The Effect of In Vitro Maturation (IVM) Protocol Changes on Measures of Oocyte/Embryo Competence

Round 1

Reviewer 1 Report

The article entitled:

The effect of in vitro maturation (IVM) protocol changes on measures of oocyte/embryo competence has used a modified protocol for oocyte collection and better embryo production.

The article is suitable for publication with minor revision.

Introduction

Lines 41 add the full words before the abbreviation at the first mention (hCG, FSH)

Materials and Methods section

Lines 82,85,86,88 please add the full words before the abbreviation at the first mention (AMH,BMI,PCO,ASRM)

Line 85 which protocol you used to score the BMI

Lines 96-102 Please add more details on the method of administration of letrozole, FSH, hCG. Add the company manufacturer of letrozole. Add a line diagram on the timing of each drug.

Line 100 start the sentence with Thirty -eight hours prior to oocyte retrival, hCG  was given (add the dose  and the unit used; Ovidrel, Serono, Merck, Darmstadt, Germany

 )

Lines 109-113 add the citation of the paragraph and move to the introduction section

Line 143 add the full words of ICSI

Results

Line 176 add the full words of OHSS

DISCUSSION

lines 212-214

please check the abbreviation of ICSI and correct the improper one

Please use the past tense throughout the discussion section you are discussing your results with previously published ones for examples Lines 232-233

Author Response

Lines 41 add the full words before the abbreviation at the first mention (hCG, FSH)

This has been done.

Lines 82,85,86,88 please add the full words before the abbreviation at the first mention (AMH,BMI,PCO,ASRM)

This has been done.

Line 85 which protocol you used to score the BMI

This has been done.

Lines 96-102 Please add more details on the method of administration of letrozole, FSH, hCG. Add the company manufacturer of letrozole. Add a line diagram on the timing of each drug.

This has been done. The timing of drug administration is now illustrated in Table 1.

Line 100 start the sentence with Thirty -eight hours prior to oocyte retrival, hCG  was given (add the dose  and the unit used; Ovidrel, Serono, Merck, Darmstadt, Germany)

This has been done.

Lines 109-113 add the citation of the paragraph and move to the introduction section

This has been done.

Line 143 add the full words of ICSI

This has been done.

Line 176 add the full words of OHSS

This has been done.

lines 212-214

please check the abbreviation of ICSI and correct the improper one

This has been done.

Please use the past tense throughout the discussion section you are discussing your results with previously published ones for examples Lines 232-233

This has been done.

Reviewer 2 Report

This paper needs strict editing work. The lecture was exhausting and fastidious.

Introduction:

 Lines 63-64: This sentence is not clear. What does the author mean by endometrial environments? Is it the endometrial environment located in the patients? Or is it a chemistry medium for embryos?

 Lines 65-68: rewrite this paragraph; the idea is unclear.

Lines 73-78: The objective is inconclusive. For what do the authors use these protocols? What are the variables in question? What is the research question? It is unclear what problem the author wants to resolve or what the knowledge that tries to find.

Material and Methods

Line 85: what is the meaning of BMI abbreviation?

Line 88: what is the meaning of SRM abbreviation?

I suggest a figure with a diagram of the protocols used in the research

Figure 1 is unnecessary

If unclear if the use of the needle was a variable of the study. If the needle was variable, the author must show an experiment where they compare this needle with the conventional needle. This is very important to clarify

Results

Lines 163-167: what was the control group? The author must show the results of the control group. Did the authors compare the results with a control group? What statistical test was used to find the significance? What was the p-value?

I observed a defective results section. These results are inconclusive.

The author should show a table or graphic with the results compared with the control group

Discussion:

Line 180: was prima facie used to compare the author's protocol?

Line 181-187: rewrite this paragraph. It is unclear

The author did not discuss their results

Line 227:  Table 1 is not a result of this research. It is unnecessarily put as a table.

In general, the discussion is not a discussion of the results obtained in the research. The author did not cite other studies with letrozole to compare their results. Also gave more importance to the needle than the protocol of letrozole. Suppose they pretend to evaluate the needle as a variable. In that case, the authors need an experiment comparing their results with a conventional needle.

Author Response

Thank you for reviewing our paper.

We respectfully have to disagree with some of the points that you made.  As indicated below, we have incorporated changes to some of the other issues.

The first concern I would like to address is the request for "extensive editing of the English language and style" used in this submission.  Both authors are native English speakers and have collectively published more than 50 peer-reviewed papers in English language journals.  We have not encountered this complaint before.  In fact, a number of referees have commented that our papers are well written.

This submission was also reviewed by a non-physician English speaking colleague whose job, in part, involves writing reports for her corporation in English. She also did not see any issues with the use of English or style of writing employed.

 Lines 63-64: This sentence is not clear. What does the author mean by endometrial environments? Is it the endometrial environment located in the patients? Or is it a chemistry medium for embryos?

We are referring to endometria in uterine environments and have now made that clear in the manuscript.

Lines 65-68: rewrite this paragraph; the idea is unclear.

We are referring to endometria in uterine environments and have now made that clear in the manuscript.

Lines 73-78: The objective is inconclusive. For what do the authors use these protocols? What are the variables in question? What is the research question? It is unclear what problem the author wants to resolve or what the knowledge that tries to find.

We have made this clearer as in what follows: "The objective of this paper was to evaluate oocyte and embryo competence measures after employing a defined protocol that utilizes all three of the forestated modifications of more common approaches to IVM, to determine if these measures were consistent with expectations after conventional IVF.  Again, these three modifications were,..."

Line 85: what is the meaning of BMI abbreviation?

This is now explained.

Line 88: what is the meaning of SRM abbreviation?

This is now explained.

I suggest a figure with a diagram of the protocols used in the research

This is now Table 1.

Figure 1 is unnecessary

This has been deleted.

If unclear if the use of the needle was a variable of the study. If the needle was variable, the author must show an experiment where they compare this needle with the conventional needle. This is very important to clarify.

The needle is not a variable in this study.  It simply makes it easier to control the location of the oocyte and get it into the laboratory more quickly.  I have clarified this in the discussion.

"Note that the benefits attributed to the use of the Steiner-Tan needle may also be obtained using a standard double lumen needle.   The surgeon needs to copiously flush each follicle and remain aware of the possible location of the retrieved oocyte at all times."

Lines 163-167: what was the control group? The author must show the results of the control group. Did the authors compare the results with a control group? What statistical test was used to find the significance? What was the p-value?...These results are inconclusive. The author should show a table or graphic with the results compared with the control group.

We took great pains in this manuscript to explain that there was not a control group.  In part, this is because our objective was to compare the outcomes to those of conventional IVF.  Also, some of our patients were not candidates for IVF given their prior experiences with OHSS.  We are in a self-pay situation and were able to offer participants reduced fees with IVM, but could not for IVF.  Some of our participants could not afford to undertake IVF.  In lieu of a control group, we utilized a "comparison groups.  Members of this group shared some of the characteristics of the study group, but are not part of a randomization.  This group reflects oocyte and embryo outcomes of patients at high risk for OHSS after IVF when all embryos were frozen and subsequently transferred in the context of the same IVF program.  I would argue that in the context of patients at risk for OHSS, it could be viewed as "not ethical" to randomize them to IVF when IVM is available.  IVM has repeatedly been shown to avoid OHSS.

There are many settings in which a randomized study with a control group is the best way to advance knowledge, but such an approach is not always appropriate.  This is discussed in the following reference:

Freiden TR. Evidence for health decision making- Beyond randomized, controlled trials. N Eng J Med 2017; 377:465-475.  DOI: 10.1056/NEJMra1614394

There is also increasing concern about suboptimal use (i.e. viewed as necessary and sufficient) of p-values as discussed in the following editorials:

Editorial. Moving to a world beyond p < 0.05. American Statistician 2019; 73(51): 1-19.  DOI.org/10.1080/0031305.2019.1583913

Editorial. New Guidelines for statistical reporting in the Journal.  2019 N Engl J Med 2019; 381-285-286  DOI: 10.1056/NEJMe1906559

Line 180: was prima facie used to compare the author's protocol?

I do not understand what you are asking.  I even looked up "prima facie" to see if I was understanding it and using it correctly in the manuscript (at first view, true, valid, or sufficient at first impression), but it did not help.

Line 181-187: rewrite this paragraph. It is unclear

This is a post hoc estimate of the size of a trial required if this were a randomized study based on the data obtained from this series of patients.  The statement is an English version of a statistical statement and is therefore intrinsically complex.  I think it is stated as simply as possible.

The author did not discuss their results

The discussion of results is in the second paragraph of the Discussion section.  It contains multiple references and I have expanded it to emphasize the difference in the implantation rate from older published series (and why this is important).

Line 227:  Table 1 is not a result of this research. It is unnecessarily put as a table.

Please see the above discussion about using a comparison group that is not a control group.

In general, the discussion is not a discussion of the results obtained in the research. The author did not cite other studies with letrozole to compare their results. Also gave more importance to the needle than the protocol of letrozole. Suppose they pretend to evaluate the needle as a variable. In that case, the authors need an experiment comparing their results with a conventional needle.

The only prior use of letrozole for IVM that I am aware of is in reference 10, which only demonstrates the feasibility of using letrozole.  Discussion of that paper will not enhance the reader's understanding.  

As noted above, the needle (itself) is not really important.  What is important is that the surgeon try to treat the oocytes as he/she would during an IVF retrieval, and minimize possible temperature changes during retrieval.  This awareness potentially corrects an iatrogenic impact of new practitioners of IVM blindly using the methods suggested in the literature for retrieval.  It is an easy change to incorporate in a new IVM program and is worthy of emphasis.

Reviewer 3 Report

Dear Authors,

Thank you for allowing me to review your manuscript.

The introduction is well documented and the purpose of the study is clearly expressed.

 At result section some correlations are always a good point.

Discussion and conclusions are well written.

For greater clarity I would add a more detailed comparison of the results reported in the literature and the results obtained with this type of stimulation and a discussion of the results based on AMH levels.

I would also like you to dedicate a paragraph on the effect of IVM  protocol changes on measures of oocyte/embryo competence in cancer patients in order to perform fertility preservation and in particular in BRCA patients.

Finally, for a multidisciplinary approach we propose to mention:

DOI10.1136/ijgc-2021-003241

DOI10.3390/biomedicines8120554

DOI10.3390/cancers14143457

Author Response

Thank you for reviewing our article.

The following changes have been made.

For greater clarity I would add a more detailed comparison of the results reported in the literature and the results obtained with this type of stimulation

My references provide much summary information of IVM series reported in the medical literature, especially reference [5].  However, I added the following as it emphasizes the variation of results related to competence factors and the difference reported here.

"An ASRM guidance committee opinion about IVM suggested that the implantation rate was the most significant reflection of the inferiority of IVM to IVF [7].  This publication noted implantation rates of  5.5% to 21.6% in the IVM medical literature, whereas; in this study, the implantation rate was 34.2%." 

and a discussion of the results based on AMH levels.

I would also like you to dedicate a paragraph on the effect of IVM  protocol changes on measures of oocyte/embryo competence in cancer patients in order to perform fertility preservation and in particular in BRCA patients.

The paragraph added with four additional references was:

"Likely the most important reason for an IVF program to have the capacity to provide IVM is to be able to treat young women diagnosed with cancer, who require a treatment for their cancer that may be toxic to their ovaries [30,31].  Unlike IVF, a plan to undertake IVM can be initiated as soon as a patient presents with this problem.  IVM can be undertaken effectively at any point in a woman’s menstrual cycle [32].  To incorporate IVM into an established IVF program, a clinician needs to understand general principles that correlate to producing competent oocytes that can be used for future reproduction.  This study suggests three principles which facilitate the production of competent oocytes.  Namely, priming based on theoretically sound principles, an aspiration process that attempts to treat oocytes as they are treated during conventional IVF, and the future use of cryopreserved blastocysts once the cancer has effectively been treated.  The use of letrozole to limit estrogen production due to exogenous FSH, has been used women with breast cancer [33]."

Round 2

Reviewer 2 Report

INTRODUCTION:

The authors continue giving more importance to the use of the needle to obtain the oocytes than the letrozole molecule, which is the variable of the study.

The introduction is inconclusive. The authors must write about the following items:

·      What is the chemical characteristic of letrozole?

·      How does the letrozole act at a cellular level?

·      Into the follicle, where is the receptor for the letrozole?

·      Does the letrozole act over cumulus cells or the oocyte?

·      What is the pharmacology of letrozole?

·      How the letrozole increase the follicle's sizes from small antral to antral follicles?

Method:

How did the author decide on the medication scheme with letrozole and FSH?

The organization of the experimental protocol is not clear:

How many patients use letrozole 2.5 mg for five days?

How many patients use letrozole 2.5 mg for seven days?

How many patients received FSH with 25 for 3 days?

How many patients received FSH with 75 IU for 3 days?

How many patients receive FSH with 25 for 5 days?

How many patients receive FSH with 75 IU for 5 days?

Most patients received 2.5 mg of letrozole for 5 days and 50m IU for four days starting on the third day of letrozole. Did authors obtain their results from this population? If the author gets the results taking all the variables of the medication scheme with different days of administration of letrozole and FSH. They should explain what day and what doses were better.

The authors mention: "In a program with greater flexibility than ours, letrozole (2.5 mg) would be started on or before cycle day 3 and low dose FSH (25-50 IU) would be added when antral follicles were sufficiently large. Letrozole would be stopped after a 2 day overlap of FSH. FSH would be stopped and hCG given with the plan to move to retrieval with a sufficient cohort of 8-12 mm follicles (Table 1)". Table 1 refers to the program with greater flexibility than the protocols of the authors. The authors should explain that if a program with greater flexibility exists, what is the importance of the authors' protocol?

The authors refer to figure 1, but figure 1 was deleted.

For the oocytes obtained, what morphologies characteristics were used to select the better oocytes?

How many coats of cumulus cells were present around the oocytes selected?

How was the tridimensional conformation of cumulus-oocyte complexes (COCs)?

The authors should show a microphotography of an example of a COCs selected.

How did the authors determine the maturity of each oocyte?

The cumulus cells were minimally disrupted to determine the maturity of each oocyte. However, this eliminated the bidirectional communication between the oocyte and cumulus cells; how could the authors be sure about the correct competence acquisition of the oocytes during the culture?

The authors should show a microphotography of a mature oocyte before the ICSI process. Also, is necessary microphotography of a blastocyst obtained.

How was evaluated the quality of the developed blastocysts?

The authors did not explain the statistical test used to evaluate the results. Never mention the character of the control group.

RESULTS:

The results should be presented in a table.

What was the control group? The author must show the results of the control group. Did the authors compare the results with a control group? What statistical test was used to find the significance? What was the p-value?

I suggest analyzing the relationship between the age of patients, BMI, AMH, number of oocytes retrieved, number of mature oocytes, number of zygotes, cleaved embryos, and blastocyst. The author could conduct a Spearmen correlation followed by a Principal Component Analysis (PCA).

DISCUSSION:

Again, Table 2 is not a result of the authors in this paper. Therefore, it is unnecessarily put as a table.

The authors continue mentioning the importance of the needle. In my opinion, the authors should write another paper about only the influence of the needle.

Author Response

The authors continue giving more importance to the use of the needle to obtain the oocytes than the letrozole molecule, which is the variable of the study.

We repeatedly focus on three issues: letrozole use, oocyte handling, and delayed transfer.  The needle is not a focus (see below).

The introduction is inconclusive. The authors must write about the following items:

·      What is the chemical characteristic of letrozole?

·      How does the letrozole act at a cellular level?

·      Into the follicle, where is the receptor for the letrozole?

·      Does the letrozole act over cumulus cells or the oocyte?

·      What is the pharmacology of letrozole?

·      How the letrozole increase the follicle's sizes from small antral to antral follicles?

This information is available from several sources (see [11] or [12]) and specifically answered by reference [11], also written by this author.  This paper is not about letrozole, although the properties of letrozole in this setting mre the reason for using letrozole.

Method:

How did the author decide on the medication scheme with letrozole and FSH?

This was suggested in reference [10], which showed the feasibility of this application.  To my knowledge, that is the only publication that has used letrozole for IVM.

The organization of the experimental protocol is not clear:

How many patients use letrozole 2.5 mg for five days?

How many patients use letrozole 2.5 mg for seven days?

How many patients received FSH with 25 for 3 days?

How many patients received FSH with 75 IU for 3 days?

How many patients receive FSH with 25 for 5 days?

How many patients receive FSH with 75 IU for 5 days?

Most patients received 2.5 mg of letrozole for 5 days and 50m IU for four days starting on the third day of letrozole. Did authors obtain their results from this population? If the author gets the results taking all the variables of the medication scheme with different days of administration of letrozole and FSH. They should explain what day and what doses were better.

The basis for the use of increased letrozole or increased FSH is the same as done for conventional IVF.   Patients believed to be more difficult, based on prior experience, received increased medications.  For example, two patients had no AFs > 7 mm by the end of our available retrieval window.    Both received a longer letrozole treatment and the more obese woman had the higher FSH doses.  As noted in the paper. “Most patients received 2.5 mg of letrozole for 5 days and 50 IU for four days starting on the third day of letrozole.”  It is unclear to me how the requested highly detailed patient specific information helps the reader understand this management approach.  I assume all readers are familiar with medication dosing for IVF.   The implicit suggestion is that the clinician should minimally increase medications where clinically indicated.

The authors mention: "In a program with greater flexibility than ours, letrozole (2.5 mg) would be started on or before cycle day 3 and low dose FSH (25-50 IU) would be added when antral follicles were sufficiently large. Letrozole would be stopped after a 2 day overlap of FSH. FSH would be stopped and hCG given with the plan to move to retrieval with a sufficient cohort of 8-12 mm follicles (Table 1)". Table 1 refers to the program with greater flexibility than the protocols of the authors. The authors should explain that if a program with greater flexibility exists, what is the importance of the authors' protocol?

Most patients used this protocol.  IVF care always requires some flexibility.  That is the standard of care.  Programmed approaches are atypical; patient individualization is not.

The authors refer to figure 1, but figure 1 was deleted.

This has been corrected.

For the oocytes obtained, what morphologies characteristics were used to select the better oocytes?

There was no selection of oocytes.  All oocytes were cultured to determine if they could become mature.

How many coats of cumulus cells were present around the oocytes selected?

There was no selection.

How was the tridimensional conformation of cumulus-oocyte complexes (COCs)?

Unremarkable.

The authors should show a microphotography of an example of a COCs selected.

This is routinely available in earlier IVM publications.

How did the authors determine the maturity of each oocyte?

The presence of two polar bodies.

The cumulus cells were minimally disrupted to determine the maturity of each oocyte. However, this eliminated the bidirectional communication between the oocyte and cumulus cells; how could the authors be sure about the correct competence acquisition of the oocytes during the culture?

Disruption was the least possible to enable visualization of polar bodies.  As is routine in IVM, this is done to maximize communication ability between oocyte and cumulus.  Competence has not been well characterized biochemically.  The routine approach to competence is to look at functional achievements of the oocytes and embryos that come from the retrieved oocytes: maturity, fertilization, blastocyst production, implantation rate, and pregnancy rates. Thus the assessment of functional competence doesn’t begin until after the oocytes have been cultured for some hours.

The authors should show a microphotography of a mature oocyte before the ICSI process. Also, is necessary microphotography of a blastocyst obtained.

This is routinely available in the IVF literature.

How was evaluated the quality of the developed blastocysts?

Standard IVF criteria were used (Gardner definitions).

The authors did not explain the statistical test used to evaluate the results. Never mention the character of the control group.

Discussed below.

RESULTS:

The results should be presented in a table.

This is in Table 2.

What was the control group? The author must show the results of the control group. Did the authors compare the results with a control group? What statistical test was used to find the significance? What was the p-value?

As noted in my last response to you:

We took great pains in this manuscript to explain that there was not a control group.  In part, this is because our objective was to compare the outcomes to those of conventional IVF.  Also, some of our patients were not candidates for IVF given their prior experiences with OHSS.  We are in a self-pay situation and were able to offer participants reduced fees with IVM, but could not for IVF.  Some of our participants could not afford to undertake IVF.  In lieu of a control group, we utilized a "comparison groups.  Members of this group shared some of the characteristics of the study group, but are not part of a randomization.  This group reflects oocyte and embryo outcomes of patients at high risk for OHSS after IVF when all embryos were frozen and subsequently transferred in the context of the same IVF program.  I would argue that in the context of patients at risk for OHSS, it could be viewed as "not ethical" to randomize them to IVF when IVM is available.  IVM has repeatedly been shown to avoid OHSS.

There are many settings in which a randomized study with a control group is the best way to advance knowledge, but such an approach is not always appropriate.  This is discussed in the following reference:

Freiden TR. Evidence for health decision making- Beyond randomized, controlled trials. N Eng J Med 2017; 377:465-475.  DOI: 10.1056/NEJMra1614394

There is also increasing concern about suboptimal use (i.e. viewed as necessary and sufficient) of p-values as discussed in the following editorials:

Editorial. Moving to a world beyond p < 0.05. American Statistician 2019; 73(51): 1-19.  DOI.org/10.1080/0031305.2019.1583913

Editorial. New Guidelines for statistical reporting in the Journal.  2019 N Engl J Med 2019; 381-285-286  DOI: 10.1056/NEJMe190655

I suggest analyzing the relationship between the age of patients, BMI, AMH, number of oocytes retrieved, number of mature oocytes, number of zygotes, cleaved embryos, and blastocyst. The author could conduct a Spearmen correlation followed by a Principal Component Analysis (PCA).

These issues may be interesting, but are not germane to the objectives of this paper.

DISCUSSION:

Again, Table 2 is not a result of the authors in this paper. Therefore, it is unnecessarily put as a table.

As noted above, this contains the results that were requested above.  It also provides a context in which to understand them using a comparison group with many similarities to the treated group in the same IVF program using the same embryologist.

The authors continue mentioning the importance of the needle. In my opinion, the authors should write another paper about only the influence of the needle.

As noted in the paper after the first revision, I explain that the presumed benefits obtained by using this needle can also be obtained by modifying surgical technique using a double lumen needle.  The use of this specific needle was a personal preference.  However, it made clearer what we were doing: namely accelerating the transit of the oocyte from patient to lab.