Review Reports - Hypo-Osmotic Swelling Test and Male Factor

Round 1

Reviewer 1 Report (Previous Reviewer 2)

The Authors have addressed my comments and the paper should be considered for publication.

Author Response

Thank you.

Reviewer 2 Report (New Reviewer)

Presently, HOST is used in the IVF laboratory to assess viability of immotile sperm cells, to be used for ICSI. In the context of semen analysis, or male infertility assessment this test is not even described in the newest 6th edition of the WHO manual, where eosin staining is preferred to assess viability of sperm cells in cases of low or absent motility. This narrative review proposes the use of HOST as another tool in analyzing of sperm claiming that it has an additive value to the other existing parameters of sperm examination. The review is difficult to read, based mainly on publication from more than two decades ago. Although the idea is plausible, the flow of ideas is somehow not clear enough, leaving the impression of a work in progress, a draft that the authors should elaborate more, try to substantiate the arguments on recent data as well and provide clearer arguments to convince the potential readers for their their proposition.

Author Response

We have painstakingly revised this manuscript many times now in defense to the reviewers. We have expanded it, then reduced it, removed almost all of the tables in question, and added multiple other references to dilute the overabundance of references to our own work.

I was asked to write a feature paper for the 3^rd Founding Year of Reproductive Medicine. The type of article was not specified but I assumed that for this special issue many experts may write a review in an area in which they have performed many original studies and thus write a review. We chose the HOS test because somehow this very important, simple test has been lost to the new Reproductive Endocrinology/Infertility specialists and thus a review, plus the addition of material from a relative recent presentation at the ASRM meeting that was not published (now our only remaining table) would be very appropriate. We added the new ways that this HOS is used today to select viable but non-motile sperm for OCSI. This was added mostly as a way to satisfy reviewers’ requesting to add more recent publications and add more references in addition to our own. Except for our recent ASRM abstract there has been nothing new, because somehow, without commercial encouragement, this very important fertility test that is correctable is not familiar to most REI’s. The pressure of this manuscript was aiming to be special for this edition that would be to discuss something old but to the readers something new.

The reviewer states that this test is not described in the newest 6^th edition of the WHO manual which led to one of the few recent publications by another HOS expert Dr. Jeyendran (We include his recent reference) pleading the infertility world that HOS is different from vitality or viability test!!

The reviewer considers our studies just a work in progress. If the editors of Reproductive Medicine carefully reads this manuscript there has been presented a very logical easily understandable presentation that supports the contention that HOS defects can cause a very interesting infertility defect, a sperm abnormality that leads to infertility not by fertilization failure but by preventing implantation of the embryo.

Almost nothing in research and medicine is so absolute that a present dogma could become a fallacy at a later time. We are convinced of the veracity of this test. It is not a work in progress as suggested by the reviewer. It is not up to our group to support all the extensive research we did on the past leading to so many publications on the subject predominantly by our group but for other clinicians or researchers in the field now aware of this test to evaluate this test then publish their experience to either corroborate or refute our findings.

Thus, our response to the reviewer is that the suggestions are not possible to complete in a timely manner. There are no new studies out there, and thus we leave it to the editors of Reproductive Medicine to accept this multiple revised manuscript as a finalized product deemed worthy of publication in the special edition or agree with the reviewer that the 30 publications from our group referenced in this manuscript as insufficient to support our conclusions and that this manuscript is not worthy to publish in this edition. We have carefully searched the literature for new publications and could not find them, but if this reviewer is familiar with some more recent publications that challenge this concept, we would be more than happy to include them in this manuscript and thus in that circumstance would revise the manuscript once again.

Based on our knowledge that sperm with low HOS test scores lead to marked reduction in implantation potential it would be considerably unethical for us to perform a study, e.g., taking couples who need IVF with males with otherwise normal sperm but low HOS test scores and try conventional insemination on ½ and ICSI on the other half and then see if we had no pregnancies following fresh embryo transfer in the conventional group vs. Good pregnancy rate in the ICSI group. However, for those doubting the veracity of the test who normally do not even evaluate the HOS test and use conventional insemination when the sperm is normal then they would be the appropriate group to just perform ICSI on the other half and then either corroborate or refute our extensive previous studies. As you can see, however, it would be highly unethical for us to perform such studies wait 2-3 years before we have accumulated more data then maybe resubmit our manuscript to Reproductive Medicine for your 5^th or 6^th anniversary edition.

In summary, this submitted manuscript does provide some new unpublished data (only presented at a recent ASRM meeting as seen in Table 1. The new data was intended to corroborate conclusions reached before by our group with extensive research published in peer reviewed journals that the HOS test is different than the vitality or viability test, is frequently seen with normal semen parameters, does not inhibit fertilization but prevents embryo implantation, and it is a forgotten very important, inexpensive, simple to perform test not evaluated by most REI’s of today. So forgotten, as pointed out by the reviewer that is not even included in the recent WHO edition. Thus, this submitted manuscript in our opinion needs no further changes and as it stands deserves to be published in the forthcoming special edition. Perhaps rekindling interest in this test assuming that this leads to other REI centers evaluating the efficacy of this test and reaching the same conclusions as we determined will generate inclusion in the next WHO manual which in our opinion and the opinion of others who have performed research involving the HOS test is a very serious omission. Thus, the concern of the reviewer is exactly one of the main reasons why this manuscript deserves to be published in Reproductive Medicine.

Round 2

Reviewer 2 Report (New Reviewer)

This narrative review is a kind of expert opinion presenting the possible value of HOST in the evaluation of male infertility. To substantiate this, one should describe the exact methodology of the test, present the existing knowledgebase-data in the literature, showing that HOST has value in predicting male infertility (or the lack of substantial evidence). How was the normal-abnormal value determined (ROC curves etc...). PPV-NPV? Is the evidence based on findings of different laboratories? Present the data related to the value of HOST in the IVF clinic. Is it based on prospective randomized studies? observational studies? expert opinions? Is there molecular evidence relating to the hypothesis presented regarding the interfering protein that is responsible for the sperm cell malfunction-bad influence on the future embryo? The HOST is probably not used in the andrology clinics because the evidence is scarce regarding its contribution to the overall assessment of male infertility. Probably with more investment in the manuscript, including remarks on the lack of sufficient evidence and offering possible methodology to evaluate its use, the review may be improved to allow reccommading t ito accept for publication.

Author Response

As per the reviewer’s suggestion we add after the first sentence in paragraph 2 a paragraph indicating that since we did not design the modification of the HOS test for humans, so the normal-abnormal value determined (ROC curves, etc.) are available in reference 5 the article by Jeyendran et al. We have made some mild modifications of the technique, so we agree with the reviewer that the actual technique should be provided, and thus the second paragraph of the introduction refers to a new table (Table 1) which provides the actual methodology.

We add at the end of the manuscript a new section entitled limitations of the manuscript. Hopefully, this additional section covers the suggestion by the reviewer, and we agree that this may strengthen the manuscript by not only convincing treating physicians to use this test in their practice, but with the new addition, encourage younger scientists seeking their masters or Ph.D. degree or fellowship in REI needing a research project to provide new research helping to find causes and new treatment for infertility especially in those couples failing to conceive despite conventional treatments and consider some of the suggested research projects in this last section.

This manuscript is a resubmission of an earlier submission. The following is a list of the peer review reports and author responses from that submission.

Round 1

Reviewer 1 Report

Summary: This review underlines the relevance of the HOS test as a measure of spermatozoa fertilization ability and pregnancy success, highlighting that it should be part of the standard semen analysis. The authors argue that spermatozoa with normal parameters from the standard spermiogram, frequently are sub fertile and pregnancy is still not possible, due to male reasons. They propose that HOS test is a unexpensive and easy to perform option to further evaluate sperm quality, particularly the functional integrity of the membrane, along with the currently performed semen analysis. Overall, the topic of this article is relevant to the field and the authors suggestion for this test to integrate the standard semen analysis is well argued. However, there are some major issues that demand attention prior to publication.

Commentary 1: Regarding the references, the articles cited are generally not very recent, mostly dating from before 2010. I suggest that more recent studies should be included in the article, to give a more updated review of the theme. Further, self-citations constitute more than half of the bibliography of this review, which is not very appropriate and should be revised.

Commentary 2: The information in the article is generally disorganized. Starting with structure, it only has two main topics, Introduction and Final comments. I suggest that 1.1 and 1.2 should constitute the Introduction of the article, while the other topics (1.3-1.13) should integrate another main topic, since they are the development of the article theme.

Commentary 3: Some sections, such as 1.3 and 1.7, approach only one study. Besides, other sections are very short (1.8 and 1.9). I advise that such sections should be joined to form major ones, in order to better integrate the information. Furthermore, I do not see the relevance of 1.4. I believe it should be withdrawn or abbreviated and included in other topic, perhaps in the first sections.

Commentary 4: In section 1.6, the difference between vitality and viability should be enlightened and the more appropriate term should be chosen and used throughout the arcticle.

Commentary 5: Concerning the language, it should be revised throughout all the article, since the writing style of the piece is confusing and lacks fluidity, sometimes being unclear and hard to understand. Punctuation is frequently missing, which further affects the fluency of the article.

Here are some samples of sentences that, in my opinion, need to be rewritten because they are long and unclear.

· Line 46 to 50

· line 151 to 154

· line 303 to 306

· line 426 to 432

· line 439 to 446

Commentary 6: There are too many tables in the main text, which hinders the article's flow, I advise that the least important ones should be included in the supplementary materials.

Author Response

Reviewer #1:

Commentary #1

We agree that this review seems lopsided with most references before 2010 and most references to our own studies. The problem is that this test has been lost and is not utilized by most infertility centers so that there are barely any studies since 2012 evaluating the HOS test as a method of diagnosing and treating male infertility. Furthermore, unfortunately most of the clinical studies relating to the HOS test were performed by our group.

Nevertheless, we agree that adding newer publications by other authors would improve the quality of our manuscript. This has been accomplished by adding section 4.5 entitled “Other possible ways to improve adverse effects on female fecundity related to male partners’ sperm with subnormal HOS tests”. We have also added “Improving IVF-ET outcome with ICSI by selecting sperm with normal HOS changes” (now section 5) and also section 6 “Possible significance of HOS subclasses as determined by different tail swelling patterns”. We have not conducted any research studies in these areas, so this allowed us to add another 40 references from groups other than our own and some of these references are from the last 2 years.

Commentary #2

We agree that the article should have more subdivisions instead of two main topics Introduction and Final Comments. Thus, we now have 7 sections – with 1.1 and 1.2 under Introduction as you suggested, but now we add:

Reduced fecundity associated with low HOS test scores with sections 2.1, 2.2 and 2.3
The association of low HOS test scores and other subnormal semen parameters with section 3.1, 3.2, 3.3, and 3.4
Hypothesized etiology of infertility from sperm with low HOS test scores leads to treatment options with sections 4.1, 4.2, 4.3, 4.4 and 4.5.
Improving outcome of IVF-ET with ICSI by selecting sperm with normal HOS changes with section 5.1.
Possible significant of HOS subclasses as determined by different tail swelling patterns with section 6.1.
Final comments is now #7. We have expanded it a bit which allowed 4 more references.

Commentary #3

Sections 1.3 and 1.7 are entirely different. 1.3 relates to the first evidence that low HOS test scores can predict poor pregnancy rates even if motile densities are normal when trying to achieve a pregnancy by natural intercourse. In contrast section 1.7 was a study to see if there was a correlation between any single sperm abnormalities, including motile density, and low HOS scores. We have a time deadline to make these changes and we do not agree with this reviewer that combining these two sections will strengthen the manuscript. It should be noted that section 1.7 is now section 3.2.

The reviewer does not see the relevance of section 1.4 and states that it should be withdrawn or abbreviated and included in other topics possibly in the first section. We disagree – It is because of the studies by other infertility centers that low HOS test scores did not corroborate with fertilization rates and morphologically normal appearing embryos that the REI community lost interest in the HOS test. They did not publish their pregnancy rates and as mentioned either they failed to look at this very important parameter or they were embarrassed to publish terrible pregnancy rates. We tried to include in our original version all pertinent publications outside of our group. Eliminating this section would eliminate references 45-47 that were from outside our group.

Commentary 4

In accordance we have added a long paragraph (started with paragraph 3 of section 4.5 and add direct quotes from Hecht and Jeyendran which is reference 81 and allowed us to add a recent publication from 2021.

Commentary 5

Line 46-50 was simplified.

Lines 151 to 154 was changed.

Lines 303-306: in line 304 we changed on to an (which may have led to confusion) it was a typo, added a comma in line 305 after scores and in line 306 changed ones to sperm. Hopefully this improves the understanding of this sentence. Time constraints preclude us from sending the finalized version to an English editor. However, we have carefully reread the article and made multiple grammatical changes and word changes and put in punctuation. Changes were made in at least 60 sentences. Nevertheless, we are sure further improvement could be made and the author would appreciate any further help from the editorial staff of Reproductive Medicine.

Lines 426-432: Has been rewritten.

Reviewer 2 Report

This review gives an overview of the relevance of the hypoosmotic swelling (HOS) test as a sperm parameter to to diagnose infertility in male patients. The HOS is commonly used to assess the quality of semen of different animal species, and its relevance in the diagnosing male infertility has already been extensively covered in numerous papers by the Authors. The Reviewer suggests that there is nothing new in the review. The major concerns of this review areas follows:

1. The title is misleading. The HOS tests is routinely used to assess the quality of bovine, dog and boar semen.

2.The Authors have presented the results from previous previously published papers in the tables (in some cases the exact copy of the Tables 1-19), without giving the appropriate original sources. I understand that the data are from the Authors’ own published papers, however, if they have been published then the appropriate attribution (sources) should be given under each table.

3. Furthermore, should consider the policy of the journals when required to use reproduced tables, even though they were from the Authors’ previous papers. To my understanding the owner of the copyright of the original tables is usually the publisher (for example, Elsevier), and permission is required to reuse published tables to be presented in another journal.

Author Response

Reviewer 2

Comment 1

Your view that the HOS test is frequently used in animals is correct, so the word human is added to the title, so it now reads “The rarely utilized sperm test named the hypo-osmotic swelling test is a very important semen parameter to evaluate human male factor as a cause of infertility”.

Comment 2

We have added under each table the fact that this table was exactly taken from a particular reference for which we were the author.

Comment 3

We are aware that you could not use tables form a previous publication from a different author without permission of the publisher, and we believed that you could not reprint figures without permission, but we did not think we could not use our own tables.

There is time sensitivity for this article, and it is targeted for a special issue. Unfortunately, we did not receive the first email with reviewer’s comments and only recently received the second email with a deadline for the revised edition by March 3^rd.

We have made a lot of changes related to comments from reviewer #1.

Thus, we are leaving the tables for now, but emphasizing as you suggested under each table that this was taken from a specific manuscript. The reference is noted, and we are immediately sending requests to the publishers for permission to use these tables. We cannot imagine that such permission would not be granted, but if they don’t there should be time for us to change some tables to make them somewhat different.

Round 2

Reviewer 1 Report

Overall, the article continues to be disorganized. I believe it is too long and frequently confusing to read. As I mentioned in my last review, the goal of the article is well defined but it is not well defended, and the principal point is lost throughout the middle sections, which in my opinion do not need to be so extensive.

There are too many tables in the main text that often refer to only one study each. I again suggest that at least half should be moved to the suplementary materials, since they are not essential to understand the arguments explained in the text and they affect its fluidity while reading.

The Introduction (1.1 and 1.2) is too long and should be abreviated. Section 3 does not need to be subdived, since the subtopics are very short. Section 4 and Section 7 are very long, I believe they can be simplified.

I advise that all of the article is revised to make it more fluid and easy to read and understand. Sentences are frequently too long and confusing. In some parts of the manuscript, the font is smaller, please revise the formatting of the article (for instance line 521 to 524).

Author Response

Thank you.

Reviewer 2 Report

I still insist that the Authors need to get the permission from the Publishers (see the copyright transfer agreement because they have re-copied the exact tables in the review paper.

Author Response