prevent the perpetuation of misinformation in the field and instead use a better description.

This is a good point. We have used ‘self correction’ as a shorthand, but the reality is more nuanced. It will often be a passive process in which aneuploid cell lineages fail to proliferate over time (‘aneuploidy depletion’). The idea of ‘self harm’ is interesting; aneuploidy arising from a healthy euploid embryo. We can also see a further situation where the gametes suffered damage (e.g. DNA fragmentation, unresolved crossovers, persistence of meiotic breaks) leading to mitotic errors. In that case, the embryo would suffer the consequences of harm in the gametes, and ‘aneuploidy rescue’ may be a useful term also. We will discuss this further and reword the terminology along these lines.

Lines 69-73: The ability to quantify aneuploidy in known admixtures of aneuploid cells is indeed well established. However, the authors claim that the translation of this to embryo biopsy samples is inferred with some confidence and that if a biopsy shows an intermediate chromosome copy number (ICN), that the biopsy and the embryo are mosaic. There are no references provided here and indeed the only evidence in the literature relating to this is to the contrary. Multifocal biopsy studies have shown that an ICN result in a single biopsy is often not seen in other biopsies from the same embryo (Capalbo et al 2021; Kim et al., 2022; Girardi et al., 2023; Marin, Xu, and Treff 2021). Multifocal biopsies showing reciprocal gain and loss which would provide stronger validation for the presence of true mosaicism are also rare. In this work, the entire manuscript is based on the accuracy of ICN in a biopsy being reflective of mosaicism in the embryo. The evidence however points to a large proportion of ICN detected in embryo biopsy potentially being technical artifacts (misdiagnosing both constitutionally normal and abnormal (meiotic aneuploid) embryos as mosaic. Therefore, although results from the modelling provide insight into theoretical results, these can not be used to inform clinical decision-making at all.

We thank the reviewer for raising this important conceptual point, which needs to be addressed. The fact that mosaicism is often not observed in serial biopsies of the same embryo is precisely an inherent feature of mosaicism and is an invalid argument to discount the original diagnosis as false. The detection of ICN is not trivial and certain PGT-A platforms might not have the capability to discern noise from true ICN, hence the need for proper validation of the technology. The most stringent validation method for mosaicism detection remains the admixture experiment, such that when ICN patterns are detected the most obvious conclusion is that the biopsy contained a mosaic mix of cells. We aim to add wording regarding these points in the manuscript.

Lines 87-89: The authors make the claim that emerging evidence is suggestive that the majority of embryos are mosaic to some degree. If in fact, mosaicism is the norm, the clinical importance may be limited.

If the majority of embryos are mosaic to some degree, it is important to understand the impacts that this may have on PGT-A biopsies and how informative such biopsies may be. Returning to the point the reviewer made above about mitotic aneuploidies as an important consideration: a mitotic nondisjunction at the first cleavage would result in a embryo that was entirely aneuploid. A mitotic nondisjunction occurring at the second cleavage would result in an embryo with 50% aneuploid cells, at the third cleavage, 25% aneuploid cells. If these aneuploid cells fail to proliferate, or are removed (either actively or passively), the level of aneuploidy will fall over time. While mosaicism is a binary (an embryo is or is not a mosaic of karyotypes), even if most embryos are mosaic, the clinical importance will depend on the level of aneuploidy.

Line 102-103: The statement that data shows that the live birth rate per ET is generally lower in mosaic embryos than euploid embryos is from retrospective cohort studies that suffer from significant selection bias. The authors have ignored non-selection study results (Capalbo et al, ajhg 2021) that suggest that putative mosaicism has limited predictive value when assessed prospectively and blinded.

We will add the referenced multifocal biopsy study, but in contrast to the reviewer we see the data it contains as supporting our position in this paper. Capalbo et al. performed rebiopsies of trophectoderm and a biopsy of inner cell mass and found that high level mosaic or aneuploid trophectoderm tended to correlate with abnormal karyotypes in the inner cell mass while low level mosaics correlated with a normal inner cell mass. This supports our point that measuring levels of aneuploidy in the trophectoderm is relevant, and that this gives useful information for ranking embryos.

Lines 94-98: The authors have misrepresented the works they have presented as evidence for biopsy result accuracy (Kim et al., 2023; Victor et al 2019; Capalbo et al., 2021; Girardi et al., 2023, and any others). These studies show that a mosaic biopsy is not representative of the whole embryo and can actually be from embryos where the remainder of the embryo shows no evidence of mosaicism. There is also a missing key reference of Capalbo et al, AJHG 2021, and Girardi et al., HR 2023 where multifocal biopsies were taken.

As above, we will add more information on these multifocal biopsy studies; we believe these studies also support our position: that individual biopsies are not predictive of aneuploidy level in an embryo. If mosaicism is detected in the biopsy, then the embryo is mosaic, but if the remainder of the embryo is euploid then that single biopsy was not an accurate representation of the embryo. This could also apply in reverse - if mosaicism is not detected in the biopsy, it does not mean there is no mosaicism in the embryo, only that mosaicism could not be identified.

Lines 371-372: "Selecting the embryo with the lowest number of aneuploid cells in the biopsy for transfer is still the most sensible decision". Where is the evidence for this other than the modeling which is affected by oversimplification and unproven assumptions? Although the statement seems logical at face value, there is no concrete evidence that the proportion of aneuploid cells within a biopsy is valuable for clinical outcomes, especially when co-evaluated with other more relevant clinical information.

We made this statement as part of a thought experiment to explain the difference between the concepts of absolute measurements versus embryo ranking. This section is not a result of the model, or clinical advice; it is a statement that in the specific example embryos given, the embryo with the fewest aneuploid cells in the biopsy would still be the embryo with the fewest aneuploid cells overall, and thus transferring this embryo (in the absence of any other differences of embryo quality) would remain sensible.

Lines 431-463: In this section, the authors discuss clinical outcome data from the transfer of putative mosaic embryos and make conclusions about the relationship between ICN level in biopsy and successful pregnancy outcomes. The retrospective and selective nature of the data used in forming the results has the potential to lead to incorrect conclusions when applied to prospective unselected data.

We believe the clinical data is a useful biological reality check, and we are discussing how to integrate it better with the modelling.

Reviewer #3 (Public Review):

Unfortunately, this study fails to incorporate the most important variable impacting the ability to predict mosaicism, the accuracy of the test. The fact is that most embryos diagnosed as mosaic are not mosaic. There may be 4 cases out of thousands and thousands of transfers where a confirmation was made. Mosaicism has become a category of diagnosis in which embryos with noisy NGS profiles are placed. With VeriSeq NGS it is not possible to routinely distinguish true mosaicism from noise. An analysis of NGS noise levels (MAPD) versus the rate of mosaics by clinic using the registry will likely demonstrate this is the case. Without accounting for the considerable inaccuracy of the method of testing the proposed modeling is meaningless.

We disagree with the reviewer that the modelling is meaningless; we disagree that mosaicism is rare (see our other points). However, if we grant that mosaicism is rare, that almost all embryos are euploid or aneuploid, and that technical noise is the primary factor generating intermediate copy number values, then it is still important to understand how to interpret such intermediate values. Low-level mosaics would more likely represent miscalled euploid embryos, and high-level mosaics would more likely represent miscalled aneuploid embryos. We demonstrate that ranking on these intermediate values correlates with implantation rates and live birth rates, supporting their use. We do agree that technical accuracy of the NGS is an important consideration, and we will be incorporating this into our modelling in the future.

Recent data using more accurate methods of identifying mosaicism indicate that the prevalence of true preimplantation embryonic mosaicism is only 2%, which is also consistent with findings made post-implantation. This model fails to account for the possibility that, because so few embryos are actually mosaic, there is actually no relevance to clinical care whatsoever. In fact, differences in clinical outcomes of embryos designated as mosaic could be entirely attributed to poor embryo quality resulting in noise levels that make NGS results fall into the "mosaic" category.

As we also wrote in the point above, we disagree; it is possible that a euploid embryo may be misinterpreted as a mosaic. It is also possible that an aneuploid embryo is misinterpreted as a mosaic. Whether the intermediate copy number values arise through biological or technical reasons, they contain information that is useful to decisions on whether to transfer. We also note a recent paper that performed single-cell dissociation of trophectoderm versus inner cell mass which found that mosaicism in human embryos is very common (Chavli et al, 2024, DOI:10.1172/JCI174483).

Additional comments:

“Indeed, as more data emerges, it appears that the majority of embryos from both healthy and infertile couples are mosaic to some degree (Coticchio et al., 2021; Griffin et al., 2022).”

This statement should be softened as all embryos will be considered mosaic when a method with a 10% false positive rate is applied to 10 more parts of the same embryo. The distinction between artifact and true mosaicism cannot be made with nearly all current methods of testing. When virtually no embryos display uniform aneuploidy in a rebiopsy study, there should be great concern over the accuracy of the testing used. The vast majority of aneuploidy is meiotic in origin.

We note that reviewer 2 wrote that mitotic aneuploidy was the key concern, whereas reviewer 3 states meiotic aneuploidy is more common; we argue that both are relevant; a recent study by McCoy et al, 2023 (DOI:10.1186/s13073-023-01231-1) found that both drive arrest of human IVF embryos.

“Experimental data provides strong evidence that, for the most part, the biopsy result obtained accurately represents the chromosome constitution of the rest of the embryo (Kim 96 et al., 2022; Navratil et al., 2020; Victor et al., 2019).”

This statement is incorrect given published systematic review of the literature indicates a 10% false positive rate based on rebiopsy results.

This shows that accurately classifying a mosaic embryo based on a single biopsy is not robust.

This is exactly why the practice of designating embryo mosaics with intermediate copy numbers should not exist.

We agree that accurately classifying a mosaic embryo based on a single biopsy is not robust. That is one of the main messages of this paper. What we show here is that biopsies from a mosaic embryo are indeed likely to disagree with each other - but we find that there is still enough information at a population level for this to be an indicator or embryo outcomes. We have not yet performed modelling to explore the effect of technical error, so we will not speculate on the impact, but we reiterate a point made earlier: the most stringent validation method for mosaicism detection remains the admixture experiment, such that when intermediate copy number patterns are detected the most obvious conclusion is that the biopsy contained a mosaic mix of cells.