Optimizing the Ovarian Tissue Cryopreservation in the ‘Oncofertility’ Institutional Program at an Italian National Cancer Institute
, Ondina Popescu 4, Anna Altavilla 4, Attilio Guarini 2, Fabio Pavone 2, Vera Loizzi 1,5, Gennaro Cormio 1,5 and Raffaella Depalo 3Round 1
Reviewer 1 Report
Authors tested two methods of cryopreservation in a very small sample size obtained from women bearing cancer, mainly those type more sensitive to hormone therapy. Their results were based on cell viability and histological analysis, concluding that slow freezing is better than URF. This manuscript is interesting but there are issues to be addressed.
Major comments:
Material and Methods
- Section 2.1: Implementing a working team for counselling patients and ovarian cortex sampling
This section does not clarify what and how many professionals were involved in the working team. Please, address and specify their function and how they served in the center (dedicated, temporary, upon demand etc).
- Sample size (n=11) is too low. Please, increase it as long as possible or show a statistical analysis stating that this is enough to find any difference.
- Lines 160-161. Please, explain why did you choose both parameters as a cutoff (anti-Müllerian 160 hormone (AMH) levels ≥ 2ng/mL and antral ultrasound follicle count ≥ 8). What did you mean by arbitrary ?
- Line 173: Please, explain why authors choose Leibovitz's L15 media and insert a proper reference. Whas it enriched with any supplements ?
- Period of cryostorage is confusing. In line 209-210 authors wrote that tissues were kept for two months. Considering that women undergoing to chemotherapy it would be interesting to evaluate longer time. If that is true, please, as long as possible, insert longer period such as years after freezing. In line 222, authors stated that they thaw after six months. Please, make a concise statement throughout the text. Also, it would be very valuable to know whether SF or URF keep cells viable after at least 5 years of cryopreservation, since this would be closer to patient condition (5 years after remission).
- Figure 1: insert bar scale in the image. Since you have only eleven samples, it would be interesting to provide all HES imagens as supplementary material.
- Authors disclosed no conflicts of interest in the proper section. However, they highligh the funding received by the Ministry of Health to conduct either the research and support the center. I recommend that this manuscrit limit their conclusion (and the main points throughout the text) to the effects of cryopreservation and place all the rest in the Funding topic.
Author Response
Answers to the Reviewer # 1
We thank are the Reviewer for comments and suggestions.
- Section 2.1: Implementing a working team for counselling patients and ovarian cortex sampling
This section does not clarify what and how many professionals were involved in the working team. Please, address and specify their function and how they served in the center (dedicated, temporary, upon demand etc).
IRCCS is a research oncology center hosting PhD students, medical and biologists students trained by the resident staff as better reported in the revised manuscript.
- Sample size (n=11) is too low. Please, increase it as long as possible or show a statistical analysis stating that this is enough to find any difference.
We thank the Reviewer for this comment. In accordance with him, we have a small sample. Nevertheless, previous authors have conducted other pilot study with similar sample size. In addition, we have showed in the new version of manuscript the statistical analysis to manifest that our results has a greater accuracy and included a new paragraph at the end of Material and Methods.
- Lines 160-161. Please, explain why did you choose both parameters as a cutoff (anti-Müllerian 160 hormone (AMH) levels ≥ 2ng/mL and antral ultrasound follicle count ≥ 8). What did you mean by arbitrary ?
“Arbitrary” Has been deleted since the cut off parameters according to reference 15. Y. Himabindu et al, J Hum Reprod Sci, 2013 2013;6(1):27-31
- Line 173: Please, explain why authors choose Leibovitz's L15 media and insert a proper reference. Whas it enriched with any supplements ?
We are grateful for this comment. Leibovitz medium was selected because it is designed to maintain
pH in the physiological range under normal atmosphere without added CO 2 and it can be used under
conditions of free gaseous exchange with the atmosphere. We have now implemented the 2.3 section.
- Period of cryostorage is confusing. In line 209-210 authors wrote that tissues were kept for two months. Considering that women undergoing to chemotherapy it would be interesting to evaluate longer time. If that is true, please, as long as possible, insert longer period such as years after freezing. In line 222, authors stated that they thaw after six months. Please, make a concise statement throughout the text. Also, it would be very valuable to know whether SF or URF keep cells viable after at least 5 years of cryopreservation, since this would be closer to patient condition (5 years after remission).
We are grateful to Reviewer for highlighting that the period of cryostorage is confusing. Therefore, we have proceeded to correct in the manuscript the two months into six months as we made a mistake. We also believe that the suggested proposal to know whether SF or URF keep cells viable after at least 5 years of cryopreservation is really interesting and considerable for a future research.
- Figure 1: insert bar scale in the image. Since you have only eleven samples, it would be interesting to provide all HES imagens as supplementary material.
As suggested by the Reviewer 1 for Figure 1 we provided the original magnification which is H&E x10. We also reported for the others Figures of the manuscript the relative magnification.
- Authors disclosed no conflicts of interest in the proper section. However, they highligh the funding received by the Ministry of Health to conduct either the research and support the center. I recommend that this manuscrit limit their conclusion (and the main points throughout the text) to the effects of cryopreservation and place all the rest in the Funding topic.
As advised, we strenghtend the objectives, results and conclutions on the available freezing techniques and limited the aspects related to Hospital accreditation. As an example, the following sentece has been cancelled from the Discussion section:
“Furthermore, based on the absence of a regional reference center for FP, we propose that in line with the oncofertility programs to be developed following the Italian Health Ministry and AIOM guidelines, a possible implementation of the OTC procedure in young patients with hormone sensitive cancers could strengthen the ongoing FP activities at the IRCCS Cancer Institute of Bari which is already funded by the Ministry of Health to develop novel methodologies to prevent the CTRI [11].”
We have also eliminated from Conclusion section:
“The "RicercaCorrente" Health Ministry funds provided helpful conditions to the IRCCS Cancer Institute of Bari for implementing the oncofertility program using the OCT as novel procedure for FP in cancer females.”
Reviewer 2 Report
Dear authors,
Thank you for the opportunity to review your article.
The topic of oncofertility is for sure interesting and with lots of debates.
A few comments:
-the abstract must be rewritten, respecting the structure of an abstract
-the introduction is well written
-the objectives of the paper is missing
-the method is well described
-the results must be discussed extensively if the purpose of the article was that one
-the discussion section and the conclusions seem to be descriptive and more like a report regarding the plans of the center and team. What are the conclusions of the paper? This section should be clear and not very long.
I don not understand the paper's purpose. Is it more like a presentation of the program, the center and the future plans. Please clarify.
Author Response
Answers to the Reviewer # 2
We are grateful to the Reviewer for the opinion regarding the manuscript and for the suggestions. Here we answer to the queries.
-The abstract must be rewritten, respecting the structure of an abstract
As suggested by the Reviewer the abstract has been rewritten respecting the structure of an abstract: an including the following sections Background, Methods, Results, Conclusions.
-The objectives of the paper is missing
Thank you for the appropriate comment giving us the possibility to spell out in the revised manuscript
that the aim of our pilot study was compare the effects of cryopreservation after SF and URF on human ovarian cortical pieces and discuss whether these strategies can be practiced for establishing
an elective OTC program in our Cancer Institute
-The results must be discussed extensively if the purpose of the article was that one.
As requested by the Reviewer the results has been improved with a statistical analysis and also we provided the original magnification of each histological images of the manuscript.
-The discussion section and the conclusions seem to be descriptive and more like a report regarding the plans of the center and team. What are the conclusions of the paper? This section should be clear and not very long.
In accordance with what was also highlighted by the Reviewer 1, we proceeded to modify the conclusions and clarify the terminal part of the manuscript.
-I don not understand the paper's purpose. Is it more like a presentation of the program, the center and the future plans. Please clarify.
The Cancer Institute of Bari (IRCCS Istituto Tumori “Giovanni Paolo II”) has launched a research program funded by the Italian Ministry of Health aimed at employing the adoption of the ovarian tissue cryopreservation (OTC) for female patients undergoing anti-cancer treatments with the final purpose to install an oncofertility reference center for the FP which would also serve other intra- and extra-regional cancer centers. In our manuscript we report our early experience in OTC including results, obtained by the comparison between two cryopreservation practices as the slow freezing (SF) to ultra-rapid freezing (URF) of ovarian cortex specimens. In the revised manuscript we have better explained the purposes of the work and we are afraid that it was not clear in the previous version.
Reviewer 3 Report
Thank you for the opportunity to review this manuscript which examines early experience with ovarian tissue cryopreservation at a clinic in Italy. The manuscript would benefit from editing with someone who better understands the English language. On a whole, it is difficult to read and understand exactly what the authors are trying to say. For example, several times, the authors use the term “female patients candidates”àcandidates is not needed; these are patients, not candidates.
There are far too many language/grammar issues to write out each one, but as it is currently written, it is not understandable.
Introduction:
“as effect of related therapies primarily including gonadotoxic drugs, thus resulting in permanent illness that 43 precludes projects in their reproductive life.”: this sentence, as written is a run on sentence and does not make sense
Material and methods
2.1
“Considering the high incidence of hormone-sensitive tumors in young female patients, it could be preferable to avoid the hormone stimulation for the oocytes' recruitment and for the relative independence from menstrual cycle phases”: as the authors know, ovarian stimulation can be initiated regardless of menstrual cycle phase when eggs/embryos are not being transferred (random start) and there is a lot of literature about the safety of ovarian stimulation, even with hormone sensitive tumors (Moravek et al)
2.2:
“occasional abdominal/pelvic surgery” what is meant by “occasional”àthis is likely another language issue
-why were immunotherapies considered a contraindication since, in theory, these should not affect ovarian reserve?
Results:
3.1: please provide a percentage of intact follicles in each group. Why were there so many fewer follicles in the URF group?
Discussion:
“provides the advantage of both avoiding the estrogenic 268 stimulation and maintainment of scheduled urgent anti-cancer treatments”: again, as mentioned above, the studies looking at safety have all shown stimulation to be safe, and the actual delay to cancer treatment to be minimal, so these statements should be tempered.
The manuscript will benefit from editing with someone who better understands the English language. On a whole, it is difficult to read and understand exactly what the authors are trying to say.
Author Response
Answers to the Reviewer # 3
The Authors thank the Reviewer for the comment. We eliminated “candidate” from the text and address the remaining issues
- Introduction: “as effect of related therapies primarily including gonadotoxic drugs, thus resulting in permanent illness that precludes projects in their reproductive life.”: this sentence, as written is a run on sentence and does not make sense.
As suggested the sentence as been clarified “The majority of female patients candidates to receive anti-cancer treatments are at risk of developing the ‘cancer treatment related infertility’ (CTRI) as effect of therapies including gonadotoxic drugs, thus resulting in permanent damages that precludes their reproductive projects.”
- 2.1 “Considering the high incidence of hormone-sensitive tumors in young female patients, it could be preferable to avoid the hormone stimulation for the oocytes' recruitment and for the relative independence from menstrual cycle phases”: as the authors know, ovarian stimulation can be initiated regardless of menstrual cycle phase when eggs/embryos are not being transferred (random start) and there is a lot of literature about the safety of ovarian stimulation, even with hormone sensitive tumors (Moravek et al).
We have rephased the sentence as follows in order to better explain the conditions in which OTC is preferrable to ovarian stimulation:
“The benefits of using OTC over ovarian stimulation may be greatest in young patients with hormone-sensitive cancers. Furthermore, some clinical conditions need a urgent anti-cancer approach and do not allow the patient to postpone the start of chemotherapy for 10-14 days. This can occurr expecially in young patients diagnosed with lymphoma: symptomatic and rapidly progressive disease, bulky mediastinal adenopathies. “
- 2.2: “occasional abdominal/pelvic surgery” what is meant by “occasional”àthis is likely another language issue.
As requested we deleted “occasional or major” because not relevant in the contest of the sentence.
- Why were immunotherapies considered a contraindication since, in theory, these should not affect ovarian reserve?
Any previous immunotherapy treatments are an exclusion criteria for the enrollement since current data concerning the consequence on the ovarian reserve is still unknown. (Minding the Bathwater: Fertility and Reproductive Toxicity in the Age of Immuno-Oncology. Anne E. Kim et al. JCO Oncol Pract. 2022 Dec;18(12):815-822. doi: 10.1200/OP.22.00469).
- 3.1: please provide a percentage of intact follicles in each group. Why were there so many fewer follicles in the URF group?
We appreciate the Reviewer comment and to this regard, now we have added more information in results section as recommend. Our data are casual principally related to the randomization of ovarian cortex strips in the URF method group and SF method group.
- Discussion: “provides the advantage of both avoiding the estrogenic stimulation and maintainment of scheduled urgent anti-cancer treatments”: again, as mentioned above, the studies looking at safety have all shown stimulation to be safe, and the actual delay to cancer treatment to be minimal, so these statements should be tempered.
We have now modified the sentence as follows: “With respect to the conventional oocyte retrieval after hormone burden and freezing of eggs or embryos, the OTC procedure based on surgical recruitment of ovarian cortex strips and subsequent reimplantation in patients after the cancer healing (5 years after obtaining disease remission), could be proposed at pre-puberal age, in hormone-sensitive cancers and when there are clinical conditions for which the anti-cancer therapy cannot be delayed for the 10-14 days necessary for ovarian stimulation. Furthermore, OTC reinplantation could restore the endocrine balance in adult cancer surviving females.”
- Comments on the Quality of English Language The manuscript will benefit from editing with someone who better understands the English language. On a whole, it is difficult to read and understand exactly what the authors are trying to say. In agreement with the suggestions of the reviewer we revised the manuscript for grammatical and spelling errors, with the contribution by a native English speaker.
Round 2
Reviewer 1 Report
Authors have much improved the manuscript and addressed most of my concerns.
Author Response
Authors have much improved the manuscript and addressed most of my concerns.
Answer: We are grateful for the favorable appreciation of our work.
Reviewer 2 Report
Dear authors,
Thanks again for the opportunity to read your article. I really appreciate the fact that you considered my comments.
First of all, I saw that you have changed the type of the paper, from "article" to "brief report". Maybe this is a better option, but please decide if you keep it, as there are slight differences between two of them.
Then, as you written, it seems that the objective of the paper is to compare the effects of cryopreservation after SF and URF in ovarian cortex. These are things already studied, and even the tentative of adding some statistics is a good one, the sample size is small.
The comments for discussion and conclusion parts were partially addressed.
Author Response
Reviewer # 2: Dear authors, thanks again for the opportunity to read your article. I really appreciate the fact that you considered my comments.
- First of all, I saw that you have changed the type of the paper, from "article" to "brief report". Maybe this is a better option, but please decide if you keep it, as there are slight differences between two of them.
Answer: We are grateful to the Reviewer for his appreciation for our manuscript and for all comments. We would like to keep the current style of the article (Brief Report) since we think is more appropriate to the content of the manuscript
- Then, as you written, it seems that the objective of the paper is to compare the effects of cryopreservation after SF and URF in ovarian cortex. These are things already studied, and even the tentative of adding some statistics is a good one, the sample size is small.
Answer: Although there are already comparative data in literature concerning SF and URF, the objective of the RC 2023 project supported by Ministry of Health, Italian Government, was to start this practice in our center, with the goal of establishing an oncofertility reference center for FP. In this brief report the objective was to report the preliminary results collected in our center in comparison to scientific literature.
Reviewer 3 Report
Thank you for allowing me to review this manuscript.
There are no studies directly comparing safety of OTC compared to ovarian stimulation. All of the published studies have shown ovarian stimulation to be safe.
The authors now state:
“The benefits of using OTC over ovarian stimulation may be greatest in young patients with hormone-sensitive cancers” Again, there is really no data backing this up. Either the authors need to provide a primary reference that would support this, or the authors should eliminate this statement.
Discussion:
“Thus, as FP method in oncofertility, the OTC is probably preferable for young female patients with hormone sensitive cancers”àagain, there is no evidence that OTC is preferable in patients with hormone sensitive cancers.
“hormone burden” please change the word burden, since this has a negative connotation.
Response to reviewers:
“Our data are casual principally related to the randomization of ovarian cortex strips in the URF method group and SF method group” what do the authors mean by “casual” in this context?
improved
Author Response
Reviewer # 3: - Thank you for allowing me to review this manuscript. There are no studies directly comparing safety of OTC compared to ovarian stimulation. All of the published studies have shown ovarian stimulation to be safe. The authors now state: “The benefits of using OTC over ovarian stimulation may be greatest in young patients with hormone-sensitive cancers” Again, there is really no data backing this up. Either the authors need to provide a primary reference that would support this, or the authors should eliminate this statement.
Answer: We remove this statement in consideration of the insufficient data and we thank the reviewer since the comparison between the safety of OTC vs ovarian stimulation could be the intuition for a future study.
- Discussion: “Thus, as FP method in oncofertility, the OTC is probably preferable for young female patients with hormone sensitive cancers”àagain, there is no evidence that OTC is preferable in patients with hormone sensitive cancers. Answer: We have replaced OTC “is preferable” with “is equally effective”
- “hormone burden” please change the word burden, since this has a negative connotation.
Answer: “Hormone burden” as replaced by “hormone stimulation”
- Response to reviewers: “Our data are casual principally related to the randomization of ovarian cortex strips in the URF method group and SF method group” what do the authors mean by “casual” in this context?
Answer: We thank the reviewer for pointing out that he had made a mistake adding the word “casual” in the response to the reviewer (Round 1) compared to the manuscript where it does not appeare
