Title: FOXO3 and PTEN expression in the ovary of girls with extra-gonadal cancer with or
without chemotherapy treatment prior to cryopreservation
Version: 1 Date: 27 Apr 2023
Reviewer's report:
Albamonte et al, expression pattern in ovaries from childhood cancer patients
This manuscript examines expression of Foxo and Pten genes in ovaries of childhood cancer
patients, with some of the patients having been administered chemotherapy drugs prior to ovary
collection. It is important to get information on ovaries of this patient group, but at present there
are some aspects of the manuscript that seem confusing, detailed below.
Major points.
1. Infant would normally refer to much younger, not above age 1. Here, the youngest patient is
7, and so the term should not be used, throughout the manuscript. (Pre- and post-pubertal ?)
2. Title. Why is the title indicating no difference when some are found? Also, why not include
Pten?
Note that if the title does end up highlighting where differences are not found then it would be
more correct to put in that way rather than that there is no difference – it would be difficult to
determine that given that sample sizes are limited in numbers, and with a notable variability in
age, cancer type, etc.
3. Abstract, Results section: again, this should mention sign results (Figs 2C and 6A).
4. Lines 113-114: information on how menarche was assessed should be included here.
5. Table 1 should also indicate what patients were assessed as post menarcheal
6. Tables. It would be much better if the information in the tables (in main manuscript and in
supp data) was coordinated so that the reader can easily see eg what fresh tissue came from
patients that had received chemo before freezing and what did not.
7. Fig 1: Arrows and arrowheads could be larger, clearer. Also, here and in other histology
images, please state whether images are of sections from patients that did or did not have chemo
before collection, ideally showing examples of both
8. Given that a lot of results are on immunos, it would be best if images of negative control
tissue was always be from pre-incubation (not just no-primary), with figure showing image of
negative control immuno of the tissue also used as positive control – so for Fig 1, blood vessel.
9. Tables S2 and S3 should be combined, since I think that statistics were carried out
comparing the groups across these tables. I would suggest having it in the main manuscript, not
supplemental.
10. Lines 230-231:  primary and secondary follicles. How many were examined in each case?
11. Fig 3. Some of images do not look healthy, with what appears to be blebbing in cytoplasm.
Are there better images that could be used?
12. Tables S4 and S5. Again, can tables be combined since data in them are being compared
13. Tables S4 and S5: Why is there an asterisk in table S4 to indicate transitional follicles? Table
is meant to be re primordials. This should be clarified, with data on transitionals removed unless
it’s clear why they are included.
Minor points.
a. Line 50: germinal. Needs re-writing since it is not clear that all drugs damage the germ cells
directly.
b. Lines 60-61: it would be better if the authors could expand on this, given that tissue
cryopreservation is the only current option available pre-puberty.
c. Line 63: ‘periodically’. Would ‘continually’ be better? Also, that sentence is not clearly
written – reads as if all follicles ovulate.
d. Line 69, ‘germ cell proper’ – what is meant by that term?
e. Lines 82-83: very vague, can anything more specific be stated? Also, ref should be clearly
cited in that sentence.
f. Line 113. Why is the name of the clinician here, and why in quotation marks?
g. Lines 126-127: what is age up to which parents/legal rep consent is required?
h. Can fig 4 just state ‘nucleus’, ‘cytoplasm’ etc? There seems no need to use letters, making it
harder to work out what you’re looking at.