Improvement in Sperm Recovery Rate and Total Motile Sperm Count Using α-Chymotrypsin in Highly Viscous Semen Sample Without Adversely Affecting Assisted Reproductive Technology Outcomes

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Dear authors, after reading your paper, here are my comments.

The post-wash microscopic analysis was done by an experienced andrologist, but it doesn't mention if the andrologist was blinded to whether they were assessing the α-chymotrypsin-treated sample or the untreated control sample from the same patient.

The given formula seems flawed or, at the very least, unconventional and confusing.  The formula computes the yield or growth factor in relation to the beginning quantity.  Standard sperm recovery rates often evaluate the proportion of motile sperm retrieved from the initial sample post-processing in relation to a theoretical maximum, or frequently compare the final total motile count (TMC) to the initial TMC, generally yielding a figure below 100% owing to losses incurred during processing.

The definition of high viscosity ("absence of a thread over 2 cm long... with repeated pipetting") involves some subjectivity. "Repeated pipetting" isn't quantified (how many times? how vigorous?). This could lead to inconsistency in sample selection. Referencing a standard protocol (like WHO guidelines) would strengthen this.

You used 5 mg of α-chymotrypsin for 5 minutes. There's no justification or reference provided for why this specific amount and duration were chosen (e.g., based on previous studies, optimization experiments). It's unclear if this is optimal. Concentration (mg/mL) might also be more relevant than absolute mass, depending on initial sample volume variations.

Author Response

Comments1: The given formula seems flawed or, at the very least, unconventional and confusing.  The formula computes the yield or growth factor in relation to the beginning quantity.  Standard sperm recovery rates often evaluate the proportion of motile sperm retrieved from the initial sample post-processing in relation to a theoretical maximum, or frequently compare the final total motile count (TMC) to the initial TMC, generally yielding a figure below 100% owing to losses incurred during processing.

Response1: Sperm recovery rate is to calculate how much motile sperm has been recovered, so post-wash TMC should be always less than pre-wash TMC. Because some portions of motile sperm will be loosed during wash process. This is common calculation in IVF lab.

Comments 2: The definition of high viscosity ("absence of a thread over 2 cm long... with repeated pipetting") involves some subjectivity. "Repeated pipetting" isn't quantified (how many times? how vigorous?). This could lead to inconsistency in sample selection. Referencing a standard protocol (like WHO guidelines) would strengthen this.

Response 2: We added WHO guideline to #13 reference

Comments 3: You used 5 mg of α-chymotrypsin for 5 minutes. There's no justification or reference provided for why this specific amount and duration were chosen (e.g., based on previous studies, optimization experiments). It's unclear if this is optimal. Concentration (mg/mL) might also be more relevant than absolute mass, depending on initial sample volume variations.

Response 3: We used commercial kit of α-chymotrypsin, QwikCheck^TM Liquefaction Kit, Medical Electronic Systems, CA, USA. This has been added in the material section. The vendor recently upgraded the kit by providing a think powder in each kit. The vendor also performed clinical experiment of no impact to human sperm motility by treatment and referenced WHO manual in their internal datasheet.

Reviewer 2 Report

Comments and Suggestions for Authors

Dear authors,

I read your paper with interest. I could consider the paper for publication but I need to clarify something. Here is my comments:

How the 5 mg dose of α-chymotrypsin was chose?

Not sure to have understand correctly the equation (I may be wrong). If we look at the equation the we assume that the number of sperm recovered after processing should be higher than before processing. In this equation, the higher the number of sperm recovered the lower the % should be. In my opinion the correct way to calculate that is:

%= ((sperm after processing – sperm before processing) / Sperm before treatment) x100

In this way if we assume that before treatment we found only 10 spermatozoa and 15 after processing, we can easily say that there’s an increment of 50%. With my equation it’s 50%. With your’s equation is 66,7%

However, as I mentioned before I may be wrong. Please comment. But if your equation is wrong all results must be reconsidered.

Looking forward for your comments to complete the revision.

Author Response

Comments 1: How the 5 mg dose of α-chymotrypsin was chose?

Response 1: We used commercial kit of α-chymotrypsin, QwikCheck^TM Liquefaction Kit, Medical Electronic Systems, CA, USA.

Comments 2: Not sure to have understand correctly the equation (I may be wrong). If we look at the equation the we assume that the number of sperm recovered after processing should be higher than before processing. In this equation, the higher the number of sperm recovered the lower the % should be. In my opinion the correct way to calculate that is:

%= ((sperm after processing – sperm before processing) / Sperm before treatment) x100

In this way if we assume that before treatment we found only 10 spermatozoa and 15 after processing, we can easily say that there’s an increment of 50%. With my equation it’s 50%. With your’s equation is 66,7%

However, as I mentioned before I may be wrong. Please comment. But if your equation is wrong all results must be reconsidered.

Response 2: Sperm recovery rate is to calculate how much motile sperm has been recovered, so post-wash TMC should be always less than pre-wash TMC. Because some portions of motile sperm will be loosed during wash process. This is common calculation in IVF lab.

Reviewer 3 Report

Comments and Suggestions for Authors

In this manuscript, Ayyar et al., report the clinical evaluation of using α-chymotrypsin to treat highly viscous semen samples in 35 patients. They used paired test comparing total motile sperm count (TMC), motile sperm recovery rate and fertilization rate between untreated and treated samples and found that α-chymotrypsin treatment led to improvement in sperm recovery rate and total motile sperm count without adversely affecting assisted reproductive technology (ART) outcomes. Overall, the study is of clinical relevance and significance, as intrauterine insemination (IUI) and in vitro fertilization (IVF) are used to overcome male infertility, but semen hyperviscosity (SHV) poses a challenge to the success of these ART methods. The experiments are designed and performed in a scientific fashion. I recommend accepting the manuscript with some minor comments and suggestions.

• In all 3 figures, it would be good to use dot plots to show values of all individual samples, in addition to the bar graph shown in the manuscript. This would allow the readers to appreciate the variability of data.
• In L141-142, the authors mention ‘this was not significantly different’. Please specify the statistical analysis used here and add the P value.

Author Response

Comments 1: In all 3 figures, it would be good to use dot plots to show values of all individual samples, in addition to the bar graph shown in the manuscript. This would allow the readers to appreciate the variability of data.

Response 1: Authors decided to use bar graph using average sperm motility and recovery rate because huge variation of motility and recovery between patients (for example, 4% to 83% of recovery rate improvement). However, treatment group consistently showed improvement of motile sperm recovery in each comparison.  

Comments 2: In L141-142, the authors mention ‘this was not significantly different’. Please specify the statistical analysis used here and add the P value

Response 2: Added in the manuscript (line 143)

Reviewer 4 Report

Comments and Suggestions for Authors

In the article titled “Improvement in Sperm Recovery Rate and Total Motile Sperm  Count Using α-chymotrypsin in Highly Viscous Semen Sample  without Adversely Affecting Assisted Reproductive Technology Outcomes” the authors  investigate the impact of α-chymotrypsin treatment on sperm recovery rate and total 20 motile sperm count (TMC) in highly viscous semen for intrauterine insemination (IUI) and in vitro 21 fertilization (IVF), particularly in cases of severely low sperm count.

I find it an interesting work but I suggest a major revision, especially since the state of the art is not up-to-date and also to try to correlate the results obtained with each other for a better understanding of the paper.

The suggestions I would make are as follows:

• Since environmental pollution is a major concern worldwide which affects male infertility, I suggest the authors to amply this concept reading and quoting the following paper: 10.3390/ijerph191711023
• Define the selection criteria of the subjects recluted in this trial. Define the characteristics of the subject ( age, smoker? Drinker? Live in polluted areas? Physical activity? Diet? Etc.
• Lines 87-88: “The experimental portion of the sample was treated with 5 mg of α-chymotrypsin for 5 minutes at 37°C.” what is the volume of ejaculate used for 5 mg of α-chymotrypsin
• sperm wash media? State the composition of this media
• After centrifugation, each group’s pellet was resuspended in 2- 4mL of sperm wash media and concentrated via centrifugation for 5 minutes at 400g ………….why at 400 g and not at a higher velocity?
• In the sperm recovery rate, is the denominator the total motility or the progressive motility?
• The results must be commented on and an explanation must also be given for the effect of α-chymotrypsin on the semen. A molecular mechanism must also be better hypothesized
• try to correlate the results obtained with each other
• The potential tossicity of α-chymotrypsin must be determined

Minor point

• I believe that to make the article more appealing you should include a nice color image that summarizes the results, the molecular mechanism and the message the authors want to give
• Better define the limitations of this study
• English revision required

Author Response

Comments 1: Since environmental pollution is a major concern worldwide which affects male infertility, I suggest the authors to amply this concept reading and quoting the following paper: 10.3390/ijerph191711023

Response 1: Although this paper that you recommended is good to understand how environmental pollution affects male infertility, there is no discussion how environmental pollution affects on semen viscosity. We think this paper has slightly different focus comparing to our paper.

Comments 2: Define the selection criteria of the subjects recluted in this trial. Define the characteristics of the subject ( age, smoker? Drinker? Live in polluted areas? Physical activity? Diet? Etc.

Response 2: As we described in the material and method, the selection criteria is only viscous semen sample for IUI treatment. Because this study is pared analysis of sperm recovery with/without α-chymotrypsin treatment mainly in IUI, we don’t typically collect further demographic or physical information of male. This suggestion will be good for next randomized control test.    

Comments 3: Lines 87-88: “The experimental portion of the sample was treated with 5 mg of α-chymotrypsin for 5 minutes at 37°C.” what is the volume of ejaculate used for 5 mg of α-chymotrypsin

Response 3: Per vendor’s protocol, 5 mg of α-chymotrypsin can be treated up to 5 ml of semen. All specimens were treated with 5 mg of α-chymotrypsin regardless of semen volume per vendor’s protocol. The range of semen volume was 1.2 ml to 3.5 ml.

Comments 4: sperm wash media? State the composition of this media

Response 4: we used commercial sperm wash media that widely used in IVF center. We added the vendor info in the material section.

Comments 5: After centrifugation, each group’s pellet was resuspended in 2- 4mL of sperm wash media and concentrated via centrifugation for 5 minutes at 400g ………….why at 400 g and not at a higher velocity? 

Response 5: This is our validated protocol. Higher velocity could impact sperm motility and viability

Comments 6: In the sperm recovery rate, is the denominator the total motility or the progressive motility? 

Response 6: Total motility

Comments 7: The results must be commented on and an explanation must also be given for the effect of α-chymotrypsin on the semen. A molecular mechanism must also be better hypothesized

Response 7: It is well known that chymotrypsin is a proteolytic enzyme that can be used to break down the proteins that contribute to semen viscosity. In this study, we focused on the clinical application of the treatment.

Comments 8: try to correlate the results obtained with each other

Response 8: Refer to line 163-173 in the discussion section.

Comments 9: The potential tossicity of α-chymotrypsin must be determined

Response 9: We used commercial kit of α-chymotrypsin, QwikCheck^TM Liquefaction Kit, Medical Electronic Systems, CA, USA. This has been added in the material section. The vendor recently upgraded the kit by providing a think powder in each kit. The vendor also performed clinical experiment of no impact to human sperm motility by treatment and referenced WHO manual in their internal datasheet.

Comments 10: I believe that to make the article more appealing you should include a nice color image that summarizes the results, the molecular mechanism and the message the authors want to give

Response 10: It is well known that chymotrypsin is a proteolytic enzyme that can be used to break down the proteins that contribute to semen viscosity. In this study, we focused on the clinical application of the treatment.

Comments 11: Better define the limitations of this study

Response 11: Refer to our comment in the end of discussion section, “Future randomized controlled trial research with large sample size should aim to refine treatment protocols and assess its broader clinical implications”.

Comments 12: English revision required

Response 12: Other 3 reviewers were ok with English, but we did minor changes of English. Authors are comfortable for the current version regarding quality of English. 

Round 2

Reviewer 4 Report

Comments and Suggestions for Authors

The authors responded sufficiently to my requests.