The Evolution of the Cryopreservation Techniques in Reproductive Medicine—Exploring the Character of the Vitrified State Intra- and Extracellularly to Better Understand Cell Survival after Cryopreservation

Round 1

Reviewer 1 Report

The manuscript is aimed to compile the available information,  up to date, about cryopreservation and how it works to maintein cell pontential. The review covered from the very begining in this field, until recent times what makes very interesting its reading. Therefore,this work has its own value in the field of animal biotechnology and deserves attention for publication. The paper is well-written with clear descriptions and appropriate figures for better understanding. I would like to highliht the very deep and clear explanations of the processes. Also, to note the vitrification procedure as an alternative to the damage of slow freezing technique seems appropiate to me.

However, the manuscript contains some flaws that need to be clarified before acceptance. In my opinion, the authors should include more references, especially from the "2.6.3.1" section. In addition, clarify the species involved in the processes or events noted in the document should provide context and a better understanding of the explanations. for example: 

line 403: "vitrification is also obtained in the cryopreservation of sperm" --> Vitrification is also obtained in the cryopreservation of equine (Álvarez et al., 2019), goat (Pradieè et al., 2018)... etc. sperm.

The authors should detail deeper the current results in several species and the state of the art in human.

I would like to recommend to organize the number sections into a easier form. To my mind, levels as "2.6.3.1." or "2.6.1" confuses and can not allow following the thread of the work. 

In my opinion this study contributes to further knowledge in its area to conform the journal requirements level.

Author Response

Review 1

The manuscript is aimed to compile the available information,  up to date, about cryopreservation and how it works to maintain cell potential. The review covered from the very begining in this field, until recent times what makes very interesting its reading. Therefore,this work has its own value in the field of animal biotechnology and deserves attention for publication. The paper is well-written with clear descriptions and appropriate figures for better understanding. I would like to highliht the very deep and clear explanations of the processes. Also, to note the vitrification procedure as an alternative to the damage of slow freezing technique seems appropiate to me.

However, the manuscript contains some flaws that need to be clarified before acceptance. In my opinion, the authors should include more references, especially from the "2.6.3.1" section.

 Answer: The section 2.6.3.1 contains two subjects.

The first part is devoted to Intracellular crowding and the colloidal vitrification. This section contains 7 references. In this first paragraph, we include an additional sentence that explain in a more better manner the crowding phenomena that occur in the intracellular compartment.

Additional sentence. “ Osmotic removal of water from the cytoplasm leads to a buildup in the concentration of intracellular solutes and an increase in the packing density of macromolecules.“

The second section is devoted to the revision of the classical equation to obtain a vitrified state. Therefore, in order to clarify in a better way , we add the section 2.6.3.2 Revision of the classical equation on the probability of achieving a vitrifying state

The reason of the additional title is because to our knowledge, this is the first approach revising the classical equation and providing additional parameters that determine the intracellular probability as a function of the degree of dehydration and the concentration of cryoprotectants.

 

In addition, clarify the species involved in the processes or events noted in the document should provide context and a better understanding of the explanations. for example: 

line 403: "vitrification is also obtained in the cryopreservation of sperm" --> Vitrification is also obtained in the cryopreservation of equine (Álvarez et al., 2019), goat (Pradieè et al., 2018)... etc. sperm.

 

Answer: “Vitrification is also obtained in the cryopreservation of sperm, even if the term vitrification applied in this context can be confusing from a methodological point of view”.

This sentence is replaced by “Intracellular vitrification is also present in the cryopreservation of sperm from different species, human (Tao, 2020), equine (Alvarez,2019), even if the term vitrification applied in this context can be confusing from a methodological point of view.

10. Tao , E. Sanger, A. Saewu, MC. Leveille., Human sperm vitrification: the state of the art. Reproductive Biology and Endocrinology. 2020, open access, https://rbej.biomedcentral.com/articles/10.1186/s12958-020-00580-5
11. Álvarez, N. González, V. Luño, L. Gil. Ejaculated compared with epididymal stallion sperm vitrification. Animal Reproduction Science,2019, 211, 106205

The authors should detail deeper the current results in several species and the state of the art in human.

Answer: This is not the objective of this article which is focused mainly on the intracellular and extracellular behaviour according to the vitrification methodology. In a second point, the objective of this paper is to show that all cryopreservation techniques applied to all biological material have as a common point in case of survival, the presence of an intracellular vitrification.

The first section” Cryopreservation: a Field of Emerging Interest” is divided in two part. We add “ in the animal field”

The current results in several species and the state of the art in human may be the subject of a future article. Such manuscript has been already published by our team (See Pubmed Vanderzwalmen, Wirleitner and Panageotidis).

 

I would like to recommend to organize the number sections into a easier form. To my mind, levels as "2.6.3.1." or "2.6.1" confuses and can not allow following the thread of the work.

Answer: We prefer to leave the section number as it is now to keep the explanations in a logical way.

( refer also in Review comments 1)

Chapter 2.6.. is devoted to the exploration of the intracellular compartment. To achieve this objective, we have approached different points in a logical way to arrive at the fact that the intracellular environment changes dynamically as a function of dehydration and exposure to cryoprotectant concentration.

In 2.6.1. it is explained that the vitrification solution allows dehydration of the cell. In 2.6.2. It is shown that the intracellular concentration of cryoprotectant is lower than that of the vitrification solution. Consequently, how can it be explained that with a lower concentration of intracellular cryoprotectant the cell survives?  In section 2.6.3. we present the arguments that explain the absence of intracellular crystallisation. In 2.6.3.1 we report the different studies showing the phenomena of crowding, in 2.6.3.2 we revised the classical equation and add two other parameters and in 2.6.3.3. we report the conclusions of studies showing that the glass transition T° inside the cell occur at a higher T° than outside the cell.

 

In my opinion this study contributes to further knowledge in its area to conform the journal requirements level.

Author Response File: Author Response.docx

Reviewer 2 Report

Reproductive Medicine   Review  8-12-2020

Title:  The Evolution of the Cryopreservation Techniques in Reproductive Medicine – Exploring the Character of the Vitrified State Intra- and Extracellularly to Better Understand Cell Survival after Cryopreservation

Professor Vanderzwalmen and his colleagues have compiled an excellent review of the subject matter as it pertains to differentiating meta-stable glass solidification (i.e., Vitrification) of extra- and intra-cellular compartments and how CPA influence cell survival.  At a time when most of the true pioneers in the field of Cryobiology have sadly departed our mortal presence, Dr. Vanderzwalmen represents one of a few next-generation Embryologists mentored in Cryobiology and capable of teaching todays’ Embryologists the foundation of Applied Cryobiology.  This manuscript educates the reader to the interrelationship between physical & chemical principles of cryopreservation that promote sustained cell survival.

    Reviews on this topic are not uncommon, however, the perspectives, examples and referencing used has offered unique and appropriate insights that I enjoyed learning about.  Overall, I found the paper to be well written and executed with useful figures. In turn, I do find the manuscript to be Acceptable for publication with the inclusion the minor revisions suggested below:

1. First and foremost there are a lot of abbreviations. I suggest the authors stick to conventional formats and possibly eliminated some:
   1. Please define and apply: “cryoprotective agents” as CPA throughout the text
   2. Use “SF” and not “SLF” , but better yet I strongly suggest simply not abbreviating “slow freezing” as it is not being consistency applied in all text as it is now.
   3. Please make sure other abbreviations like H2O and LN2 properly superscript the number character in all text
   4. If other Abbreviations are only used a couple times (e.g., OHSS, POF) I suggested removing them and spell-out
2. To complete the thought process of the useful applications of embryo cryopreservation in the introduction, the authors may want to include a reference to the conservation of wildlife germplasm. A possible reference could include:

Schiewe, MC.  The science and significance of embryo cryopreservation. 

J Zoo Wildlf Med 22:6-22, 1991.  {L42}

3. Some phases/Word uses seem to be excessively used (e.g., Thereby) and I have made suggestions throughout the text to remove or replace them

 

Specific Line revisions:

L45  end sentence after “…..treatment.”  ; Delete “and the” ; Cap “Modern” IVF ….

L45/46  Delete …. “. Thereby ;  Replace with the word “where” cryopreservation of …..

L49  Add “was”  to …gametes was originally implemented…..

L50  Replace “experiences a perpetual extension of” with “and then extended to” indications such as ……

L60   Add comma to …..(e.g., fertility ….

L66  Add “complete” before ….survival;  Replace ……… “one of the” with “a” main reason

L67  Add “s”  to ……cryopreservation techniques……..

L69   Replace …..”lower”  with “low”

L70  Introduce the abbreviation “CPA” defining ……permeable cryoprotective agents

L70/71 confusing word usage …. “Hence, probes are” ….. suggest using “Embryos were”

L73  Delete  “will” and simply state ………cells continue to ….

L76  Replace “may” with “can”   stating …….factors that can occur……

L77  No CAP of “The” ……(i) the intracellular ….

L79  No CAP again and semicolon after …….and;  (ii) the formation ….

L101  Add “did”  and replace “developed”  ….onwards “did” cryobiologists develop ….

L130  Delete space between 0 and the Centigrade symbol

L199  Figure 2.   Add “, the” to read   .  In solutions, the content of salts, …..

L200  Delete cryoprotectants” and simply use “CPA”;  Add “s” to … CPA leads to an …..

L221/L320  there is a coupe places were an acceptable ranges of cooling and warming is inferred as 2,000C/min to 20,000; since we know some closed straw systems have validated 1000 to 1500C/min as effective and other open microdrop method claims rates in excess of 100,000C/min I suggest a new range of 1,000 to 100,000C/min be used

L223  I do not understand the use of the word “solved” before ….salt; Is this correct?

L243  Add comma after ……For example, …..

L278  Replace parenthesis with commas   …was, and still is, the ….

L284  Delete and modify “There we could demonstrate” with “It was demonstrated”….

L284   Replace “around”  with “approximately”

L285  Add “of “ and a Parenthesis  to say ….concentration of the VS solution (6.4M) …

L305  Replace “Thereby, the” viscosity  with “Therefore, viscosity …..

L306 Change “leading” to “leads” ..

L310  This new paragraph needs to describe the subject and can not begin with “This” suggest you modify to “The molecular crowding” hypothesis …..

L311/312  Move “whereas” to ….whereas intracellularly…; CAP “Extracellularly, …; Delete “mainly” ; Add “primarily” before …dictate the to read “Extracelluarly, CPA concentrations primarily dictate the viscosity.  Whereas, intracellular cytoplasmic dehydration “ …..

L312/313  Delete …….”itself is essentially” and replace with “are dynamically” involved..

L313  Replace  “Therefore, “ with “Thus,” the amphorous ….

L316  Replace …”may take to different”  with …”is influenced by cellular” architecture …

L317 Add comma and replace “and” with “with” to say…..architecture, with the ….

L317  Delete “will depends” and “the importance of the” and replace with …… …..vitrification “being dependent” on different factors ….

L327/331 Figure 4 is too wordy,

L327/328 Delete ….…state “(intra and extracellular).  The probability to obtain a vitrified state”  thus merging sentence 1 and 2

L329  Add  “Whereas,” before a lower case “inside”

L330  Delete …..”(IC, crowding) has to be and as another” ; Delete the period at the line end ; Adding …crowding “is an added factor”…….. continuing into a modify L331

L331 Replace “The influence of IC crowding”  with “that influences viscosity, but metastable glass formation” fluctuates…..

L343/347   Replace CP with CPA in Figure 5 and text

L346/347  Delete “cryoprotectants” and the extra period at the end of the sentence

L359  Replace “a” with “achieve” and “to” with “in the”……to achieve successful vitrification in the whole system.

L361  Replace …..is “a main key factor to ensure”  with ….is “the main factor ensuring”

L362  Delete …..material “that has been” subjected to …..

L363   Delete “the”  in …..theory regarding “the” differences…..

L364  Replace “there is also a difference of”  with ….whether “differences also exist in” the cryopreserved states……

L365  Delete “in the” and Add “of”

L369-375  correct formatting to reflect semicolons used before the next  numbered example and the addition of……”; and” (v) …… before the last example

L381  Delete “Thereby,” and do not start a new paragraph. ; CAP “The” term ….

L383/384  Replace …..”applied. This includes also the slow freezing technique. “  with“ applied, including slow freezing processes.”

L385/393  Lower case “v/v”

L391     Replace … ,”and thereby the”  with “, resulting in an” intracellular ….

L400  Please clarify and change : “factor that creates” or “factors that create” an ….

L401/402  Modify to read:  “In slow freezing, the latter occurs due to external ice crystal formation, whereas with vitrification it is” the exposure ……

L403 Suggest modifying beginning sentence to read:  “Vitrification can also be obtained in sperm cryopreservation”, even if ….

L404   Add …… The “kinetic” Vitrification of ….

L405  Replace  “compared to” and “we apply for”  with ….different “than” the protocol “applied to” oocytes ….

L406   Delete …..”in this protocol is” ;  Add ….“are” very low

L407   Replace …..”display” with “possess”

L408   Change ……CPA extracellularly will    with “extracellular CPA do” not …..

L409  Replace “here also”  and “we assume”  with moving “in”  and …...  What can “be assumed” about

L410  Add comma after “In sperm,”

L412  Replace “We have seen here above that cell dehydration is a” common …..with    “Thus, cell dehydration is the” common phenomenon …..

L413  Add ….cryopreservation techniques “which promote cell survival.”

L415  Replace and add  “Understanding” with “Grasping the physical-chemical” principles …… ; Change …to “understand”  to “understanding” how and why

L416  Delete the sentence “Thereby, ………essential.”

L418  Delete “but also” and Add a comma

L419   “amphibians” plural;  Add “to preserve cellular integrity” after ….polysaccharides

L420  Delete “as”

L421  Modify :…and outflow of water.  The result is” to say : ….and “the outflow of water occurs, resulting in an” .....

L422  Add “phenomenon,” after ….cryopreservation

L424   Delete “will”;  Change “different’ to “differently”

L427   Replace “imminent for the” success.  With “imminently factor into” success.

L428   Delete “Thereby,”; Start sentence with “Regardless” of the ……

L429   Delete “can be named.  That is the” and replace with  ….cryopreservation “is the” achievement

L430  Delete  “where”

L431   Add a period after “ultra-fast cooling.”  ;  CAP “The” intracellular

L431  Delete “will” ; Change “vitrified” to “vitrifies”

L434  Change “Further” to “Furthermore,”

L436  Replace “procedure” with “application”; Delete “would not”; and Add …in ART, “for which the success of” many “other” therapies….

L437   Delete “without this technique”;  Replace “understanding of”  with “knowledge questioning”

L438   Replace “is “ with “will be”  and ….understand “and to discuss”  with “understanding, optimizing and discussing the”

L439  Replace “for” with “on” ; make “outcome” plural….”outcomes.”

Author Response

Review 2

 

Tittle:  The Evolution of the Cryopreservation Techniques in Reproductive Medicine – Exploring the Character of the Vitrified State Intra- and Extracellularly to Better Understand Cell Survival after Cryopreservation

Professor Vanderzwalmen and his colleagues have compiled an excellent review of the subject matter as it pertains to differentiating meta-stable glass solidification (i.e., Vitrification) of extra- and intra-cellular compartments and how CPA influence cell survival.  At a time when most of the true pioneers in the field of Cryobiology have sadly departed our mortal presence, Dr. Vanderzwalmen represents one of a few next-generation Embryologists mentored in Cryobiology and capable of teaching todays’ Embryologists the foundation of Applied Cryobiology.  This manuscript educates the reader to the interrelationship between physical & chemical principles of cryopreservation that promote sustained cell survival.

    Reviews on this topic are not uncommon, however, the perspectives, examples and referencing used has offered unique and appropriate insights that I enjoyed learning about.  Overall, I found the paper to be well written and executed with useful figures. In turn, I do find the manuscript to be Acceptable for publication with the inclusion the minor revisions suggested below:

inclusion the minor revisions suggested below:

1. First and foremost there are a lot of abbreviations. I suggest the authors stick to conventional formats and possibly eliminated some:
   1. Please define and apply: “cryoprotective agents” as CPA throughout the text – Answer: was done
   2. Use “SF” and not “SLF” , but better yet I strongly suggest simply not abbreviating “slow freezing” as it is not being consistency applied in all text as it is now. Answer: we used now the abbreviation SF instead of SLF through the whole text
   3. Please make sure other abbreviations like H2O and LN2 properly superscript the number character in all text Answer: was done for LN2, H2O for water should be known as an established abbrevation
   4. If other Abbreviations are only used a couple times (e.g., OHSS, POF) I suggested removing them and spell-out Answer: abbreviation for POF and OHSS were deleted
2. To complete the thought process of the useful applications of embryo cryopreservation in the introduction, the authors may want to include a reference to the conservation of wildlife germplasm. A possible reference could include:

Schiewe, MC.  The science and significance of embryo cryopreservation. J Zoo Wildlf Med 22:6-22, 1991.  {L42}

3. Some phases/Word uses seem to be excessively used (e.g., Thereby) and I have made suggestions throughout the text to remove or replace them

 Answer: We have acknowledged this and modified this according your suggestions

4. Some phases/Word uses seem to be excessively used (e.g., Thereby) and I have made suggestions throughout the text to remove or replace them

 Answer: We have revised the

Specific Line revisions:

L45  end sentence after “…..treatment.”  ; Delete “and the” ; Cap “Modern” IVF ….

Answer: was done (see line 63)

L45/46  Delete …. “. Thereby ;  Replace with the word “where” cryopreservation of ….

Answer: was deleted  (see line 64)

 

L49  Add “was”  to …gametes was originally implemented…..

Answer: was done (see line 64)

 

L50  Replace “experiences a perpetual extension of” with “and then extended to” indications such as ……

Answer: was done (see line 68-69)

L60   Add comma to …..(e.g., fertility …. Answer: was done (see line 78)

 

L66  Add “complete” before ….survival;  Replace ……… “one of the” with “a” main reason

Answer: one was replaced by a (see line 66. The adding of the term “complete” does not make sense from our point of view.

 

L67  Add “s”  to ……cryopreservation techniques……..

Answer: was done (see line 84)

L69   Replace …..”lower”  with “low”  

Answer: was done (see line 87)

L70  Introduce the abbreviation “CPA” defining ……permeable cryoprotective agents Answer: was done (throughout the manuscript)

L70/71 confusing word usage …. “Hence, probes are” ….. suggest using “Embryos were”

Answer: was done (see line 88-89)

L73  Delete  “will” and simply state ………cells continue to ….

Answer: was done (see line 91)

L76  Replace “may” with “can”   stating …….factors that can occur……

Answer: was done (see line 94)

L77  No CAP of “The” ……(i) the intracellular ….

Answer: was corrected (see line 95)

L79  No CAP again and semicolon after …….and;  (ii) the formation ….

 Answer: was corrected (see line 97)

L101  Add “did”  and replace “developed”  ….onwards “did” cryobiologists develop ….

Answer: was done (see line 119)

L130  Delete space between 0 and the Centigrade symbol

Answer: was done (see line 130)

L199  Figure 2.   Add “, the” to read   .  In solutions, the content of salts, …..

 Answer: was done (see line 218)

L200  Delete cryoprotectants” and simply use “CPA”;  Add “s” to … CPA leads to an …..

Answer: was done (see line 219) CPA lead…leads does not make sense in our view as it is referred to the plural form

L221/L320  there is a coupe places were an acceptable ranges of cooling and warming is inferred as 2,000C/min to 20,000; since we know some closed straw systems have validated 1000 to 1500C/min as effective and other open microdrop method claims rates in excess of 100,000C/min I suggest a new range of 1,000 to 100,000C/min be used

Answer: the reviewer is right, there are a few systems below or above the range 2,000-20,000°C/min, however they might be the minority and not applied in routine. Therefor we just inserted a short phrase mentioning the range between 1,000-100,000°C/min (line 239,240)

L223  I do not understand the use of the word “solved” before ….salt; Is this correct? ….. Answer: the term “salt” is now deleted (see line 242)

L243  Add comma after ……For example, ….. Answer: was done (see line 262)

L278  Replace parenthesis with commas   …was, and still is, the …. Answer: was done (see line 297)

L284  Delete and modify “There we could demonstrate” with “It was demonstrated”….

Answer: was done (see line284)

L284   Replace “around”  with “approximately” Answer: was done (see line 303)

L285  Add “of “ and a Parenthesis  to say ….concentration of the VS solution (6.4M) … Answer: was done (see line 304)

L305  Replace “Thereby, the” viscosity  with “Therefore, viscosity …..… Answer: was done (see line 326)

L306 Change “leading” to “leads” .. Answer: was replaced to –which leads (see line 327)

 

L310  This new paragraph needs to describe the subject and can not begin with “This” suggest you modify to “The molecular crowding” hypothesis …..

Answer: This paragraph was revised (see line 332-335)

 

L311/312  Move “whereas” to ….whereas intracellularly…; CAP “Extracellularly, …; Delete “mainly” ; Add “primarily” before …dictate the to read “Extracelluarly, CPA concentrations primarily dictate the viscosity.  Whereas, intracellular cytoplasmic dehydration “ …..

Answer: was corrected (see line 334)

L312/313  Delete …….”itself is essentially” and replace with “are dynamically” involved..

Answer: we did not replace it, because this would alter the meaning.

L313  Replace  “Therefore, “ with “Thus,” the amphorous …. Answer: was done (see line 336)

L316  Replace …”may take to different”  with …”is influenced by cellular” architecture

L317 Add comma and replace “and” with “with” to say…..architecture, with the ….

L317  Delete “will depends” and “the importance of the” and replace with …… …..vitrification “being dependent” on different factors ….

Answer: was corrected (Lines 341-343)

L327/331 Figure 4 is too wordy

Answer: This is now revised

L327/328 Delete ….…state “(intra and extracellular).  The probability to obtain a vitrified state”  thus merging sentence 1 and 2

L329  Add  “Whereas,” before a lower case “inside”

L330  Delete …..”(IC, crowding) has to be and as another” ; Delete the period at the line end ; Adding …crowding “is an added factor”…….. continuing into a modify L331

L331 Replace “The influence of IC crowding”  with “that influences viscosity, but metastable glass formation” fluctuates…..

Answer: was revised (Lines 356-360)

L343/347   Replace CP with CPA in Figure 5 and text

Answer: was done is the complete manuscript and figures

L346/347  Delete “cryoprotectants” and the extra period at the end of the sentence

Answer: was done

L359  Replace “a” with “achieve” and “to” with “in the”……to achieve successful vitrification in the whole system.

Answer: was done (Line 388)

L361  Replace …..is “a main key factor to ensure”  with ….is “the main factor ensuring”

Answer: was done (Line 390)

L362  Delete …..material “that has been” subjected to …..…..

Answer: was done (see line 391)

L363   Delete “the”  in …..theory regarding “the” differences…..

Answer: was done (see line 392)

L364  Replace “there is also a difference of”  with ….whether “differences also exist in” the cryopreserved states……

L365  Delete “in the” and Add “of”

Answer: was done (Lines 390-393)

L369-375  correct formatting to reflect semicolons used before the next  numbered example and the addition of……”; and” (v) …… before the last example

Answer: was done (Lines 398-404)

L381  Delete “Thereby,” and do not start a new paragraph. ; CAP “The” term …. Answer: was done (see line 410)

 

L383/384  Replace …..”applied. This includes also the slow freezing technique. “  with“ applied, including slow freezing processes.”

Answer: was done (Lines 411-413)

L385/393  Lower case “v/v”

Answer: was done (see line 414)

L391     Replace … ,”and thereby the”  with “, resulting in an” intracellular ….

Answer: was done (Line 420)

L400  Please clarify and change : “factor that creates” or “factors that create” an ….

Answer: was done (Line 429)

 

L401/402  Modify to read:  “In slow freezing, the latter occurs due to external ice crystal formation, whereas with vitrification it is” the exposure ……

Answer: The paragraph was revised (Lines 428-434)

L403 Suggest modifying beginning sentence to read:  “Vitrification can also be obtained in sperm cryopreservation”, even if ….

Answer: The paragraph was revised (Lines 428-436)

L404   Add …… The “kinetic” Vitrification of ….

 L405  Replace  “compared to” and “we apply for”  with ….different “than” the protocol “applied to” oocytes ….

L406   Delete …..”in this protocol is” ;  Add ….“are” very low

Answer: We changed the sentence to:” The process of vitrification of spermatozoa is far different compared to the protocols applied for oocytes and embryos, as for sperm CPA is applied in very low concentrations or even absent (see Lines 434-436)”

L407   Replace …..”display” with “possess”

Answer: was done (see line 437)

L408   Change ……CPA extracellularly will    with “extracellular CPA do” not ….. Answer: was done (see line 409)

L409  Replace “here also”  and “we assume”  with moving “in”  and …...  What can “be assumed” about Answer: was done (see line 438)

L410  Add comma after “In sperm,” Answer: was done (see line 440)

L412  Replace “We have seen here above that cell dehydration is a” common …..with    “Thus, cell dehydration is the” common phenomenon ….. Answer: was done (see line 442)

L413  Add ….cryopreservation techniques “which promote cell survival.” Answer: was done (see line 443)

 

L415  Replace and add  “Understanding” with “Grasping the physical-chemical” principles …… ; Change …to “understand”  to “understanding” how and why

L416  Delete the sentence “Thereby, ………essential.”

 

Answer: The sentence was changed to: “Grasping the physical-chemical” principles of cryopreservation is essential for the understanding how and why gametes and embryos survive cryopreservation.” (Lines 445-446)

L418  Delete “but also” and Add a comma. Answer: was done (see line 446)

 

L419   “amphibians” plural;  Add “to preserve cellular integrity” after ….polysaccharides.

L420  Delete “as”

Answer: was done (see line 448-450)

L421  Modify :…and outflow of water.  The result is” to say : ….and “the outflow of water occurs., resulting in an” .....

Answer: was done (Line 451)

L422  Add “phenomenon,” after ….cryopreservation –

Answer: was done (Line 452)

L424   Delete “will”;  Change “different’ to “differently” Answer: was done (see line 454)

L427   Replace “imminent for the” success.  With “imminently factor into” success.

Answer: was revised (Line 457)

 

L428   Delete “Thereby,”; Start sentence with “Regardless” of the ……

L429   Delete “can be named.  That is the” and replace with  ….cryopreservation “is the” achievement 

Answer: Sentence was revised  (Lines 458-462) ” Regardless of the protocol used for cryopreservation, the common denominator for cell survival after cryopreservation is the achievement of an intracellular colloidal vitrified state”

L430  Delete  “where”

L431   Add a period after “ultra-fast cooling.”  ;  CAP “The” intracellular

 

Answer: paragraph was revised  (Lines 458-462) ” In an aqueous solution, a glassy vitrified state can be observed following ultra-fast cooling”

 

L431  Delete “will” ; Change “vitrified” to “vitrifies” Answer: the replacement of vitrified  to vitrifies does not make sense from our point of view as vitrified describes see therefore…

https://onlinelibrary.wiley.com/doi/abs/10.1111/j.1365-2818.1983.tb04225.x

“…..the vitrified state is characterized in the electron diffractogram by two broad rings with radius
corresponding to 0.37 and 0.214nm respectively (Fig. 1, insert)…….. “

L434  Change “Further” to “Furthermore,” Answer: was done (see line 463)

 

L436  Replace “procedure” with “application”; Delete “would not”; and Add …in ART, “for which the success of” many “other” therapies….

Answer: was done (Lines 466-467)

L437   Delete “without this technique”;  Replace “understanding of”  with “knowledge questioning” Answer: was done (see line 488)

L438   Replace “is “ with “will be”  and ….understand “and to discuss”  with “understanding, optimizing and discussing the”

Answer: was done (see line 469)

L439  Replace “for” with “on” ; make “outcome” plural….”outcomes.”

Answer: was replaced (see line 470)- The in depth exploration and understanding of the underlying chemical and physical principles presented in this review will be the basis for understanding, optimizing and discussing the potential harmful effects of cryopreservation on pregnancy and neonatal outcomes.

 

 

.

 

 

We hope that all our revisions will fulfill the reviewer’s satisfaction and we hope that our manuscript can be now suggested for publication it its revised form

 

Author Response File: Author Response.docx

Reviewer 3 Report

accept. The comments of other reviewers should be taken into account. The possibility of printing should be considered, including the linguistic correction. So acceptance but after corrections. 

Author Response

Review 2

 

Tittle:  The Evolution of the Cryopreservation Techniques in Reproductive Medicine – Exploring the Character of the Vitrified State Intra- and Extracellularly to Better Understand Cell Survival after Cryopreservation

Professor Vanderzwalmen and his colleagues have compiled an excellent review of the subject matter as it pertains to differentiating meta-stable glass solidification (i.e., Vitrification) of extra- and intra-cellular compartments and how CPA influence cell survival.  At a time when most of the true pioneers in the field of Cryobiology have sadly departed our mortal presence, Dr. Vanderzwalmen represents one of a few next-generation Embryologists mentored in Cryobiology and capable of teaching todays’ Embryologists the foundation of Applied Cryobiology.  This manuscript educates the reader to the interrelationship between physical & chemical principles of cryopreservation that promote sustained cell survival.

    Reviews on this topic are not uncommon, however, the perspectives, examples and referencing used has offered unique and appropriate insights that I enjoyed learning about.  Overall, I found the paper to be well written and executed with useful figures. In turn, I do find the manuscript to be Acceptable for publication with the inclusion the minor revisions suggested below:

inclusion the minor revisions suggested below:

1. First and foremost there are a lot of abbreviations. I suggest the authors stick to conventional formats and possibly eliminated some:
   1. Please define and apply: “cryoprotective agents” as CPA throughout the text – Answer: was done
   2. Use “SF” and not “SLF” , but better yet I strongly suggest simply not abbreviating “slow freezing” as it is not being consistency applied in all text as it is now. Answer: we used now the abbreviation SF instead of SLF through the whole text
   3. Please make sure other abbreviations like H2O and LN2 properly superscript the number character in all text Answer: was done for LN2, H2O for water should be known as an established abbrevation
   4. If other Abbreviations are only used a couple times (e.g., OHSS, POF) I suggested removing them and spell-out Answer: abbreviation for POF and OHSS were deleted
2. To complete the thought process of the useful applications of embryo cryopreservation in the introduction, the authors may want to include a reference to the conservation of wildlife germplasm. A possible reference could include:

Schiewe, MC.  The science and significance of embryo cryopreservation. J Zoo Wildlf Med 22:6-22, 1991.  {L42}

3. Some phases/Word uses seem to be excessively used (e.g., Thereby) and I have made suggestions throughout the text to remove or replace them

 Answer: We have acknowledged this and modified this according your suggestions

4. Some phases/Word uses seem to be excessively used (e.g., Thereby) and I have made suggestions throughout the text to remove or replace them

 Answer: We have revised the

Specific Line revisions:

L45  end sentence after “…..treatment.”  ; Delete “and the” ; Cap “Modern” IVF ….

Answer: was done (see line 63)

L45/46  Delete …. “. Thereby ;  Replace with the word “where” cryopreservation of ….

Answer: was deleted  (see line 64)

 

L49  Add “was”  to …gametes was originally implemented…..

Answer: was done (see line 64)

 

L50  Replace “experiences a perpetual extension of” with “and then extended to” indications such as ……

Answer: was done (see line 68-69)

L60   Add comma to …..(e.g., fertility …. Answer: was done (see line 78)

 

L66  Add “complete” before ….survival;  Replace ……… “one of the” with “a” main reason

Answer: one was replaced by a (see line 66. The adding of the term “complete” does not make sense from our point of view.

 

L67  Add “s”  to ……cryopreservation techniques……..

Answer: was done (see line 84)

L69   Replace …..”lower”  with “low”  

Answer: was done (see line 87)

L70  Introduce the abbreviation “CPA” defining ……permeable cryoprotective agents Answer: was done (throughout the manuscript)

L70/71 confusing word usage …. “Hence, probes are” ….. suggest using “Embryos were”

Answer: was done (see line 88-89)

L73  Delete  “will” and simply state ………cells continue to ….

Answer: was done (see line 91)

L76  Replace “may” with “can”   stating …….factors that can occur……

Answer: was done (see line 94)

L77  No CAP of “The” ……(i) the intracellular ….

Answer: was corrected (see line 95)

L79  No CAP again and semicolon after …….and;  (ii) the formation ….

 Answer: was corrected (see line 97)

L101  Add “did”  and replace “developed”  ….onwards “did” cryobiologists develop ….

Answer: was done (see line 119)

L130  Delete space between 0 and the Centigrade symbol

Answer: was done (see line 130)

L199  Figure 2.   Add “, the” to read   .  In solutions, the content of salts, …..

 Answer: was done (see line 218)

L200  Delete cryoprotectants” and simply use “CPA”;  Add “s” to … CPA leads to an …..

Answer: was done (see line 219) CPA lead…leads does not make sense in our view as it is referred to the plural form

L221/L320  there is a coupe places were an acceptable ranges of cooling and warming is inferred as 2,000C/min to 20,000; since we know some closed straw systems have validated 1000 to 1500C/min as effective and other open microdrop method claims rates in excess of 100,000C/min I suggest a new range of 1,000 to 100,000C/min be used

Answer: the reviewer is right, there are a few systems below or above the range 2,000-20,000°C/min, however they might be the minority and not applied in routine. Therefor we just inserted a short phrase mentioning the range between 1,000-100,000°C/min (line 239,240)

L223  I do not understand the use of the word “solved” before ….salt; Is this correct? ….. Answer: the term “salt” is now deleted (see line 242)

L243  Add comma after ……For example, ….. Answer: was done (see line 262)

L278  Replace parenthesis with commas   …was, and still is, the …. Answer: was done (see line 297)

L284  Delete and modify “There we could demonstrate” with “It was demonstrated”….

Answer: was done (see line284)

L284   Replace “around”  with “approximately” Answer: was done (see line 303)

L285  Add “of “ and a Parenthesis  to say ….concentration of the VS solution (6.4M) … Answer: was done (see line 304)

L305  Replace “Thereby, the” viscosity  with “Therefore, viscosity …..… Answer: was done (see line 326)

L306 Change “leading” to “leads” .. Answer: was replaced to –which leads (see line 327)

 

L310  This new paragraph needs to describe the subject and can not begin with “This” suggest you modify to “The molecular crowding” hypothesis …..

Answer: This paragraph was revised (see line 332-335)

 

L311/312  Move “whereas” to ….whereas intracellularly…; CAP “Extracellularly, …; Delete “mainly” ; Add “primarily” before …dictate the to read “Extracelluarly, CPA concentrations primarily dictate the viscosity.  Whereas, intracellular cytoplasmic dehydration “ …..

Answer: was corrected (see line 334)

L312/313  Delete …….”itself is essentially” and replace with “are dynamically” involved..

Answer: we did not replace it, because this would alter the meaning.

L313  Replace  “Therefore, “ with “Thus,” the amphorous …. Answer: was done (see line 336)

L316  Replace …”may take to different”  with …”is influenced by cellular” architecture

L317 Add comma and replace “and” with “with” to say…..architecture, with the ….

L317  Delete “will depends” and “the importance of the” and replace with …… …..vitrification “being dependent” on different factors ….

Answer: was corrected (Lines 341-343)

L327/331 Figure 4 is too wordy

Answer: This is now revised

L327/328 Delete ….…state “(intra and extracellular).  The probability to obtain a vitrified state”  thus merging sentence 1 and 2

L329  Add  “Whereas,” before a lower case “inside”

L330  Delete …..”(IC, crowding) has to be and as another” ; Delete the period at the line end ; Adding …crowding “is an added factor”…….. continuing into a modify L331

L331 Replace “The influence of IC crowding”  with “that influences viscosity, but metastable glass formation” fluctuates…..

Answer: was revised (Lines 356-360)

L343/347   Replace CP with CPA in Figure 5 and text

Answer: was done is the complete manuscript and figures

L346/347  Delete “cryoprotectants” and the extra period at the end of the sentence

Answer: was done

L359  Replace “a” with “achieve” and “to” with “in the”……to achieve successful vitrification in the whole system.

Answer: was done (Line 388)

L361  Replace …..is “a main key factor to ensure”  with ….is “the main factor ensuring”

Answer: was done (Line 390)

L362  Delete …..material “that has been” subjected to …..…..

Answer: was done (see line 391)

L363   Delete “the”  in …..theory regarding “the” differences…..

Answer: was done (see line 392)

L364  Replace “there is also a difference of”  with ….whether “differences also exist in” the cryopreserved states……

L365  Delete “in the” and Add “of”

Answer: was done (Lines 390-393)

L369-375  correct formatting to reflect semicolons used before the next  numbered example and the addition of……”; and” (v) …… before the last example

Answer: was done (Lines 398-404)

L381  Delete “Thereby,” and do not start a new paragraph. ; CAP “The” term …. Answer: was done (see line 410)

 

L383/384  Replace …..”applied. This includes also the slow freezing technique. “  with“ applied, including slow freezing processes.”

Answer: was done (Lines 411-413)

L385/393  Lower case “v/v”

Answer: was done (see line 414)

L391     Replace … ,”and thereby the”  with “, resulting in an” intracellular ….

Answer: was done (Line 420)

L400  Please clarify and change : “factor that creates” or “factors that create” an ….

Answer: was done (Line 429)

 

L401/402  Modify to read:  “In slow freezing, the latter occurs due to external ice crystal formation, whereas with vitrification it is” the exposure ……

Answer: The paragraph was revised (Lines 428-434)

L403 Suggest modifying beginning sentence to read:  “Vitrification can also be obtained in sperm cryopreservation”, even if ….

Answer: The paragraph was revised (Lines 428-436)

L404   Add …… The “kinetic” Vitrification of ….

 L405  Replace  “compared to” and “we apply for”  with ….different “than” the protocol “applied to” oocytes ….

L406   Delete …..”in this protocol is” ;  Add ….“are” very low

Answer: We changed the sentence to:” The process of vitrification of spermatozoa is far different compared to the protocols applied for oocytes and embryos, as for sperm CPA is applied in very low concentrations or even absent (see Lines 434-436)”

L407   Replace …..”display” with “possess”

Answer: was done (see line 437)

L408   Change ……CPA extracellularly will    with “extracellular CPA do” not ….. Answer: was done (see line 409)

L409  Replace “here also”  and “we assume”  with moving “in”  and …...  What can “be assumed” about Answer: was done (see line 438)

L410  Add comma after “In sperm,” Answer: was done (see line 440)

L412  Replace “We have seen here above that cell dehydration is a” common …..with    “Thus, cell dehydration is the” common phenomenon ….. Answer: was done (see line 442)

L413  Add ….cryopreservation techniques “which promote cell survival.” Answer: was done (see line 443)

 

L415  Replace and add  “Understanding” with “Grasping the physical-chemical” principles …… ; Change …to “understand”  to “understanding” how and why

L416  Delete the sentence “Thereby, ………essential.”

 

Answer: The sentence was changed to: “Grasping the physical-chemical” principles of cryopreservation is essential for the understanding how and why gametes and embryos survive cryopreservation.” (Lines 445-446)

L418  Delete “but also” and Add a comma. Answer: was done (see line 446)

 

L419   “amphibians” plural;  Add “to preserve cellular integrity” after ….polysaccharides.

L420  Delete “as”

Answer: was done (see line 448-450)

L421  Modify :…and outflow of water.  The result is” to say : ….and “the outflow of water occurs., resulting in an” .....

Answer: was done (Line 451)

L422  Add “phenomenon,” after ….cryopreservation –

Answer: was done (Line 452)

L424   Delete “will”;  Change “different’ to “differently” Answer: was done (see line 454)

L427   Replace “imminent for the” success.  With “imminently factor into” success.

Answer: was revised (Line 457)

 

L428   Delete “Thereby,”; Start sentence with “Regardless” of the ……

L429   Delete “can be named.  That is the” and replace with  ….cryopreservation “is the” achievement 

Answer: Sentence was revised  (Lines 458-462) ” Regardless of the protocol used for cryopreservation, the common denominator for cell survival after cryopreservation is the achievement of an intracellular colloidal vitrified state”

L430  Delete  “where”

L431   Add a period after “ultra-fast cooling.”  ;  CAP “The” intracellular

 

Answer: paragraph was revised  (Lines 458-462) ” In an aqueous solution, a glassy vitrified state can be observed following ultra-fast cooling”

 

L431  Delete “will” ; Change “vitrified” to “vitrifies” Answer: the replacement of vitrified  to vitrifies does not make sense from our point of view as vitrified describes see therefore…

https://onlinelibrary.wiley.com/doi/abs/10.1111/j.1365-2818.1983.tb04225.x

“…..the vitrified state is characterized in the electron diffractogram by two broad rings with radius
corresponding to 0.37 and 0.214nm respectively (Fig. 1, insert)…….. “

L434  Change “Further” to “Furthermore,” Answer: was done (see line 463)

 

L436  Replace “procedure” with “application”; Delete “would not”; and Add …in ART, “for which the success of” many “other” therapies….

Answer: was done (Lines 466-467)

L437   Delete “without this technique”;  Replace “understanding of”  with “knowledge questioning” Answer: was done (see line 488)

L438   Replace “is “ with “will be”  and ….understand “and to discuss”  with “understanding, optimizing and discussing the”

Answer: was done (see line 469)

L439  Replace “for” with “on” ; make “outcome” plural….”outcomes.”

Answer: was replaced (see line 470)- The in depth exploration and understanding of the underlying chemical and physical principles presented in this review will be the basis for understanding, optimizing and discussing the potential harmful effects of cryopreservation on pregnancy and neonatal outcomes.

 

 

.

 

 

We hope that all our revisions will fulfill the reviewer’s satisfaction and we hope that our manuscript can be now suggested for publication it its revised form

 

Author Response File: Author Response.docx