Testicular and Haematological Cancer Induce Very High Levels of Sperm Oxidative Stress

Round 1

Reviewer 1 Report

In this study attempts were made to show that patients affected with testicular and hematological cancers are more susceptible to increased levels of oxidative stress (OS) compared with normozoospermic patients. The Reviewer suggests that the following comments would be helpful to improve the quality of the manuscript.

1. A major drawn back of this this study is the poor analysis of the data. The statistical analysis is incomplete and the poor choice of statistical tests would give unreliable results, and finally their poor interpretations. According to the study design, the data do not follow a normal distribution (L175) and the Kruskal-Wallis test (ANOVA) should be used to detect differences among the groups  because there are five main groups (CP, HD, TCP, NSP and HD, Table 1). If there are differences among he groups then use a nonparametric test, for example, Wilcoxon Mann-Whitney, for pairwise comparisons. The same applies for the data shown in Figures 2 to 4.

2. Data show in Table 1 and Figures 2 to 4 are unreadable, making it very difficult to analyze the  findings of this study. For Figure 4, show the coefficient and p values.

3. Without a re-visit of the statistical analysis, the manuscript is incomplete.

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Reviewer 2 Report

The manuscript entitled “Cancer induces very high levels of sperm oxidative stress” evaluates sperm oxidative stress and sperm DNA fragmentation in patients of testicular and haematological cancer. Oxidative stress was assessed in total sperm and in viable sperm fraction using double staining with MitoSOX Red and LIVE/DEAD Fixable Green Dead Cell Stain (LD-G), coupled with flow cytometry. The study is interesting and novel and deserves to be published. However, there are some minor concerns and suggestions for the authors.

1.             The main shortcoming of this paper is availability of data of different analyses in different numbers of subjects (testicular cancer patients, hematological cancer patients, normozoospermic men, healthy controls) that is sometimes difficult to follow. For example, study included 79 testicular cancer patients, 44 haematological cancer patients (i.e., 123 cancer patients), 52 normozoospermic man and 19 healthy donors (i.e., 71 controls). Oxidative stress was determined in 96 and sDF in 85 cancer patients and in 71 controls. However, according to the lines 23 and 276, sDF correlated with viable oxidative stress in all subjects, cancer patients and control, N=134 subjects. How did you calculate 134 subjects (85 cancer patients with sDF and 71controls = 156)? I suggest to Authors to add a flowchart illustrating the distribution of patients/control subjects according to different analyses.

2.             The title “Cancer induces very high levels of sperm oxidative stress” implies that all cancer induces very high levels of sperm oxidative stress. However, in this study, only patients with testicular and haematological cancer were included. Therefore, maybe the title could be more precise.

3.             It is not clear why testicular and haematological patients were selected. Is there any reason?

4.             Abstract, line 16, “ … and total (ROS production in viable sperm fraction/total spermatozoa) …”: “viable sperm fraction” should be replaced by “total sperm”.

5.             In the study, testicular patients with and without orchiectomy were included. How samples were collected - particular in the subjects with orchiectomy (by masturbation)? Is only one testicle removed in patients with orchiectomy (unilateral orchiectomy)?

6.             Table 1: What data are presented? Median [IQR]? Please, add the number of subjects in various study groups (HD, TCP, NSP). HD group was presented twice (in two rows). Please, correct it.

7.             Table 1, Morphology (%): For those who are not familiar with the subject, it would be clearer if “% of normal forms” is added to Morphology.

8.             Suggestion: Please, please explain the abbreviations for those readers who are not familiar with the subject (e.g., line 160, please add “sperm chromatin dispersion (SCD) test”; PI and FR region in the Supplemental Figure 1).

9.             Lines 274-285: Maybe it might be clearer to add that the results for total oxidative stress are not shown.

10.         Lines 306-308, “In seminoma, we found a sharp, albeit not significant (p=.120), decrease of viable oxidative stress following surgery (without orchiectomy: 39.05[26.77-68.72]%, n=14, vs with orchiectomy: 20.47[14.00-45.90]%, n= 11)”.: Orchiectomy is a surgical removing of one or both testicles. Therefore, there is a mistake: It should probably be “ … following surgery (with orchiectomy: 20.47[14.00-45.90]%, n= 11), vs without orchiectomy: 39.05[26.77-68.72]%, n=14)”. Please, check the results and number of subjects.

11.         Discussion, line 318, “In this study we observed dramatic levels of sperm oxidative stress”: Suggestion: please, replace “dramatic” with “increased”.

12.          Line 330/331, “We also found that cancer patients prior to therapy showed increased levels of sDF, … ”: It is not clear. What therapy? This study did not compare the sDF data in patients before and after any therapy.

13.         Line 96 and 330, Supplemental Table 2: Please, correct units for sperm number (and concentration) in 10 exp 6 (not 106).

14.         Supplemental Table 2: N=? Data are presented as median [IQR]. Please, replace “+/-“ with “-”.

I think that only minor editing of English language is required. 

Author Response

Please see the attachment

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

The Authors have  satisfactorily addressed my comments, except to a minor  clarification.

Place Table 1 on one page and put the significant differences as"<0.001" instead of "0.000"

 

 

Author Response

Please see the attachment.

Author Response File: Author Response.pdf