Impact of coenzyme Q10 as an adjuvant therapy to letrozole on spermiogram results and sex hormone levels in Iraqi men with infertility; randomized open label comparative study

Background: Worldwide, infertility affects about 15% of reproductive-age couples. In many cases, infertility can't be treated, however new treatment options with promising value have been involved in recent clinical trials. The aim of this clinical trial was to evaluate the impacts of adding coenzyme Q10 (CoQ10) to letrozole on the results of spermiogram and sex hormone tests in men diagnosed with idiopathic oligoasthenoteratozoospermia (iOAT) syndrome, which is a type of male defective spermatogenesis of unknown etiology. Methods: This randomized, open-label, parallel two-arm interventional study included 67 adult male patients aged 18-60 years with a confirmed diagnosis of iOAT syndrome recruited from The High Institute for Infertility Diagnosis & Assisted Reproduction Technologies/Nahrain University. Patients were randomly separated into two groups, Group A included 29 patients treated with letrozole 2.5 mg tablet orally twice a week, Group B included 38 patients treated with a combination of letrozole 2.5 mg tablet orally twice a week plus CoQ10 400 mg per day. Both groups completed treatment for three months. Semen samples, serum follicle-stimulating hormone (FSH), estradiol (E 2), and testosterone (T) were analyzed at day one, and at the end of month one, two and three. Results: Both groups showed that sperm concentration, normal morphology, total sperm count and motility, serum testosterone and FSH levels, and T/E 2 ratio were significantly increased, while estradiol levels significantly decreased after three months of treatment. Seminal fluid volume changed significantly in group A only. In comparing between the two groups, all measured parameters, apart from sperm motility and FSH level, demonstrated a significant difference after three months of treatment, while sperm volume reached significant value after only two months of therapy. Conclusions: CoQ10 as adjuvant treatment to letrozole effectively improved most of the tested sperm parameters in Iraqi men with iOAT. Registration: ClinicalTrials.gov ( NCT05847257, May 6, 2023).


Introduction
The World Health Organization (WHO) defines infertility as "the incapability of a couple to conceive in spite of having regular sexual activity for at least one year without using any contraceptive methods". 1Around the world, approximately 15% of reproductive-age couples are struggling with infertility, 2 about half of these issues are related to male factors. 3here are many causes contributing to male infertility, including infection, 4 alteration in organ function, 5 environmental factors, genetic factors, 6 and sex hormone disturbance. 7diopathic male infertility is referred to the impairment of sperm parameters without clear male-associated cause, although physical examination, endocrine, genetic, and biochemical laboratory results for these men are normal, semen analysis can show abnormal findings, they do not have a history of disorders that influence fertility".8 Idiopathic oligoasthenoteratozoospermia (iOAT) is associated with faulty spermatogenesis and is characterized by unusually low sperm count, motility, and a large number of dysmorphic spermatozoa in the ejaculate, The etiology of which is unclear and is frequently considered undetectable by standard laboratory techniques.About 30% of OAT patients are identified as idiopathic, and idiopathic testicular abnormalities cause the most severe cases of OAT. 9 Although established disorders such as varicocele, cryptorchidism, and hypogonadism are identifiable causes of OAT and infertility, approximately 25% of these individuals do not have a known explanation behind their irregular semen analysis.10,11 A complex interaction of hormones that act centrally and intratesticular is necessary for spermatogenesis.In response to the activity of gonadotropin-releasing hormone (GnRH) from the hypothalamus, the anterior pituitary secretes luteinizing hormone (LH) and follicle-stimulating hormone (FSH).In the testis, FSH operates on Sertoli cells to stimulate the maturation of spermatogonia.LH has an influence on Leydig cells promoting testosterone synthesis.Local testosterone concentrations must be significantly greater than serum levels for effective spermatogenesis. T, by its effects on Sertoli cells, this intratesticular testosterone indirectly promotes the development of germ cells.7 Many hormones participate in regulating spermatogenesis.LH stimulates Leydig cells to release testosterone to promote sperm production and virilization.Additionally, it has negative feedback that suppresses the release of LH and FSH from the pituitary gland.FSH promotes Sertoli cells to support spermatogenesis, which releases inhibin B, which regulates FSH secretion by its negative feedback.FSH is required to establish spermatogenesis and it's very important to understand that testes will produce lower numbers and poorer quality of sperm with only FSH stimulation, while LH is necessary to obtain both quantity and quality of sperm production, as a result neither FSH alone nor LH alone is sufficient to produce high quality sperm.12 Aromatase is a cytochrome p-450 enzyme present in the adipose tissue, testes, liver, and female reproductive organs that has roles in converting testosterone (T) to estradiol (E 2 ) and androstenedione to estrone.Aromatase inhibitors block the conversion of T to E 2 ; therefore, the level of testosterone is increased while the estrogen level is suppressed.As the endogenous testosterone levels rise, and in combination with the reduction in the estrogen suppressing role on the hypothalamic-pituitary-gonadal (HPG) axis, spermatogenesis is further stimulated.13 In men, the modification of plasma E 2 levels to the normal physiological range results in a positive impact on FSH, LH and testosterone levels mediated by an effect on the pituitary gland derived by this reduction.14 Therefore in men with low testosterone levels, aromatase inhibitors enhance testosterone secretion.15 Letrozole belongs to third-generation aromatase inhibitors that inhibit estrogen biosynthesis reversibly and act as an anticancer agent for advanced breast cancer.16 The medication dosage form is tablet 2.5 mg and when taken orally it is absorbed readily (bioavailability 99.9%); food has no effects on its absorption.Letrozole is rapidly distributed, its excreted mainly through the urine (about 90%) and its half-life is about two days.17 Oxidative stress (OS) and reactive oxygen species (ROS) are believed to damage the spermatozoa and account for 30 to 80% of infertility cases in men.18 ROS in semen originates from different endogenous and exogenous sources,

REVISED Amendments from Version 2
We have improved some of the errors in the tables, improved the clarity of our research, and corrected some errors like improvement in sperm motility (which we did not achieve when comparing both groups; however, there was improvement in each group throughout the three months of follow-up).
Any further responses from the reviewers can be found at the end of the article the endogenous sources include round cells, epithelial cells and leukocytes, while lifestyle factors including drinking alcohol, smoking and environmental sources (such as radiation and toxins) are considered sources of exogenous ROS. 19ncreased ROS production causes oxidative stress and reduces the antioxidant capacity of spermatozoa. 20Spermatozoa have plasma membranes that are made up of lipids and polyunsaturated fatty acids, increased levels of ROS make the membrane vulnerable to lipid peroxidation and damage. 21The lower motility and reduced fluidity of membranes in sperm occur due to lipid peroxidation and are associated with reduced ability of sperm to fertilize. 22Coenzyme Q10 (CoQ10) is a fat soluble vitamin-like molecule naturally found in cell membranes in the human body and it is naturally found in our diet and can be synthesized endogenously. 23CoQ10 has antioxidant effects and is involved in the production of mitochondrial energy, which is important for maintaining the power source of spermatozoa and provide protection to their membranes from damage through lipid peroxidation.Therefore, it's considered among the most extensively utilized antioxidants as an option to treat idiopathic infertility in men. 24The aim of the current clinical trial was to evaluate the impacts of adding CoQ10 to letrozole on spermiogram results and sex hormone levels in men diagnosed with iOAT syndrome.

Ethical statement
Written informed consent was obtained from each participant to participate and publish clinical information.The study was approved by both the institutional regulation board of the Department of Clinical Pharmacy/College of Pharmacy/ Mustansiriyah University (ID number: 3983, date: 14 th December 2021), and the High Institute for Infertility Diagnosis & Assisted Reproductive Technologies/Nahrain University (Date: 3 rd November 2021; Ethical code: A21024). 25The recruitment period started on the 1 st of January 2022 after both faculties approved the study.The study followed the Declaration of Helsinki (2008) for research on human subjects and its later amendments.This trial was registered with ClinicalTrials.gov(NCT05847257) on May 6, 2023.Due to the recent introduction of required clinical trial registration in Iraq, we registered the study retrospectively to assure its transparency.This study adhered to the CONSORT guidelines, no harms or unintended effects have been reported for this study.

Trial design
This study was a randomized, open-label, parallel two-arm interventional study.The study initially included 74 patients; on follow-up, seven cases were lost, and the final analysis involved 67 cases.These patients were further divided into two groups randomly: Group A (29) patients received letrozole 2.5 mg (Letrozole ® 2.5 mg, Accord Healthcare Limited, UK) twice weekly for three months, and Group B (38) patients received letrozole 2.5 mg twice a week plus CoQ10 400 mg (CoQ-10 ® , Natrol, USA) once per day (200 mg twice daily) for three months as illustrated in Figure 1.

Study settings
Patients with iOAT syndrome were selected and diagnosed during their visit to The High Institute for Infertility Diagnosis & Assisted Reproduction Technologies/Nahrain University.Before taking part, each subject provided their written, informed consent.The duration of this study was from the 1 st of December 2021 until 1 st of October 2022.

Exclusion criteria
1. Patients who have been found to have additional infertility causes, such as varicocele or obstruction of the ejaculatory duct.
2. Those who have had surgery for male factor infertility.
3. Patients with infections such as sexually transmitted infections (STIs).
4. Patients with renal or liver disease.

Seminal fluid analysis
The samples were collected via masturbation after abstinence of 3 to 4 days directly into a dry, clean disposable widemouth plastic container in a private room close to the laboratory in the institute.Immediately after that the samples were carried to laboratory of semen examination then put in an incubator at 37°C for 30 minutes.After complete liquefaction, the semen analysis was done by macroscopic and microscopic examination according to standard criteria of WHO (2021). 26

Randomization
The randomization process was done utilizing the online software Research Randomizer.First, the patients were sequentially numbered during the interview and then randomly assigned to one of two groups using the online software the block randomization design was implemented in which we divided the participants into 14 blocks (sets) each with a block size of 6 patients stratified by treatment groups).The proportion of the control and intervention groups was planned to be 1:1 (balanced design); however, due to the loss of some cases, the final proportion became 0.76:1.

Primary outcomes
Seminal fluid analysis Assessment of semen volume, sperm concentration, total sperm count, progressively motile sperm (%), non-progressively motile sperm (%), immotile sperm (%), morphologically normal sperm (%).Time Frame: values changes from baseline to the end of the first, second or third month for three consecutive months of treatment.

Hormone profile
Assessment of concentration of serum FSH, estradiol, and testosterone.Time Frame: change in value from baseline to the end of the first, second and third month for three consecutive months of treatment.

Sample size
A previous study by Peivandi et al., 27 showed that letrozole 2.5 mg increases sperm concentrations over three months and demonstrated an in improvement by 33% for sperm parameters (concentration after three months of therapy) so we assumed that the use of CoQ10 will increase this value to 55%.Using MedCalc (RRID:SCR_015044) software version 14.8.1, we arrived at 64 patients for both groups to confirm the null hypothesis (with type I error 5% and type II error of 5%), we increased the sample to 74 to account for maximum possible loss in patients being around 15% without affecting the accuracy of the results.

Statistical analysis
All analyses were carried using IBM SPSS Statistics (RRID:SCR_016479) version 28.1 (Chicago, USA), repeated measures ANOVA was used to assess the differences in each group across four time periods with post hoc Tukey's test to assess the pair wise comparisons.An independent t-test was used to assess the difference between each treatment group.A chi-squared test was used to assess the difference in categorical variables.The level of significance was 0.05 (alpha level), and all p-values were two tailed.For sample size calculations MedCalc (RRID:SCR_015044) software version 14.8.1 was used.

Results
The demographic data and baseline characteristics of all participants are summarized in Table 1. 41In this table, there were non-significant variations among all parameters between the two groups.
Table 2 shows the results of letrozole effects alone and with CoQ10 on sperm production after one, two and three months.The effects on both groups showed a significant growth in the mean value of sperm concentration and total sperm count in respect to the baseline (p<0.01).However, significant (p<0.05) and non-significant (p>0.05)changes in seminal fluid volume in group A and B were reported, respectively.At the end of the current study, the results revealed significant (p<0.05)differences between the two groups for all parameters (sperm concentration and total sperm counts) except seminal fluid volume, which was significant only after two months (p>0.05).
Table 3 illustrates the effects of letrozole alone and with CoQ10 on sperm motility and morphology after one, two and three months.Statistically, significant improvement was reported in both groups regarding sperm motility and morphology after three months of treatment.However, comparison between the groups, showed no significant differences (p>0.05) in all parameters except the only sperm morphology was significantly improved in group B compared to that of group A (p<0.05).
Results of comparing the effect of letrozole alone and in combination with CoQ10 on sex hormones after one, two and three months are demonstrated in Table 4.In both groups, a highly significant (p<0.01)increase in testosterone and FSH levels was noticed.Conversely, estradiol levels were significantly decreased (p<0.01).Between the two groups, significant (p<0.05) and highly significant (p<0.001)changes in testosterone and estradiol levels were noticed, respectively, with no significant (p>0.05)changes for FSH levels.Regarding the ratio of T/E 2 , the growth was about 190% and 312% for group A and B from the base line, respectively, the statistical significancy was achieved between the two groups (p<0.05).

Discussion
The male reproductive capacity is affected by many demographic factors, with age seemingly the most sensitive factor since aging is negatively correlated with spermatogenesis. 28In the current study, most participants were in their thirties; consequently, a correlation may be present relating to the quantity and quality of sperm.The second important factor is smoking, a study by Jin-Bo Dai et al., (2015) confirmed that tobacco smoking is considered a risk factor for developing infertility, smoking cigarettes, water pipes or vapes may contain many harmful chemicals that disrupt the antioxidant status of testes and consequently lead to the interruption of spermatogenesis. 29However, in spite of the adverse consequences of smoking on male fertility, many men are still fertile but they are at risk for becoming infertile. 30More than half of the patients in the current study were smokers and this may have a great influence on the quality of sperms.
In this study, most of the patients were overweight and obese, this may have an association with low quality of spermatozoa.Belloc et al., (2014) showed a correlation between high BMI and low semen quality, whereas sperm morphology is not affected. 31In previous clinical trials, letrozole was used alone at a dose of 2.5 mg per day to treat infertility in men, 32,33 while another previous study used a weekly dose of 2.5 mg letrozole to improve testosterone. 34owever, the dose of letrozole used in the current study was 2.5 mg twice a week for all patients, this came from the experience of consultants in an attempt to decrease the expected side effects, including loss of libido, which was reported in earlier studies. 33,35study by Kooshesh et al., (2020) concluded that the use of letrozole in the treatment of men with iOAT and T: E2 ratio ≤ 10, can successfully increase sperm quality and chromatin integrity, and consequently increase spontaneous pregnancy.13 To a large extent, these finding are in agreement with the current results.
In men, estrogen is mainly produced by the conversion of testosterone to E 2 via aromatase enzymes, 36 an excess of estrogens can block the HPG axis, therefore, infertility occurs due to reduced release of FSH and LH.Administration of letrozole can lead to increased testosterone and decreased estradiol production.So, by improving the T/E 2 ratio, this can have positive effects on spermatogenesis and obtaining high quality and quantity of sperm. 7e addition of CoQ10 to letrozole showed notable improvement in the ratio of T/E 2 (312%) compared to that of using letrozole alone (190%) after three months of treatment.Peivandi et al., (2019) showed a correlation between the improvement in spermatogenesis to the increase in the ratio of T/E 2. 27 Appasamy et al., (2007) suggested that the increased ROS levels may have potential to disrupt hormonal balance and reduce the levels of male sex hormones, therefore resulting in infertility. 37However, administration of CoQ10, which has notable antioxidant properties to scavenge ROS and reverse oxidative stress, results in the improvement of spermiogram parameters. 38afarinejad (2009) illustrated the effects of CoQ10 on sperm parameters, using 300 mg CoQ10 a day for six months to show enhancement in sperm count, motility and morphology, while decreasing the levels of FSH. 39In the present study, a combination of CoQ10 and letrozole was used for three months.The results indicated improvement in sperm count, morphology, and concentration.Furthermore, an assessment of sex hormone levels showed an increase in testosterone, a decrease in E 2 levels, and an improvement in their ratios.
Spermatozoa's plasma membrane comprises lipids and polyunsaturated fatty acids; therefore, excessive ROS makes the membrane vulnerable to lipid peroxidation damage. 40Since ROS attacks the double bonds in the unsaturated fatty acid, forming a lipid peroxide radical, which starts to interact with the adjacent lipid molecule and trigger a chain reaction that compromises the cell membrane, ultimately lipid peroxidation causes DNA fragmentation and sperm apoptosis.The antioxidant effect of Co-Q10 will protect the cell member and stop sperm death; consequently, it will enhance spermatogenesis, as evidenced by enhanced motility and morphology.
The combined administration of letrozole and CoQ10 led to significant improvements of all spermiogram parameters, this combination appears to have a synergistic effect to improve spermatogenesis.

Study limitations
A limitation of this study includes the small sample size for both groups due to missing many patients during follow-up periods and exclusion criteria mentioned above.In addition, there was a lack of control group due to the difficulty in convincing men who have not been diagnosed with infertility to undertake SFA and hormone analysis.Thirdly, no sensitivity analyses were conducted for the confounding variables.Finally, ROS in seminal fluid was not measured due to the lack of availability of tools to measure ROS in semen.

Conclusions
The combined use of letrozole and CoQ10 was found to improve sperm parameters and patients showed a boosted improvement in some of the spermiogram parameters compared to that of patients administered letrozole alone.Based on the results of this study, letrozole plus CoQ10 is recommended to treat patients with iOAT syndrome who have high estradiol and low testosterone levels.
condensation in male infertility are some of my focuses about my studies.

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard. Version 2
Reviewer Why is the antioxidant usage period determined as three months?3.
How was Coenzyme Q10 concentration used by the participants determined? 4.
In statistical analyses, the tests used to analyze the dependent variables should be added along with the analyzes of the independent variables.

5.
It is known that smoking causes oxidative stress and it is also stated in the article.However, smoking was not analyzed in the study other than as demographic data.Researchers may have evaluated the parameters of smoker and non-smoker participants separately.

6.
It should be mentioned in the discussion why semen volume increased in the group using only letrozole compared to the group using letrozole + coQ10.

7.
The reason for the apparent effect of CoQ10 on sperm morphology is not mentioned in the discussion, but should be mentioned.

If applicable, is the statistical analysis and its interpretation appropriate? Partly
Are all the source data underlying the results available to ensure full reproducibility?Yes

Are the conclusions drawn adequately supported by the results? Partly
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Epigenetics, DNA damage, ROS, antioxidant capacities, sperm chromatin condensation in male infertility are some of my focuses about my studies.

Qinghua Shi Division of Reproduction and Genetics, University of Science and Technology of China, Hefei, China
This manuscript presents the results of letrozole alone and with CoQ10 on semen parameters after one, two and three months-treatment on patients with iOAT, and achieved a promising conclusion.However, the number of patients receiving the treatment is small, research from more patients is needed to confirm its conclusion.Besides, some points need to be fixed: 1.Recent and original research papers related to this topic should be cited.2. "LH stimulates Leydig cells to release testosterone to promote the production of sperm and virilization and exerts negative feedback to suppress ......FSH promotes Sertoli cells to support spermatogenesis to release inhibin B .....", Too many "to".The subject before "exerts" is not clear.3. "......ecological factors (such as radiation and toxins) are.....", radiation and toxins are not and ecological factors.4. "The proportion of the control and intervention groups was 1:1 (balanced design)" is not correct.5. "Assessment of sperm volume/ ml, sperm concentration/ ml, total sperm count/ml, progressive sperm motility (%), non-progressive sperm motility (%), immotile sperm (%), normal sperm morphology (%)" should be: "Assessment of semen volume, sperm concentration, total sperm count, progressively motile sperm (%), non-progressively motile sperm (%), immotile sperm (%), morphologically normal sperm (%)".6. "Assessment of concentration of serum FSH, concentration of serum estradiol, and concentration of serum testosterone" should be "Assessment of concentration of serum FSH, estradiol, and testosterone."7. Table 2, the "/ml" in the first column must be deleted.8.In several Table legends, ""between (Time-wise values)" within each group" should be rephrased."Different small case letters indicate significant difference." is not clear.9. Table 3, The name in the first column is inaccurate.10.Discussion, "the results indicated improvement in sperm motility, ....." , ".....levels showed an increase in FSH .....levels", "The combined administration of letrozole and CoQ10 led to significant improvements of all spermiogram parameters," are not true.In "This combination appears to have a synergistic effect to obtain higher quality spermatogenesis", should be changed to "......to improve spermatogenesis".11.Conclusion, "The combined use of letrozole and CoQ10 was found to improve sperm parameters and patients showed a boosted improvement in all spermiogram parameters compared to that of patients administered letrozole alone.".The "all" should be removed.12. Acknowledgments, "Dr Hayder A. Fawzi for its support and encouragement to", the "its" should be "his".

If applicable, is the statistical analysis and its interpretation appropriate? Partly
Are all the source data underlying the results available to ensure full reproducibility?Yes

Are the conclusions drawn adequately supported by the results? Partly
Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Spermatogenesis, reductive genetics, meiosis, infertility I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

Introduction:
The content is well-written and easy to understand.Although the mechanisms of sperm protection by CoQ10 and letrozole are elucidated, a thorough review of published articles on the effects of CoQ10 and letrozole treatment on OAT is more important.The rationale for using both drugs in this study should be rigorously discussed.Did the author expect the synergistic function?The present introduction is better summarized to avoid a lengthy introduction.
Cited references are usually those that provide evidence or information for the first time, rather than articles that reuse that information.
Reference 1 needs to be the statement of WHO.Please check it carefully.
References 2 and 3 are not the original articles that reported the prevalence of 15% or the prevalence of malefactors.Please cite the original article and authors who reported this information.
Reference 6 is a minireview, not the original article that provided the information as cited.Please cite the original article and authors who reported this information.
Reference 7 is a review of the significant aspects of irisin actions and its involvement in energy homeostasis and male reproduction.Please cite a more relevant article.
"Idiopathic oligoasthenoteratozoospermia (iOAT) is associated with faulty spermatogenesis and is characterized by unusually low sperm count, motility, and a large number of dysmorphic Q/ The first two sentences are not relevant to the heading.It would be better to describe what is known, such as the effect of CoQ10 on sperm quality or male sex hormones; and what aspects need further exploration.Are there any similar studies published?It is better to summarize the results here.
A/ This article focuses first and most on infertility, so we started our article in defining the main issue based on well-known definition of WHO, and after we defined infertility, we introduced the intervention as you mentioned.Next the new background.
"Infertility is common problem affecting many couples, still many interventions directed to treat infertility with varying outcomes.The aim of this clinical trial was to evaluate the impacts of adding coenzyme Q10 (CoQ10) to letrozole on the results of spermiogram and sex hormone tests in men diagnosed with idiopathic oligoasthenoteratozoospermia (iOAT) syndrome, which is a type of male defective spermatogenesis of unknown etiology."

Introduction:
Q/ The content is well-written and easy to understand.Although the mechanisms of sperm protection by CoQ10 and letrozole are elucidated, a thorough review of published articles on the effects of CoQ10 and letrozole treatment on OAT is more important.The rationale for using both drugs in this study should be rigorously discussed.Did the author expect the synergistic function?The present introduction is better summarized to avoid a lengthy introduction.
A/ During our initial proposal of the research, we kept in mind possible additive effect at least and at best synergic effect.
Cited references are usually those that provide evidence or information for the first time, rather than articles that reuse that information.A/ New definition: "Idiopathic male infertility is referred to the impairment of sperm parameters without clear male-associated cause, Although physical examination, endocrine, genetic, and biochemical laboratory results for these men are normal, semen analysis can show abnormal findings, they do not have a history of disorders that influence fertility" [ We gave our explanation inside the article, please refer to the designated section inside "ethical statement", " Due to the recent introduction of required clinical trials registration in Iraq, we registered the study retrospectively to assure its transparency."Q/ The authors are required to provide the details of methods to make the process reproducible.We included all possible methodologies and we adhered to the CONSORT checklist, again if something specific is/are unclear please ask specifically and we will provide an answer.Q/ How to randomize the participants?Sealed envelope?How many sets?The proportion of the control and intervention groups is better stated.I suppose it was 1:1?We implemented a block randomization design in which we divided the participants into 14 blocks (sets) each with a block size of 6 patients stratified by treatment groups, see Link table .The proportion of the control and intervention groups was 1:1 (balanced design).Q/ How to determine the sample size?Did you estimate the sample size based on the semen concentration?What is the initial semen concentration and the expected concentration after treatment?If the authors used the semen concentration of Peivandi's study, did you include the same kind of participants Peivandi included male patients with abnormal parameters of semen fluid based on WHO criteria, which were serum T/E2 ratio < 10 (testosterone mg/dl and estradiol pg/ml).We estimated the sample by A previous study by Peivandi et al., showed that letrozole 2.5 mg increases sperm concentrations over three months and demonstrated an in improvement by 33% for sperm parameters (concentration after three months of therapy) so we assumed that the use of CoQ10 will increase this value to 55%.Using MedCalc (RRID:SCR_015044) software version 14.8.1, we arrived at 64 patients for both groups to confirm the null hypothesis (with type I error 5% and type II error of 5%), we increased the sample to 74 to account for maximum possible loss in patients being around 15% without affecting the accuracy of the results.Keep in mind that sample size is a method to estimate the sample size, within certain levels of certainty and best to relay on the trial outcomes.Q/ For sample estimation: If the authors assessed multiple parameters, concentration, motility, and morphology, the largest sample size estimated from each parameter will be used.That means if the sample size needed based on concentrations is less than the sample size calculated based on motility, the sample size used for motility should be used.Could you please provide the process of sample calculation?Yes, the largest sample size for sperm concentrations was used.And attached is the output of the MedCal program.See linked figure .Q/ It is better to provide the definition of OAT? How many semen samples per patient were used?How much is the lag time between each sample collection?We included a definition of OAT in the introduction The semen sample was collected four times per patient (once at baseline, after 1 month, after 2 months, and after 3 months) each time we took a single semen sample at each visit.All this information is stated in the article already! Q/ VIDAS® automated quantitative tests are used for plasma and serum.Semen has different components.Using the same instrument may produce different results compared to plasma.Could you please show the evidence to support to use of this instrument?VIDAS is used for assessing serum markers which we mentioned in the "variable measurement" section, it can not measure the semen parameters we measured which include: "sperm volume/ ml, sperm concentration/ ml, total sperm count/ml, progressive sperm motility (%), non-progressive sperm motility (%), immotile sperm (%), normal sperm morphology (%)."We measured using the WHO methods for assessing semen parameters, we added the details inside the new version of the article under the new section "semen fluid analysis".The samples were collected via masturbation after abstinence of 3 to 4 days directly into a dry, clean disposable wide-mouth plastic container in a private room close to the laboratory in the institute.Immediately after that the samples were carried to laboratory of semen examination then put in an incubator at 37ºC for 30 minutes.After complete liquefaction, the semen analysis was done by macroscopic and microscopic examination according to standard criteria of WHO (2021) Ref: World Health Organization.WHO laboratory manual for the examination and processing of human semen Sixth Edition.2021.Q/ How to measure the semen fluid volume, concentration, motility, etc. Please describe the procedures.We add a new section that explains seman measurement.Q/ The technique of semen preparation for hormonal measurement is crucial.Could you please state the procedures, such as any dilution or centrifugation?Were cryopreservation needed?How? Regarding these points, we only used VIDIS to assess the hormonal levels, we used the manual methods for assessing the semen parameters and we separated these details inside the article.Q/ For hormone measurements, please describe how the instrument was used, the settings used, and positive/negative control samples.How to interpret the results of the read.How about the CV of the measurement?What is the measurement range and sensitivity?This information will provide the validity of the study.FSH [VIDAS®FSH, bioMerieux SA, France Cat no.:30 407-01, (Measurement range: 0.1-110 mIU/mL, Detection limit: ≤0.1, Intra-assay CV: ≤5%, Inter-assay CV: ≤6%)" estradiol [VIDAS®E2II, bioMerieux SA, France Cat no.: 30 431, "Measurement range: 9 -3000 pg/mL, Detection limit: 9 pg/mL, Intra-assay CV: ≤7.5%, and Inter-assay CV: ≤9.5%)"] testosterone [VIDAS® Testosterone II, bioMerieux SA, France Cat no.:414320], "Measurement range: 0.1 -13 ng/mL, Detection limit: 0.1 ng/mL, Intra-assay CV: ≤10%, and Inter-assay CV: ≤4.5%)"] VIDAS is an automated device that gives you the interpretation directly, please refer to the manufacturer's website (https://www.biomerieux-nordic.com/product/vidasr-fertility-panel).Results: Q/ CONSORT FLOW CHART Thirty-four and 40 patients were randomized in groups A and B, respectively.There were 5 and 2 patients lost in each group.Why were only 29 patients/group analyzed?Where were the other 9 patients in Group B? The authors presented the protocol analysis.If possible, ITT analysis will be more useful.Thank you for your note we had an error in filling the flow chart and we updated it in the article, see linked figure .Discussion: Well written.

Figure 1 .
Figure 1.Flow chart of the trial.
Report 08 February 2024 https://doi.org/10.5256/f1000research.157556.r237937© 2024 Hekim N.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Neslihan Hekim Ondokuz Mayis University, Samsun, Turkey Researchers aimed to evaluate the impacts of adding coenzyme Q10 to letrozole on the results of spermiogram and sex hormone tests in men diagnosed with idiopathic OAT.My suggestions and questions regarding the study are as follows: It is not stated where participants received their OAT diagnosis.1.Some cytogenetic and molecular genetic analysis results, which are commonly used in the evaluation of infertile patients, are not included in the exclusion criteria.Have participants' Y chromosome microdeletion or CFTR gene mutation analyzes been evaluated?2.
scientific standard, however I have significant reservations, as outlined above.Reviewer Report 05 February 2024 https://doi.org/10.5256/f1000research.157556.r237934© 2024 Shi Q.This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Table 2 .
Effect of letrozole alone and in combination with CoQ10 on sperm concentration.

Table 1 .
Demographic data and disease characteristics.
N: Number of patients, % Percentage.BMI, body mass index.

Table 3 .
Effect of letrozole alone and in combination with CoQ10 on sperm motility and morphology.

Table 4 .
Effect of letrozole alone and in combination with CoQ10 on sex hormones.
Q/ Reference 1 needs to be the statement of WHO.Please check it carefully.A/ We changed Ref 1 to more appropriately, as listed below: World Health Organisation.Infertility [Internet].www.who.int.2022.Available from: https://www.who.int/health-topics/infertility#tab=tab_1[Accessed: February 2022] Q/ References 2 and 3 are not the original articles that reported the prevalence of 15% or the prevalence of malefactors.Please cite the original article and authors who reported this information.A/ Ref 2, changed to the following: "Sharlip ID, Jarow JP, Belker AM, Lipshultz LI, et al.Best practice policies for male infertility.Fertility and sterility.2002;77(5):873-82.DOI:10.1016/S0015-0282(02)03105-9"Ref 3, changed to the following: Q/ Reference 6 is a minireview, not the original article that provided the information as cited.Please cite the original article and authors who reported this information.A/ Ref 6, changed to the following: Ma Y, He X, Qi K, Wang T, et al.Effects of environmental contaminants on fertility and reproductive health.Journal of Environmental Sciences.2019;77:210-7.DOI: https://doi.org/10.1016/j.jes.2018.07.015Q/ Reference 7 is a review of the significant aspects of irisin actions and its involvement in energy homeostasis and male reproduction.Please cite a more relevant article.A/ Ref 7, changed to the following: "Kim HH, Schlegel PN.Endocrine manipulation in male infertility.The Urologic clinics of North America.2008;35(2):303-18, x.DOI:10.1016/j.ucl.2008.01.003"Q/ "Idiopathic oligoasthenoteratozoospermia (iOAT) is associated with faulty spermatogenesis and is characterized by unusually low sperm count, motility, and a large number of dysmorphic spermatozoa in the ejaculate" Please define iOAT clearly with proper reference.

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Minhas S, Bettocchi C, Boeri L, Capogrosso P, et al.European Association of Urology Guidelines on Male Sexual and Reproductive Health: 2021 Update on Male Infertility.