Recent Developments in In Vitro Spermatogenesis and Future Directions

Round 1

Reviewer 1 Report

Thank you for the opportunity to review your manuscript on recent advancements and challenges in in vitro spermatogenesis. This is a very important and rapidly evolving field of study, and your comprehensive review will no doubt be beneficial to many. Below are my comments and suggestions which, if addressed, may enhance the clarity and impact of your manuscript.

1. Title: Your title is clear and to the point, capturing the essence of your manuscript. However, considering that a considerable part of your review discusses future directions and challenges in the field, it might be worth reflecting this in your title as well.

2. Abstract: The abstract provides a succinct summary of your manuscript. However, it would be beneficial if you could add a bit more detail about the novel methods discussed in your paper.

3. Introduction: You've provided a good background of the topic. I would suggest including a brief section explaining why in vitro spermatogenesis is such an important area of research, especially for non-specialist readers.

4. Methods: The detailed discussion on various methods used for in vitro spermatogenesis is impressive. However, it would be useful to briefly explain the principles behind some of the key techniques. This will help readers who may not be familiar with these techniques to understand them better.

5. Results and Discussion: Your critical analysis and discussion of the results from various studies are commendable. However, it would be beneficial to provide explicit context about how these findings are propelling the field forward. It might also be useful to delve deeper into discussing the limitations of these studies.

6. Future Direction: Your insights into the future directions and challenges in the field are well-articulated. Further discussion on the potential impact of solving these challenges, particularly from a clinical perspective, could enhance this section.

7. Conclusion: Your conclusion effectively summarizes the main points of your review. You may consider adding a few lines on the overall significance or potential impact of the research in this field to make it more compelling.

8. References: Please check all your references for accuracy to ensure the readers can reliably trace back the original works.

9. Tables/Figures: The tables and figures in your manuscript are clear and well-presented. It would be helpful if they were referred to more explicitly in the text.

10. Writing Style: While your paper is generally well-written, there are instances where the language could be simplified for accessibility.

11. It would be interesting if you could incorporate studies on in vitro spermatogenesis from non-human models. Comparative insights could inform future research directions in humans and strengthen your manuscript.

In summary, your manuscript is well-structured and offers a comprehensive overview of the current state of in vitro spermatogenesis. Addressing these comments should further enhance the quality and impact of your work. 

Thank you for your contribution to the field, and I look forward to seeing the revised manuscript.

I appreciate your efforts in putting together this comprehensive manuscript. The quality of English is generally good, and your ideas are well-structured and easy to follow. However, there are a few areas where improvements could be made to ensure the language is clear, concise, and accessible to a broad audience. Here are my comments:

1. Grammar and Syntax: There are a few instances of grammatical errors and awkward sentence constructions that disrupt the flow of reading. I would recommend a thorough proofreading of the manuscript to correct these.

2. Terminology: Although the technical terms you have used are accurate and relevant to the topic, their frequent use may be intimidating to readers not intimately familiar with this field. Where possible, consider simplifying the language or provide brief explanations of complex terms.

3. Clarity and Conciseness: Some sentences in your manuscript are quite lengthy and could be broken down into simpler sentences for better readability. Additionally, certain parts of the manuscript contain redundant information which could be omitted to make the text more concise.

4. Consistency: Please ensure that the use of terms, abbreviations, and the tense of verbs is consistent throughout the manuscript. This will greatly enhance the overall quality of your writing.

5. Punctuation: There are a few instances where punctuation marks have been incorrectly used or omitted, which could potentially lead to confusion. Please review these to ensure correct usage.

6. Writing Style: The manuscript is somewhat formal in style, which may be a deterrent for some readers. Consider adopting a more engaging writing style, while still maintaining scientific rigor.

7. Coherence: While your manuscript is largely coherent, there are some sections that need better transitions to smoothly guide the reader from one idea to the next.

To address these issues, it would be beneficial to enlist a professional language editing service or a native English-speaking colleague for a thorough review of the manuscript.

Thank you for your commitment to excellence in scientific writing, and I look forward to reviewing your revised manuscript.

Author Response

We thank each reviewer for their thorough evaluation of our manuscript, and we appreciate the opportunity to revise the manuscript. There are some valuable concerns raised by the reviewers that we have addressed which has improved the quality of the paper. We addressed comments made by reviewers, and here are the responses to each concern raised by each reviewer.

Reviewer 1

1. Title: Your title is clear and to the point, capturing the essence of your manuscript. However, considering that a considerable part of your review discusses future directions and challenges in the field, it might be worth reflecting this in your title as well.
   1. Added and future directions

2. Abstract: The abstract provides a succinct summary of your manuscript. However, it would be beneficial if you could add a bit more detail about the novel methods discussed in your paper.
   1. Following sentence has been added in abstract.
   2. Advances in tissue engineering and regenerative medicine have introduced techniques like ex vivo tissue culture and technologies such as bioreactors, microfluidic systems, and organoids. Bioreactors and microfluidic systems replicate physiological conditions for tissue and cell cultivation, while organoids model organ functionality. Meanwhile, scaffolds, made from various materials, provide essential structural support, guiding the growth and organization of cells into functional tissues.

3. Introduction: You've provided a good background of the topic. I would suggest including a brief section explaining why in vitro spermatogenesis is such an important area of research, especially for non-specialist readers.
   1. Following statement has been added in introduction of ex vivo culuture.
   2. In vitro spermatogenesis is a key research area due to its potential in treating male infertility by artificially inducing sperm maturation outside the body. It can aid young cancer patients who risk sterility from treatments by using previously harvested spermatogonial stem cells to produce sperm later in life. The study of this process also offers valuable insights into developmental biology and genetic disorders. Additionally, it provides a platform for drug and toxicology testing without live subjects. Lastly, there's potential for treating genetic diseases by modifying genes during the process before conception.

4. Methods: The detailed discussion on various methods used for in vitro spermatogenesis is impressive. However, it would be useful to briefly explain the principles behind some of the key techniques. This will help readers who may not be familiar with these techniques to understand them better.
   1. Following statements have been added in the beginning of ex vivo culture, organoid, and scaffold sections.
      1. Ex vivo culture involves maintaining and studying cells or tissues outside of their original organism in a controlled environment.
      2. An organoid is a miniaturized and simplified version of an organ produced in vitro in three-dimensional culture with micro-anatomy. In in vitro differentiation studies, organoids serve as a bridge between traditional two-dimensional cell cultures and whole-organ experiments. They can be derived from stem cells or dissociated tissues and can self-organize into structures that closely resemble the functionality and architecture of organs. In this context, organoids are utilized to study organ development, disease modeling, drug testing, and potentially for regenerative medicine applications, offering a more accurate representation of in vivo conditions than traditional cell culture methods.

• Scaffolds used in in vitro studies offer structural supports that mimic the in vivo environment, aiding cell attachment, proliferation, and differentiation. These scaffolds support three-dimensional cell interactions, guide cell growth, allow efficient nutrient and waste diffusion, and can be modified to release specific growth factors or drugs. In in vitro spermatogenesis research, the extracellular matrix (ECM) maintains the necessary complex interactions for germ cell maturation. Commonly utilized ECM components include collagen, which offers a natural environment for cell adhesion; laminin, which supports cell differentiation and migration; fibronectin, promoting cell attachment and growth; gelatin, often used as a hydrogel for cell encapsulation; and Matrigel, a protein mixture resembling the intricate extracellular environment in tissues. Thus, selecting the right scaffold and ECM components is vital for replicating in vivo conditions and ensuring accurate differentiation studies.

5. Results and Discussion: Your critical analysis and discussion of the results from various studies are commendable. However, it would be beneficial to provide explicit context about how these findings are propelling the field forward. It might also be useful to delve deeper into discussing the limitations of these studies.
   1. The following statement has been added to the conclusion section.
   2. So far, ex vivo organ/tissue culture has achieved full spermatogenesis in vitro in non-rodent species (Figure 3). However, even with an ex vivo organ/tissue culture system, it is not certain that continuous spermatogenesis can be achieved in vitro. Since the ex vivo organ/tissue culture system is the oldest approach to in vitro spermatogenesis, the newest approaches, such as organoids, bioprinting, and microfluidics, will only improve the differentiation efficiency.

6. Future Direction: Your insights into the future directions and challenges in the field are well-articulated. Further discussion on the potential impact of solving these challenges, particularly from a clinical perspective, could enhance this section.
   1. The following statement has been added to the conclusion section.
   2. The research on in vitro spermatogenesis holds transformative potential across various domains of medicine, social sciences, and ethics. Its most immediate application is in the realm of reproductive medicine, where it could provide new avenues for treating male infertility. By creating mature sperm cells outside the body, men with low sperm counts, poor sperm quality, or even those rendered infertile due to medical treatments could have an alternative means to father biological children. Additionally, it could be invaluable for preserving the fertility of young men undergoing treatments like chemotherapy, which may compromise their reproductive capabilities. This could alleviate not just the physical but also the emotional and psychological challenges that come with infertility, offering hope to millions of families. The research also has significant implications for drug discovery and toxicology testing. Being able to generate sperm in vitro would allow for high-throughput screening of substances that could affect male fertility, speeding up the safety profiling of new drugs and identifying potential reproductive hazards among existing ones. The overall significance of in vitro spermatogenesis research is immense. It stands to revolutionize reproductive medicine, contribute significantly to genetic research, and offer new pathways in drug discovery.
7. Conclusion: Your conclusion effectively summarizes the main points of your review. You may consider adding a few lines on the overall significance or potential impact of the research in this field to make it more compelling.
   1. The above statement has been added to the conclusion section.

We also consulted a professional editing service here at UGA to address comments on the quality of the English Language. We highlighted the edited parts.

Author Response File: Author Response.docx

Reviewer 2 Report

This review provides a nice summary of the advances in in vitro gametogenesis that is well-written, organized appropriately, and has sufficient depth in each section.  The focus on in vitro rather than overall spermatogenesis is soundly worked around by referencing recent reviews on the latter topics to remove redundancy.

However, I do like the overview of male germ cell development and spermatogenesis that is included.  It would be good to include in the text or legend if a specific species is intended to be the subject of the illustrated pathways, or if it's general and conserved pathways among all mammals, etc.

The text does do a good job describing species differences.  However, the comparison creates a few tricky nomenclature issues.  For example, on lines 153-55, perhaps it might be better to use protein names (unless existing data is only from mRNA profiling) as showing human gene names, but mentioning mouse expression is confusing.

However, in general, the manuscript does pay good attention to nomenclature rules and the current gene names, etc.

There were a few references at the introduction that I was curious about the senior authors but they were truncated by et al requiring me to go to Pubmed.  You might consider using one of the NIH-style EndNote formatting options that include all authors unless the journal requires truncation.

It is probably a PDF issue, but the first column of Table 1 should be formatted so that the final letter of the label such as "human" doesn't go on a line by itself.

The attention to species other than just mice and man is welcomed.

Author Response

We thank each reviewer for their thorough evaluation of our manuscript, and we appreciate the opportunity to revise the manuscript. There are some valuable concerns raised by the reviewers that we have addressed which has improved the quality of the paper. We addressed comments made by reviewers, and here are the responses to each concern raised by each reviewer.

Reviewer 2

This review provides a nice summary of the advances in in vitro gametogenesis that is well-written, organized appropriately, and has sufficient depth in each section.  The focus on in vitro rather than overall spermatogenesis is soundly worked around by referencing recent reviews on the latter topics to remove redundancy.

We thank you for the kind words.

However, I do like the overview of male germ cell development and spermatogenesis that is included.  It would be good to include in the text or legend if a specific species is intended to be the subject of the illustrated pathways, or if it's general and conserved pathways among all mammals, etc.

The diagram (Figure 1) is intended for general and conserved pathways. We added

“:conserved germ cell developmental markers are presented in Figure 1” in line 84 of the manuscript

“Conserved expression markers during male” on the figure legend of Figure1.

The text does do a good job describing species differences.  However, the comparison creates a few tricky nomenclature issues.  For example, on lines 153-55, perhaps it might be better to use protein names (unless existing data is only from mRNA profiling) as showing human gene names, but mentioning mouse expression is confusing.

The referenced study (reference 49) only looked at gene expressions. We followed how authors used the names on the paper: they used human nomenclature when referring to genes expressed in all three and only used mouse gene names when only referring to mouse genes.

However, in general, the manuscript does pay good attention to nomenclature rules and the current gene names, etc.

There were a few references at the introduction that I was curious about the senior authors but they were truncated by et al requiring me to go to Pubmed.  You might consider using one of the NIH-style EndNote formatting options that include all authors unless the journal requires truncation.

We followed MDPI style for the publication.

It is probably a PDF issue, but the first column of Table 1 should be formatted so that the final letter of the label such as "human" doesn't go on a line by itself.

The table should be inserted into the publication with no truncation.

The attention to species other than just mice and man is welcomed.

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

Accept in present form