Studying the Safety of Femtosecond Laser Applications in Assisted Hatching Technology

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

The manuscript provides a comprehensive overview of the fundamental principles and potential benefits of femtosecond laser-assisted hatching (fs-LAH), as well as its promising impact on embryo hatching rates. To further enhance the academic rigor and persuasive strength of the paper, the following improvements are recommended:
1. The optimization of laser parameters employed in the study remains uncertain. It is advisable to conduct additional experimental evaluations to determine the most effective parameter settings.
2. The current safety assessment of femtosecond laser application appears insufficient. It is recommended to incorporate a more comprehensive evaluation of relevant safety indicators to ensure procedural reliability.
3. The study focuses solely on in vitro hatching rate outcomes without supporting in vivo animal experimentation. To better assess developmental potential and long-term effects, it would be valuable to transplant treated embryos into animal models (e.g., mice) for follow-up observation.
4. The introduction lacks a clear articulation of the existing challenges in conventional hatching techniques and fails to sufficiently highlight the distinctive advantages of femtosecond lasers. A more thorough contextualization of these aspects is recommended to strengthen the rationale for the proposed methodology.

Author Response

Answers to Reviewers' comments

First of all, we would like to thank the Reviewers for their efforts and time spent in analyzing the manuscript. We have followed their recommendations and provide the responses point by point.

 

Reviewer 1

We thank the Reviewer for these insightful questions.

The manuscript provides a comprehensive overview of the fundamental principles and potential benefits of femtosecond laser-assisted hatching (fs-LAH), as well as its promising impact on embryo hatching rates. To further enhance the academic rigor and persuasive strength of the paper, the following improvements are recommended:

Q: The optimization of laser parameters employed in the study remains uncertain. It is advisable to conduct additional experimental evaluations to determine the most effective parameter settings.

A: Thank you for the suggestion. Indeed, the rationale behind choosing the laser parameters is not well described. In our previous studies we conducted experimental evaluation of admissible parameters of laser radiation for fs-LAH [1, 2]. In this paper, the parameters of laser radiation (beam velocity and laser intensity) were chosen to produce a similar cut width to that obtained with fs-LAH using visible laser pulses in [3]. This similarity provides a basis for the direct comparison of possible biological effects induced by laser radiation. We have clarified this idea and expanded the text of the article.

Accordingly, we selected intermediate parameters from the permissible range—υ = 10 μm/s and I=6.5 TW/cm² (E=155 nJ)—as indicated by the red circle in Figure 7. The embryo translation velocity was the same as for the visible fs-LAH. In combination with velocity υ, laser intensity I enabled us to obtain a similar cut width D~1.7 μm to that achieved with an intensity of I=2.5 TW/cm² at a wavelength of λ=514 nm [33]. Selecting laser parameters for both radiation wavelengths, which result in similar ZP microsurgery outcomes, allows the biological effects of IR and visible fs-LAH to be compared.

1. Sitnikov, D. S., & Mukhdina, D. E. (2024). Influence of the Cryopreservation Protocol on the Results of Femtosecond Laser Microsurgery of Zona Pellucida. High Temperature, 62(5), 663–668. https://doi.org/10.1134/S0018151X25700257
2. Sitnikov, D. S., Mukhdina, D. E., Filatov, M. A., & Silaeva, Y. Y. (2024). Determination of the Optimal Impact Parameters for Microdissection of Zona Pellucida Using Femtosecond IR Laser Pulses. High Temperature, 62(1), 102–109. https://doi.org/10.1134/S0018151X24700184
3. Filatov, M. A., Kubekina, M. V., Tvorogova, A. V., Ilchuk, L. A., Zhuravlev, A. D., Sazonova, E. A., Sitnikov, D. S. (2025). Biological effects of femtosecond and millisecond lasers application for assisted hatching in mouse embryos. Journal of Assisted Reproduction and Genetics. https://doi.org/10.1007/s10815-025-03499-1

 Q: The current safety assessment of femtosecond laser application appears insufficient. It is recommended to incorporate a more comprehensive evaluation of relevant safety indicators to ensure procedural reliability.

A: We agree with the Reviewer that a more comprehensive evaluation of biological effects would strengthen the study. However, the methods and indicators were chosen to match our previous study on the biological effects of visible femtosecond laser pulses. Our focus on HSP and ROS responses exclusively is indicated as a limitation of the study. HSPs are known to be an indicator of thermal stress in cells; a justification for choosing ROS as an indicator is also presented in the study:

Previous studies have indicated that exposure to femtosecond laser pulses can potentially cause cell damage or even death through the generation of reactive oxygen species (ROS) [37]. While cells tightly regulate ROS levels under normal conditions, stress (oxidative or thermal) can increase ROS production, resulting in DNA, lipid, and protein damage.

Q: The study focuses solely on in vitro hatching rate outcomes without supporting in vivo animal experimentation. To better assess developmental potential and long-term effects, it would be valuable to transplant treated embryos into animal models (e.g., mice) for follow-up observation.

A: We completely agree with the Reviewer that transferring embryos to a pseudopregnant mouse would allow us to better assess developmental potential and long-term effects. However, the main goal of this study was to compare biological effects induced by visible and infrared femtosecond laser pulses applied for laser-assisted hatching. We have conducted a comprehensive analysis of ROS and HSP expression levels, as well as key developmental metrics such as hatching rate for IR laser pulses, and compared our findings with those for visible radiation, previously published. This enabled us to draw some conclusions on the efficacy of IR fs-LAH. We understand that the lack of data on birth rate is a limitation of this study and have updated the corresponding paragraph:

A limitation of this study is its focus on HSP and ROS responses exclusively. Future investigations should expand the spectrum of biological effect monitoring to include additional markers, particularly apoptosis indicators, in order to further validate the safety profile of femtosecond LAH procedures. Studying the results of LAH-treated embryo transfers in pseudopregnant mice would help us better understand the embryos’ developmental potential and the long-term effects of fs-LAH treatments. This remains the subject of our ongoing research.

We agree that additional experiments with embryo transplantation to the uterus are required. And we will perform that in our future work.

 

Q: The introduction lacks a clear articulation of the existing challenges in conventional hatching techniques and fails to sufficiently highlight the distinctive advantages of femtosecond lasers. A more thorough contextualization of these aspects is recommended to strengthen the rationale for the proposed methodology.

A: Thank you for this insightful question. The main challenges of conventional (non-laser) assisted hatching techniques are related to high labor costs, strict requirements for the qualifications of embryologists, and the risk of human error. These factors are outside the scope of our study. As for the conventional millisecond LAH, the main challenge is related to high risk of thermal damage to the cells adjacent to the ZP. Millisecond thermal exposure poses risks to embryo viability and could potentially trigger cellular damage through apoptotic pathways.

We have added the following text to the Introduction to highlight the ms-LAH challenges and distinctive advantages of femtosecond lasers:

To minimize the risk of thermal damage, LAH should be performed at an early stage of development (day 2 or 3), when there is sufficient perivitelline space between the z.p. and blastomeres. However, this approach hinders subsequent trophectoderm biopsy due to the inability to control whether the embryo hatches “TE-forward” or “ICM-forward” [29] (here, TE and ICM stand for trophectoderm and inner cell mass, respectively). Furthermore, z.p.’s opening during the late stages of development results in blastocyst collapse for an indefinite period of time, which also complicates TE biopsy.

Visible-wavelength fs lasers have been used successfully in the microsurgery of the z.p. at late blastocyst stages, resulting in increased hatching rates and successful implantation onto an adhesive plastic surface used for modelling in vitro processes [32]. Fs-LAH does not cause blastocyst collapse, significant reactive oxygen species (ROS) production, or heat-shock protein (HSP) overexpression [33]. Thus, the aforementioned fs lasers can serve as a convenient alternative, making the work of embryologists easier and simplifying routine processes in clinical practice.

Reviewer 2 Report

Comments and Suggestions for Authors

Dmitry S. Sitnikov et al. reported an interesting work about the safety test of fs-scale LAH, which was to some degree interesting. The topic of a certain significance nowadays, and might arouse some discussion in its field. Overall, the manuscript was well written. The reviewer suggested a Minor Revision for this paper. Detailed comments:

• How did the authors confirm the selection of the wavelength 1028 and 514 nm? Were there any clinical or experimental evidence?
• The subsection Statistics should be moved to the end of the Method Section.
• Regarding Table 2, were there parallel reproducible tests?
• Table 3 could be set as a Supplementary file.

Author Response

Answers to Reviewers' comments

First of all, we would like to thank the Reviewers for their efforts and time spent in analyzing the manuscript. We have followed their recommendations and provide the responses point by point.

 

Reviewer 2

We thank the Reviewer for deeply delving into the topic of the article.

Dmitry S. Sitnikov et al. reported an interesting work about the safety test of fs-scale LAH, which was to some degree interesting. The topic of a certain significance nowadays, and might arouse some discussion in its field. Overall, the manuscript was well written. The Reviewer suggested a Minor Revision for this paper. Detailed comments:

Q: How did the authors confirm the selection of the wavelength 1028 and 514 nm? Were there any clinical or experimental evidence?

A: Thank you for this interesting question. The choice of wavelengths was determined by the laser radiation source. Yb-doped diode-pumped solid-state lasers became very popular last decade. Equipped with an integrated fiber seed oscillator, they are ideal for industrial applications such as micromachining, as well as for scientific research. The fundamental frequency of the emitted radiation can easily be doubled in a nonlinear crystal, thus shifting the radiation wavelength from the infrared (λ = 1028 nm) to the visible (λ = 514 nm) region of the spectrum. As we found no studies on femtosecond laser microsurgery of the zona pellucida, one of the aims of our paper was to determine the most suitable spectral region for this purpose. Alternative femtosecond laser sources using Ti:sapphire crystals (λ = 780 nm) and Cr:forsterite (λ = 1240 nm) are bulky and are more suited to scientific than industrial or medical applications. Since the manipulation medium for embryos contains no chromophores, such as melanin or hemoglobin, there are no strong limitations on radiation wavelength.

Q: The subsection Statistics should be moved to the end of the Method Section.

A: The text was modified according to this recommendation.

Q: Regarding Table 2, were there parallel reproducible tests?

A: For the data presented in Table 2 (embryo hatching rate), we performed contingency tables 2x2 and Yates's chi-square test, as the data presented in Table 2 are numbered. A detailed description of the current analysis is given in the section Statistics in M&Ms chapter.

Q: Table 3 could be set as a Supplementary file.

A: Thank you for this suggestion. We decided to move the table to the appendix.

 

Reviewer 3 Report

Comments and Suggestions for Authors

This manuscript 

presents a well-designed study evaluating the safety of femtosecond infrared laser application in assisted hatching technology using a mouse embryo model. The work is highly relevant to the field of reproductive biotechnology and laser-assisted microsurgery, offering robust experimental evidence that supports the safety of femtosecond laser use in embryo manipulation. The structure of the paper is clear, and the introduction effectively situates the research within the broader context of assisted reproductive technologies. The detailed description of the materials and methods ensures reproducibility, which is a major strength of the work. The results are clearly presented, and the statistical analyses appear appropriate for the data type and sample sizes used. The discussion is thorough, logically connecting the experimental outcomes with previous literature and emphasizing the biological significance of the findings.

However, some areas could be strengthened to further enhance the scientific clarity and impact of the paper. First, while the study convincingly demonstrates the absence of oxidative and thermal stress responses, the authors may consider addressing the potential for longer-term developmental outcomes, even as a limitation or direction for future research. Second, the comparison between visible and infrared femtosecond wavelengths is very informative but could benefit from a more explicit discussion of how these findings translate to clinical or human embryo applications. The graphical representations of gene expression and ROS data are appropriate but could be improved by standardizing axes and ensuring uniform statistical annotation for ease of interpretation. Additionally, the manuscript would benefit from minor linguistic refinement to improve readability, as several sentences are quite long and dense, which might hinder comprehension for non-specialist readers. Overall, the study makes a valuable contribution to the understanding of laser safety in embryology and supports the feasibility of femtosecond systems in clinical contexts with strong experimental justification.

Author Response

Answers to Reviewers' comments

First of all, we would like to thank the Reviewers for their efforts and time spent in analyzing the manuscript. We have followed their recommendations and provide the responses point by point.

Reviewer 3

We thank the Reviewer for carefully reading and positively evaluating our results.

This manuscript presents a well-designed study evaluating the safety of femtosecond infrared laser application in assisted hatching technology using a mouse embryo model. The work is highly relevant to the field of reproductive biotechnology and laser-assisted microsurgery, offering robust experimental evidence that supports the safety of femtosecond laser use in embryo manipulation. The structure of the paper is clear, and the introduction effectively situates the research within the broader context of assisted reproductive technologies. The detailed description of the materials and methods ensures reproducibility, which is a major strength of the work. The results are clearly presented, and the statistical analyses appear appropriate for the data type and sample sizes used. The discussion is thorough, logically connecting the experimental outcomes with previous literature and emphasizing the biological significance of the findings. However, some areas could be strengthened to further enhance the scientific clarity and impact of the paper.

Q: First, while the study convincingly demonstrates the absence of oxidative and thermal stress responses, the authors may consider addressing the potential for longer-term developmental outcomes, even as a limitation or direction for future research.

A: Thank you for the suggestion. We have extended the paragraph on limitations; it now reads as follows:

A limitation of this study is its focus on HSP and ROS responses exclusively. Future investigations should expand the spectrum of biological effect monitoring to include additional markers, particularly apoptosis indicators, in order to further validate the safety profile of femtosecond LAH procedures. Studying the results of LAH-treated embryo transfers in pseudopregnant mice would help us better understand the embryos’ developmental potential and the long-term effects of fs-LAH treatments. This remains the subject of our ongoing research.

Q: Second, the comparison between visible and infrared femtosecond wavelengths is very informative but could benefit from a more explicit discussion of how these findings translate to clinical or human embryo applications.

A: We have added the following text with our considerations of translating our results to a novel object in clinics —a human embryo.

From the perspective of potential clinical applications of the obtained results, the following considerations can be made. The application of fs-LAH to human embryos has its own peculiarities: i) The human z.p. is wider than that of the mouse model. Therefore, cutting through 80-90% of its thickness is expected to have less impact on adjacent cells than in a mouse embryo. ii) Analyses of the zona pellucida images of human embryos have shown that its structure is more non-uniform. This means there is an increased risk of absorption at these heterogeneities and of optical breakdown. Therefore, it may be advisable to set the intensity closer to the lower limit of the acceptable range, which would further lower the I_max/I_min value for IR radiation. Therefore, using laser radiation in the visible spectral range seems preferable.

Q: The graphical representations of gene expression and ROS data are appropriate but could be improved by standardizing axes and ensuring uniform statistical annotation for ease of interpretation.

A: We standardized the Y-axes in Figure 6 in the range 0-90. Moreover, brief statistical annotations were added at the end of figure captions.

Q: Additionally, the manuscript would benefit from minor linguistic refinement to improve readability, as several sentences are quite long and dense, which might hinder comprehension for non-specialist readers.

A: The article has been professionally proofread using MDPI’s service.

R: Overall, the study makes a valuable contribution to the understanding of laser safety in embryology and supports the feasibility of femtosecond systems in clinical contexts with strong experimental justification.

A: Thank you for your positive evaluation of our results.