RNA Transcripts in Human Ovarian Cells: Two-Time Cryopreservation Does Not Affect Developmental Potential

Round 1

Reviewer 1 Report

Zhou et al. has nicely depicted the results of RNA transcription in human ovarian cells and how cryopreservation affects it. 

One of the major concerns is that there is no validation of any single result from the data obtained in silico. The authors should validate some gene targets as this would help to further consolidate the findings that they see. 

In the result section 2.1, it is a little bit confusing how the authors have phrased the findings. They should make it more clear with the language and also do some changes in the figure to clearly depict which groups are being compared. 

in section 2.2, rephrase the first sentence. It is not very clear. 

There are some sentences that are not grammatically correct and they should be rephrased - line 177-178, 273-274. Check properly for more corrections as well.  

The panels in figure 2 are not aligned properly. 

It is very difficult to read the gene names in figure 3. 

In section 2.5, authors say that the average loss of follicles was observed after 7 days of in vitro culture in group 2 in contrast with follicles of group 1 and then they comment that it is only a tendency as the differences are not statistically significant. It is not correct to even comment on the tendency as these groups are not significantly different to each other. Either they should show it by some other method or rephrase the sentence to not claim on the tendencies. 

Line 306 starting with nuclear and .... - it is not clear what the authors want to say. 

The section 4.5  should be rephrased so that it is easier to understand. 

The authors should check for the abbreviations in the text - sometimes they say DEG and sometimes DGE. 

The conclusions should be elaborated a little bit more to clearly show the message of the study. 

Author Response

Reviewer1

Zhou et al. has nicely depicted the results of RNA transcription in human ovarian cells and how cryopreservation affects it. 

Answer of authors:

Thank you very much for your careful work with our manuscript. We accept all your remarks and respective changes were done in the text.

1. One of the major concerns is that there is no validation of any single result from the data obtained in silico. The authors should validate some gene targets as this would help to further consolidate the findings that they see. 

Answer of authors:

Yes, you are right, but less ovarian tissue was collected at that time. To ensure sufficient RNA quality for sequencing, there was no remaining ovarian tissue to verify the expression of the target gene.  And actually, for the data obtained from transcriptome sequencing, we have already submitted three samples for each group, and all the experiments were repeated for three times. Thank you very much for your valuable suggestions.

2. In the result section 2.1, it is a little bit confusing how the authors have phrased the findings. They should make it more clear with the language and also do some changes in the figure to clearly depict which groups are being compared. 

Answer of authors:

Thank you very much for your valuable comments. This part has been rewritten, and the legend to FIGURE1 has been rewritten eaders can understand it clearly as follows:

At the beginning of our research DEGs analysis was performed. Compared to Group1(one-time cryopreserved), 85 genes were differentially expressed, among them 79 genes were up-regulated, and 6 genes were down-regulated in Group 2(two-time cryopreservation) (FIgure.1A).Then we compared Group3(one-time cryopreserved and in vitro cultured cells) to Group1, there are 2084 genes upregulated and 2340 genes downregulated (Figure.1B). Compared to Group2, 4307 genes were upregulated and 4201 genes were downregulated in Group4 (two-time cryopreservation and in vitro cultured cells) (Figure.1C)

3. in section 2.2, rephrase the first sentence. It is not very clear. 

Answer of authors:

Thank you. We have deleted the first sentence: “Through the enrichment analysis of KEGG pathways, the expression of differential genes can beter understood.”

4. There are some sentences that are not grammatically correct and they should be rephrased - line 177-178, 273-274. Check properly for more corrections as well.  

Answer of authors:

Thank you. We have rewritten these two sentences.

177-178: It were analyzed functions of biological process (BP), cellular component (CC), and molecular function (MF). 

273-274: Protein-protein interactions (PPI) among DEGs were performed using the STRING database. It was firstly ranked the DEGs for determination of their P-value significance. Then top genes were adopted for subsequent analysis.

5. The panels in figure 2 are not aligned properly. 

Answer of authors:

Thanks for your suggestion, we have modified FIGURE2 to be more aligne.

It is very difficult to read the gene names in figure 3. 

Answer of authors:

Original Figure 2 and 3 are attached separately from the text. After acceptance of our manuscript, the production department of MDPI will published these figures with high resolution. If it will be not possible, they will contact us and we will put these two figures in the text as well as AS SUPPLEMENTED MATERIALS. These figures are only for evaluation of reviewers.

6. In section 2.5, authors say that the average loss of follicles was observed after 7 days of in vitro culture in group 2 in contrast with follicles of group 1 and then they comment that it is only a tendency as the differences are not statistically significant. It is not correct to even comment on the tendency as these groups are not significantly different to each other. Either they should show it by some other method or rephrase the sentence to not claim on the tendencies. 

Answer of authors:

Thank you. It is accepted. Now we have deleted the info about tendency as not clear.

This figure is not a table or a graph and it is not necessary to present a statistical treatment

7. Line 306 starting with nuclear and .... - it is not clear what the authors want to say. 

Answer of authors:

What we want to express is that after two times of cryopreservation, the ovarian tissue is still in good condition after thawing, and can be developed into an artificial ovary Now it is written: "Nuclear and cytoplasmic STAINING…

8. The section 4.5  should be rephrased so that it is easier to understand. 

Answer of authors:

Thank you for your suggestion. We have rewritten this part“Twelve ovarian tissue samples were used for RNA extraction. It was detected which cells were used for library preparation with an Illumina compatible kit and sequencing by the DNBSEQ platform [19]. The raw data of this project (in fq.gz format) can be found in the Sequence Read Archive of the National Center for Biotechnology Information (Project No:  PRJNA932020 (http://www.ncbi.nlm.nih.gov/bioproject/932020 accessed date: 07 02 2023). Raw data are kind of original data of transcriptome sequencing. 

The original sequenced reads or raw reads obtained by sequencing contain low-quality reads with adapters. In order to ensure the quality of information analysis, raw reads must be filtered to obtain clean reads. Subsequent analysis is based on clean reads. The filtering procedure was as follows: (1) removing reads containing adapters; (2) removing reads containing N > 10% (N repre-sents bases that could not be determined); (3) remove low-quality reads. The Q score (quality value) of >50% of the bases of the read is ≤5”

Here we would like to also mention that the raw data from transcriptome sequencing samples can only be analyzed when matched with existing bioinformatics data to provide scientists with useful information to draw novel scientific conclusions. So, this section described a common protocol for getting transcriptome data.

9. The authors should check for the abbreviations in the text - sometimes they say DEG and sometimes DGE. 

Answer of authors:

Thank you very much for reading the article carefully, we have checked the full text abbreviations to “DEGs”.

10. The conclusions should be elaborated a little bit more to clearly show the message of the study. 

Answer of authors:

We fully agree with your suggestion and have substantially rewritten the conclusion section as well as in abstract:

Analysis of gene expression in cryopreserved ovarian cells indicates that two-time (repeated) cryopreservation does not affect significantly the developmental potential of these cells. For medical reasons, when ovarian tissue is thawed but cannot be transplanted, it can be immediately re-frozen again.

Reviewer2

This paper analysis the transcript changes for two-time cryopreservation. It’s an interesting study.

Below are some comments that will improve the quality of this manuscript.

Authors: Thank you very much for reading this article carefully. We have carefully revised your suggestion point by point as follows.

1. There are too many paragraphs of the introduction part, I suggest to merge some paragraphs.

Thank you. We have revised the introduction section to focus on the general steps of tissue cryopreservation and the different methods of freezing and why we need to do a second time freezing in some cases. The aim of this paper is therefore to explore whether the quality of second time frozen ovarian tissue is compromised and whether there are subsequent growth and developmental defects.

2. The length-to-width ratio of Figure 1D,E,F is peculiar.

Answer of authors:

We have adjusted the legend scale of FIGURE1.

I would like also to underline that Original Figure 1 is attached separately from the text. After acceptance of our manuscript, the production department of MDPI will published these figures with high resolution. If it will be not possible, they will contact us and we will put these two figures in the text as well as AS SUPPLEMENTED MATERIALS. These figures are only for evaluation of reviewers.

3. The authors should provide more details about some analysis software, such as GO, KEGG, and PPI analysis.

Thank you.

We describe what is GO, KEGG, and PPI on Page17, before Conclusion:

“GO analysis has been widely used in the field of bioinformatics, which covers three aspects of biology: cellular components, molecular functions, and biological processes. KEGG is an annotation for understanding the advanced functions and utilities of cells and organisms.

Differential genes from each group were also analyzed using Protein-protein interaction (PPI), which could systematically analyze the interaction relationships of a large number of proteins in biological systems, which is important for understanding how proteins work in samples, understanding the response mechanisms of biological signals and energy-matter metabolism in specific freezing conditions, and understanding the functional connections between proteins.”

Then normally, it is recommended to use KOBAS-I intelligence tools for KEGG online analysis. And “clusterProfiler” package in R for GO analysis. PPI analysis was based on the STRING database, with known and Predicted Protein-Protein Interactions.

Of course, there are many other methods to do GO, KEGG, and PPI analysis. Most scientists would like to choose R and R studio for data analysis, which is open access language with many useful packages with free of charge. For detailed GO, KEGG or PPI analysis using R language with representative data packages, kind please search teaching video via google.

4. Some abbreviations are explained more than once, please check through the paper.

Answer of authors:

Thanks for your suggestion, which shown that demonstrates that you have read our manuscriot very carefully. I can explain why we have repeated the means of some abbreviatios more than one time.

I have done it especially.

Yes, by guidelines of majority of journals an explanation of abbreviation must be presented one time only, for example in Materials and Methods. However, I know exactly that majority of readers begin to read the article not from Introduction, but from Conclusion, Figures with legends or from Discussion. In fact, the article is written for readers, not only for authors of guidelines. In accordance with this point of view, sections of article must have a certain autonomy. It will be "comfortable" for Readers. That is way, I have repeated explanation of abbreviations more than one time. In the same time, some "popular" abbreviations need not to be explained (for example, DNA).

Reviewer 2 Report

This paper analysis the transcript changes for two-time cryopreservation. It’s an interesting study.

Below are some comments that will improve the quality of this manuscript.

1.     There are too many paragraphs of the introduction part, I suggest to merge some paragraphs.

2.     The length-to-width ratio of Figure 1D,E,F is peculiar.

3.     The authors should provide more details of some analysis software, such as GO, KEGG, and PPI analysis.

4.     Some abbreviations are explained more than once, please check through the paper.

Author Response

Reviewer1

Zhou et al. has nicely depicted the results of RNA transcription in human ovarian cells and how cryopreservation affects it. 

Answer of authors:

Thank you very much for your careful work with our manuscript. We accept all your remarks and respective changes were done in the text.

1. One of the major concerns is that there is no validation of any single result from the data obtained in silico. The authors should validate some gene targets as this would help to further consolidate the findings that they see. 

Answer of authors:

Yes, you are right, but less ovarian tissue was collected at that time. To ensure sufficient RNA quality for sequencing, there was no remaining ovarian tissue to verify the expression of the target gene.  And actually, for the data obtained from transcriptome sequencing, we have already submitted three samples for each group, and all the experiments were repeated for three times. Thank you very much for your valuable suggestions.

2. In the result section 2.1, it is a little bit confusing how the authors have phrased the findings. They should make it more clear with the language and also do some changes in the figure to clearly depict which groups are being compared. 

Answer of authors:

Thank you very much for your valuable comments. This part has been rewritten, and the legend to FIGURE1 has been rewritten eaders can understand it clearly as follows:

At the beginning of our research DEGs analysis was performed. Compared to Group1(one-time cryopreserved), 85 genes were differentially expressed, among them 79 genes were up-regulated, and 6 genes were down-regulated in Group 2(two-time cryopreservation) (FIgure.1A).Then we compared Group3(one-time cryopreserved and in vitro cultured cells) to Group1, there are 2084 genes upregulated and 2340 genes downregulated (Figure.1B). Compared to Group2, 4307 genes were upregulated and 4201 genes were downregulated in Group4 (two-time cryopreservation and in vitro cultured cells) (Figure.1C)

3. in section 2.2, rephrase the first sentence. It is not very clear. 

Answer of authors:

Thank you. We have deleted the first sentence: “Through the enrichment analysis of KEGG pathways, the expression of differential genes can beter understood.”

4. There are some sentences that are not grammatically correct and they should be rephrased - line 177-178, 273-274. Check properly for more corrections as well.  

Answer of authors:

Thank you. We have rewritten these two sentences.

177-178: It were analyzed functions of biological process (BP), cellular component (CC), and molecular function (MF). 

273-274: Protein-protein interactions (PPI) among DEGs were performed using the STRING database. It was firstly ranked the DEGs for determination of their P-value significance. Then top genes were adopted for subsequent analysis.

5. The panels in figure 2 are not aligned properly. 

Answer of authors:

Thanks for your suggestion, we have modified FIGURE2 to be more aligne.

It is very difficult to read the gene names in figure 3. 

Answer of authors:

Original Figure 2 and 3 are attached separately from the text. After acceptance of our manuscript, the production department of MDPI will published these figures with high resolution. If it will be not possible, they will contact us and we will put these two figures in the text as well as AS SUPPLEMENTED MATERIALS. These figures are only for evaluation of reviewers.

6. In section 2.5, authors say that the average loss of follicles was observed after 7 days of in vitro culture in group 2 in contrast with follicles of group 1 and then they comment that it is only a tendency as the differences are not statistically significant. It is not correct to even comment on the tendency as these groups are not significantly different to each other. Either they should show it by some other method or rephrase the sentence to not claim on the tendencies. 

Answer of authors:

Thank you. It is accepted. Now we have deleted the info about tendency as not clear.

This figure is not a table or a graph and it is not necessary to present a statistical treatment

7. Line 306 starting with nuclear and .... - it is not clear what the authors want to say. 

Answer of authors:

What we want to express is that after two times of cryopreservation, the ovarian tissue is still in good condition after thawing, and can be developed into an artificial ovary Now it is written: "Nuclear and cytoplasmic STAINING…

8. The section 4.5  should be rephrased so that it is easier to understand. 

Answer of authors:

Thank you for your suggestion. We have rewritten this part“Twelve ovarian tissue samples were used for RNA extraction. It was detected which cells were used for library preparation with an Illumina compatible kit and sequencing by the DNBSEQ platform [19]. The raw data of this project (in fq.gz format) can be found in the Sequence Read Archive of the National Center for Biotechnology Information (Project No:  PRJNA932020 (http://www.ncbi.nlm.nih.gov/bioproject/932020 accessed date: 07 02 2023). Raw data are kind of original data of transcriptome sequencing. 

The original sequenced reads or raw reads obtained by sequencing contain low-quality reads with adapters. In order to ensure the quality of information analysis, raw reads must be filtered to obtain clean reads. Subsequent analysis is based on clean reads. The filtering procedure was as follows: (1) removing reads containing adapters; (2) removing reads containing N > 10% (N repre-sents bases that could not be determined); (3) remove low-quality reads. The Q score (quality value) of >50% of the bases of the read is ≤5”

Here we would like to also mention that the raw data from transcriptome sequencing samples can only be analyzed when matched with existing bioinformatics data to provide scientists with useful information to draw novel scientific conclusions. So, this section described a common protocol for getting transcriptome data.

9. The authors should check for the abbreviations in the text - sometimes they say DEG and sometimes DGE. 

Answer of authors:

Thank you very much for reading the article carefully, we have checked the full text abbreviations to “DEGs”.

10. The conclusions should be elaborated a little bit more to clearly show the message of the study. 

Answer of authors:

We fully agree with your suggestion and have substantially rewritten the conclusion section as well as in abstract:

Analysis of gene expression in cryopreserved ovarian cells indicates that two-time (repeated) cryopreservation does not affect significantly the developmental potential of these cells. For medical reasons, when ovarian tissue is thawed but cannot be transplanted, it can be immediately re-frozen again.

Reviewer2

This paper analysis the transcript changes for two-time cryopreservation. It’s an interesting study.

Below are some comments that will improve the quality of this manuscript.

Authors: Thank you very much for reading this article carefully. We have carefully revised your suggestion point by point as follows.

1. There are too many paragraphs of the introduction part, I suggest to merge some paragraphs.

Thank you. We have revised the introduction section to focus on the general steps of tissue cryopreservation and the different methods of freezing and why we need to do a second time freezing in some cases. The aim of this paper is therefore to explore whether the quality of second time frozen ovarian tissue is compromised and whether there are subsequent growth and developmental defects.

2. The length-to-width ratio of Figure 1D,E,F is peculiar.

Answer of authors:

We have adjusted the legend scale of FIGURE1.

I would like also to underline that Original Figure 1 is attached separately from the text. After acceptance of our manuscript, the production department of MDPI will published these figures with high resolution. If it will be not possible, they will contact us and we will put these two figures in the text as well as AS SUPPLEMENTED MATERIALS. These figures are only for evaluation of reviewers.

3. The authors should provide more details about some analysis software, such as GO, KEGG, and PPI analysis.

Thank you.

We describe what is GO, KEGG, and PPI on Page17, before Conclusion:

“GO analysis has been widely used in the field of bioinformatics, which covers three aspects of biology: cellular components, molecular functions, and biological processes. KEGG is an annotation for understanding the advanced functions and utilities of cells and organisms.

Differential genes from each group were also analyzed using Protein-protein interaction (PPI), which could systematically analyze the interaction relationships of a large number of proteins in biological systems, which is important for understanding how proteins work in samples, understanding the response mechanisms of biological signals and energy-matter metabolism in specific freezing conditions, and understanding the functional connections between proteins.”

Then normally, it is recommended to use KOBAS-I intelligence tools for KEGG online analysis. And “clusterProfiler” package in R for GO analysis. PPI analysis was based on the STRING database, with known and Predicted Protein-Protein Interactions.

Of course, there are many other methods to do GO, KEGG, and PPI analysis. Most scientists would like to choose R and R studio for data analysis, which is open access language with many useful packages with free of charge. For detailed GO, KEGG or PPI analysis using R language with representative data packages, kind please search teaching video via google.

4. Some abbreviations are explained more than once, please check through the paper.

Answer of authors:

Thanks for your suggestion, which shown that demonstrates that you have read our manuscriot very carefully. I can explain why we have repeated the means of some abbreviatios more than one time.

I have done it especially.

Yes, by guidelines of majority of journals an explanation of abbreviation must be presented one time only, for example in Materials and Methods. However, I know exactly that majority of readers begin to read the article not from Introduction, but from Conclusion, Figures with legends or from Discussion. In fact, the article is written for readers, not only for authors of guidelines. In accordance with this point of view, sections of article must have a certain autonomy. It will be "comfortable" for Readers. That is way, I have repeated explanation of abbreviations more than one time. In the same time, some "popular" abbreviations need not to be explained (for example, DNA).