The IGF1/FSH Ratio Correlates with Sperm Count and Testicular Volume
Round 1
Reviewer 1 Report
Dear authors congratulations for your effort. It is well established that male infertility consists an area not well explored, even nowdays and therefore any research addressing this matter is of great importance. If I may I would like to share some thoughts with you regarding your research, hoping you'll find them helpful.
IGF1 plays a multivariant role in many processes (growth, apoptosis, wound healing and so on). If we want to be accurate we have to examine it's isoforms (Ea, Eb and Ec). In order to prove a possitive correlation between IGF1 levels and any process we have to study its levels in serum and in the tissue or fluid we investigate ( for exmple seminal plasma and testicular tissue). regarding your study i believe it would be beneficial to measure the levels of IGF1 in seminal plasma also and study the correlaltion between all the parameters you chose to investigate. If you could also obtain testicular tissue from volunteers also it would be better, performing a Western Blot, PCR and H&E stains . I understand the limitations of your study, but at least you could have measured IGF1 levels in seminal plasma.
Nevertheless, congratulations again for your effort. Looking forward to see your next study regarding this promising field of investigation.
Author Response
Manuscript ID endocrines-1885787
Comment 1. IGF1 plays a multivariant role in many processes (growth, apoptosis, wound healing and so on). If we want to be accurate we have to examine it's isoforms (Ea, Eb and Ec). In order to prove a possitive correlation between IGF1 levels and any process we have to study its levels in serum and in the tissue or fluid we investigate (for example seminal plasma and testicular tissue). Regarding your study i believe it would be beneficial to measure the levels of IGF1 in seminal plasma also and study the correlaltion between all the parameters you chose to investigate. If you could also obtain testicular tissue from volunteers also it would be better, performing a Western Blot, PCR and H&E stains. I understand the limitations of your study, but at least you could have measured IGF1 levels in seminal plasma.
Answer to comment 1. We appreciated the time you spent in reviewing the present manuscript and your constructive criticisms.
We understand your point. However, we believe that the primary target of IGF1 (by which it would influence the effects of FSH on spermatogenesis) is represented by Sertoli cells. Indeed, Sertoli cells express the IGF1 receptor (IGF1R) and we have studied the crosstalk between IGF1 and FSH in porcine Sertoli cells (doi: 10.3390/jcm8050577. PMID: 31035547; PMCID: PMC6571966). In particular, IGF1 appears to amplify the effects of FSH by enhancing the autophosphorylation of IGF1R and triggering FSH/IGF1 common signaling pathway (doi: 10.1111/andr.12444. Epub 2017 Dec 1. PMID: 29195026). The following cartoon, taken from this line of research, summarizes what we found (please see the attached file).
Since Sertoli cells lie in the basal compartment of tubules, due to the presence of the blood-testis barrier (BTB), it is more likely that blood IGF1 reaches these cells acting on them, rather than IGF1s present in the seminal fluid. Therefore, the rationale for measuring IGF1 in blood was to provide the amount of IGF1 that can reach and exert its effects on Sertoli cells. Furthermore, this study was conducted many months ago and we are unable to reach the patients and ask them to provide their seminal fluid. Finally, it is not possible to obtain testicular tissue because it is an invasive procedure, which is seldom used for diagnostic purposes. It is performed in patients with azoospermia for sperm retrieval to be used in assisted reproductive techniques. Therefore, it is not possible to perform such kind of studies on humans in Italy.
Lastly, it is a good suggestion to evaluate each single isoform. For this study, we used a kit, which is not able to evaluate the single IGF1 isoforms. We have included this point in the study’s limitations.
Reviewer 2 Report
Endocrines
COMMENTS TO THE EDITORS AND THE AUTHORS
endocrines-1885787 “Is there a role for insulin-like growth factor 1 in male fertility?
Dear the Editors and the Authors,
Please find enclosed the comments for the above-mentioned manuscript.
A SUMMARY OF THE CONTENT
The authors stated that the objective of the study is to assess whether insulin-like growth factor 1 (IGF1) is associated with testicular function. Results showed that IGF1 serum levels did not correlate with sperm concentration, total sperm count (TSC), and testicular volume (TV). The authors stated that the IGF1/FSH ratio showed a positive correlation with sperm concentration (r= 0.408; p=0.004), TSC (r= 0.468; p=0.001), and TV (0.463; p=0.002). Patients with oligozoospermia (Group 1, 23.7%, n=14) had a significantly lower IGF1/FSH ratio (57.9±9.5 vs. 94.1±8.7; 20p= 0.03) compared to those with TSC >39 million/ejaculate (Group 2, 76.3%, n=45). Groups were not significantly different in IGF1 or FSH serum levels. The authors concluded that IGF1/FSH ratio was positively correlated with sperm production and TV and that the IGF1/FSH ratio can represent an index of the quantity of IGF1 in relation to the FSH levels to which Sertoli cells are exposed.
THE OVERALL OPINION OF THE MANUSCRIPT
The strengths.
(1) The manuscript addresses the interesting question.
The limitations.
(1) The title is not supported by the data since the authors did not assess fertility. As it is well known, fertility is the capability to produce offspring through reproduction following the onset of sexual maturity. The fertility rate is the average number of children born by a female during her lifetime and is quantified demographically. Moreover, the authors stated that “IGF1 serum levels did not correlate with sperm concentration, TSC, and TV”.
(2) The objectives were not addressed in the manuscript. Namely, the authors stated that “the objective of the study is to assess whether insulin-like growth factor 1 (IGF1) is associated with testicular function”. Testicular function is very complex and include many cells (Leydig cells, Sertoli cells, peritubular myoid cells, two types of macrophages, different cells of immune system, germinative cells in different stadium of differentiation, fibroblast, etc.). Moreover, it is very well known that IGF1 is associated with testicular function in many important ways (please see https://pubmed.ncbi.nlm.nih.gov/?term=IGF1+testicular+function&sort=date&size=100 ).
(3) The experimental design is not accurate to answer the objectives. The number of participants is very low. The types of spermiograms were not stated. The number of methods is limited.
(4) The amount of results is not satisfactory and does not support the title, the objectives and the conclusions.
(5) The discussion is not supported by the results.
(6) The conclusions are not supported by the results.
(7) The original, and important pioneered results, as well as recent advance in the field focusing on the subject of the study are not cited, although it is very well known that that IGF1 is associated with testicular function in many important ways (please see https://pubmed.ncbi.nlm.nih.gov/?term=IGF1+testicular+function&sort=date&size=100). The authors cited (page 2, line 48) as “recent” article published 4 years ago (2018).
(8) The statement (page 5, lines 135-137; “Despite the huge amount of in vitro data showing the involvement of IGF1 in testicular development and function (Cannarella et al., 2018), only one study has investigated the role of this hormone in human sperm output so far.”). Please see PubMed. Just one example https://pubmed.ncbi.nlm.nih.gov/34273055/.
Accordingly, major and substantial revision addressing/solving the all above mentioned limitation is required for further consideration.
Good luck and all the best J
Author Response
Manuscript ID endocrines-1885787
Comment 1. The title is not supported by the data since the authors did not assess fertility. As it is well known, fertility is the capability to produce offspring through reproduction following the onset of sexual maturity. The fertility rate is the average number of children born by a female during her lifetime and is quantified demographically. Moreover, the authors stated that “IGF1 serum levels did not correlate with sperm concentration, TSC, and TV”.
Answer to comment 1. We appreciate the time you spent reviewing the present manuscript and the constructive criticisms given.
Based on your comment, we decided to change the title, which now is “The IGF1/FSH ratio correlates with sperm count and testicular volume”.
Comment 2. The objectives were not addressed in the manuscript. Namely, the authors stated that “the objective of the study is to assess whether insulin-like growth factor 1 (IGF1) is associated with testicular function”. Testicular function is very complex and include many cells (Leydig cells, Sertoli cells, peritubular myoid cells, two types of macrophages, different cells of immune system, germinative cells in different stadium of differentiation, fibroblast, etc.). Moreover, it is very well known that IGF1 is associated with testicular function in many important ways (please see https://pubmed.ncbi.nlm.nih.gov/?term=IGF1+testicular+function&sort=date&size=100 ).
Answer to comment 2. There is evidence for a role of IGF1 in Sertoli cells, Leydig cells, and germ cells. Although, the repercussion of basic information in clinical practice is unknown. Indeed, IGF1 is not currently used in any of the diagnostic algorithms used for the management of infertile patients. Therefore, we aimed to provide data that can have a direct repercussion in clinical practice. Therefore, based on your suggestion, the objective was rewritten in the revised manuscript, where it reads as follows:
“Although there is evidence of a role of IGF1 in Sertoli cells, Leydig cells, and germ cells, the repercussions of this basic information in clinical practice are unknown. IGF1 may influence FSH signaling in human SCs, as recently suggested (Cannarella et al., 2018), and, thus, also human sperm output. No studies so far have attempted to evaluate the possible influence of the IGF1/FSH ratio (which may be used as a marker to simultaneously assess IGF1 and FSH levels), on human sperm count. Therefore, the present study aimed to evaluate whether IGF1/FSH ratio affects testicular function and, in particular, sperm number and TV, in a cohort of unselected men”.
Comment 3. The experimental design is not accurate to answer the objectives. The number of participants is very low. The types of spermiograms were not stated. The number of methods is limited.
Answer to comment 3. Thank you for your comments.
The title and the objectives have been clarified (please see the replies to the previous comment). The IGF1/FSH ratio was clearly mentioned among the objectives/endpoint measures, thus providing a better adherence to the study.
The number of participants is 59. According to power analysis, the sample size is large enough to allow statistically significant conclusions. Power analysis has been included in the revised version of the manuscript. It reads as follows “TSC was chosen for power calculation. The study required a total of 28 participants (14 per group) to preserve a 90% power to detect a 5% absolute difference for a 5% two-sided test while accounting for a 5% dropout rate”.
The section on sperm analysis was completely revised and more information was provided, as detailed below.
“Sperm parameters were analyzed in the Seminology Unit of the Division of Endocrinology, Metabolic Diseases and Nutrition, University of Catania (Catania, Italy). For each patient, semen samples were collected by masturbation into a sterile container after 2-7 days of sexual abstinence and were analyzed immediately after liquefaction. According to the 2010 WHO guidelines, each sample was evaluated for conventional sperm parameters. More specifically, the outcomes of the study were: sperm concentration, total sperm count, sperm progressive motility, sperm total motility, sperm morphology, and semen leukocyte concentration. For the assessment of sperm concentration and sperm motility, 10 µL of well-mixed semen sample was loaded into a clean Makler chamber, kept at 37°C, gently covered with a cover glass, and examined using an X200 magnification. Ten of the 100 squares in the microscope field were randomly skimmed. Sperm morphology was evaluated using the Papanicolaou staining procedure, while vitality was assessed using eosin staining. Finally, the leukocyte concentration was assessed using cell peroxidase staining by ortho-toluidine, according to the manual recommendations of the WHO 2010 (WHO, 2010)”.
Comment 4. The amount of results is not satisfactory and does not support the title, the objectives, and the conclusions.
Answer to comment 4. The title and objective were rewritten to make them more adherent to the study. The conclusion was rephrased.
Comment 5. The discussion is not supported by the results.
Answer to comment 5. The discussion has been extensively reviewed in an attempt to make our point clearer. Each change is reported in red.
Comment 6. The conclusions are not supported by the results.
Answer to comment 6. Please see the answer to comment 4.
Comment 7. The original, and important pioneered results, as well as recent advance in the field focusing on the subject of the study are not cited, although it is very well known that that IGF1 is associated with testicular function in many important ways (please see https://pubmed.ncbi.nlm.nih.gov/?term=IGF1+testicular+function&sort=date&size=100). The authors cited (page 2, line 48) as “recent” article published 4 years ago (2018).
Answer to comment 7. Additional studies were included in the revised Discussion. Also, we replaced “recently” with “already”.
Comment 8. The statement (page 5, lines 135-137; “Despite the huge amount of in vitro data showing the involvement of IGF1 in testicular development and function (Cannarella et al., 2018), only one study has investigated the role of this hormone in human sperm output so far.”). Please see PubMed. Just one example https://pubmed.ncbi.nlm.nih.gov/34273055/.
Answer to comment 8. Several studies have already investigated the relationship between IGF1 and sperm parameters. However, evidence from clinical studies on the existence of a relationship between IGF1 and sperm number is scarce and, therefore, whether the measurement of IGF1 serum levels has a practical value in the diagnostic work-up of patients with oligozoospermia is still unclear. We have extensively revised the Discussion to make clearer this point. Thank you for your helpful comment.
Reviewer 3 Report
There are some flaws that must be improved.
Patient selection: group I are oligozoospermics and Group II are normozoospermic. They come to the Centre for a study or they are fertile or infertile?
In the discussion: Line 136, "only one study.....". There is one study published (Fertility and Sterility: Vol. 62, No.5, November 1994. Rajmil et al. GH response to GH-RH in oligospermic men page 1043). It deserves to be comment and compare results. Seems that it was a publication with different methods but similar results calling attention on the implications of growth factors over sperm regulation. Be sure to conduct a complete literature search, including old ones.
Table III: the addition to the head of the columns (Group I) Oligozoospermic and (Group II) normozoospemic will improve the understanding by the readers (it is explained previously).
Author Response
Manuscript ID endocrines-1885787
Comment 1. Patient selection: group I are oligozoospermics and Group II are normozoospermic. They come to the Centre for a study or they are fertile or infertile?
Answer to comment 1. We appreciated your comment, which gave us the chance to improve the “Patient selection” section of the present study.
The enrolling center is the Division of Endocrinology, Metabolic Diseases and Nutrition of the University-Teaching Hospital Policlinico “G. Rodolico – San Marco”, University of Catania (Catania, Italy). This division is devoted to the care of patients with infertility, andrology care and well-being (including prevention and follow-up of healthy men), and patients with endocrine diseases (e.g. patients with thyroid nodular disease, obesity, etc., which can be fertile).
Specifically, patients were recruited based on their sperm count. Information on fertility was not collected, though patients with normozoospermia are more likely to be fertile. Please see the revised “Patient selection” section.
Comment 2. In the discussion: Line 136, "only one study.....". There is one study published (Fertility and Sterility: Vol. 62, No.5, November 1994. Rajmil et al. GH response to GH-RH in oligospermic men page 1043). It deserves to be comment and compare results. Seems that it was a publication with different methods but similar results calling attention on the implications of growth factors over sperm regulation. Be sure to conduct a complete literature search, including old ones.
Answer to comment 2. We appreciated your comment. Several studies have already investigated the relationship between IGF1 and sperm parameters. However, evidence from clinical studies on the existence of a relationship between IGF1 and sperm number is scarce, and, therefore, whether the measurement of IGF1 serum levels has a practical value in the diagnostic work-up of patients with oligozoospermia is still unclear. We have extensively revised the Discussion to make clearer this point. Thank you for your helpful comment. Changes are reported in red. Furthermore, the study by Rajmil et al., 1994 has been quoted. Thank you.
Comment 3. Table III: the addition to the head of the columns (Group I) Oligozoospermic and (Group II) normozoospemic will improve the understanding by the readers (it is explained previously).
Answer to comment 3. Done, as requested.
Round 2
Reviewer 2 Report
COMMENTS TO THE EDITORS AND THE AUTHORS
endocrines-1885787R1 “The IGF1/FSH ratio correlates with sperm count and testicular volume”
Dear the Editors and the Authors,
Please find enclosed the comments for the above-mentioned manuscript.
The revised version of the manuscript is written better then the original one, but significant limitations are still present.
(1) The part of the text in the abstract (lines 33-34) is not proven by the results i.e. presented results could not suggest what is written (“This suggests that, adulthood, IGF1 could affect spermatogenesis by modulating FSH signaling on Sertoli cells. This finding could pave the way for new diagnostic/therapeutic strategies in the work-up of patients with impaired spermatogenesis. However, further studies on larger cohorts and intervention studies should be encouraged to confirm the results of this study.”). It is better to be removed.
(2) Introduction is not improved at all. Please describe the original, and important pioneered results, as well as recent advance in the field focusing on the subject of the study.
(3) The part of the text in the conclusions (lines 273-276) is not proven by the results i.e. presented results could not suggest what is written (“This suggests that, in adulthood, IGF1 could influence spermatogenesis by modulating FSH signaling and effect in SCs. This may pave the way for novel diagnostic/therapeutic strategies in the work-up of patients with reduced sperm production. However, further studies on larger cohorts and intervention studies should be encouraged to confirm our findings.”). It is better to be removed.
(4) Please consider adding one paragraph stating the limitation of the study in relation to the number of the subject, limitation related to the types of spermiograms as well as methodologies.
Accordingly, minor revision addressing/solving the all above mentioned limitation is required for further consideration.
Good luck and all the best ?
Author Response
Answers to Reviewer #2 comments
Manuscript ID endocrines-1885787
Comment 1. The part of the text in the abstract (lines 33-34) is not proven by the results i.e. presented results could not suggest what is written (“This suggests that, adulthood, IGF1 could affect spermatogenesis by modulating FSH signaling on Sertoli cells. This finding could pave the way for new diagnostic/therapeutic strategies in the work-up of patients with impaired spermatogenesis. However, further studies on larger cohorts and intervention studies should be encouraged to confirm the results of this study.”). It is better to be removed.
Answer to comment 1. Dear Reviewer, we highly appreciate the time spent in reviewing our manuscript, as well as your valuable feedback. We understand that the results of the study don’t prove neither the existence of an influence of IGF1 on spermatogenesis, nor the link between the IGF1 and the FSH signaling pathway. However, they do not negate these aspects. Following your comment, we decided to restructure the sentences you referred to, and to lower the tones. The revised sentences are reported below:
“Our results may reflect the existence of a molecular pathway to which IGF1 and FSH belongs. However, further studies are needed”.
Comment 2. Introduction is not improved at all. Please describe the original, and important pioneered results, as well as recent advance in the field focusing on the subject of the study.
Answer to comment 2. Pioneer studies on the effect of IGF1 on spermatogenesis have now been included. Regarding the recent advance in the field, the most important novelty – which supports the rational of the study – is the given by the molecular data highlighting the presence of a common pathway to which FSH and IGF1 belong, which has already been discussed.
Comment 3. The part of the text in the conclusions (lines 273-276) is not proven by the results i.e. presented results could not suggest what is written (“This suggests that, in adulthood, IGF1 could influence spermatogenesis by modulating FSH signaling and effect in SCs. This may pave the way for novel diagnostic/therapeutic strategies in the work-up of patients with reduced sperm production. However, further studies on larger cohorts and intervention studies should be encouraged to confirm our findings.”). It is better to be removed.
Answer to comment 3. Please see the answer to comment 1. We rephrased the conclusions as follows: “Our results may reflect the existence of a molecular pathway to which IGF1 and FSH be-longs, and though which IGF1 might exert its influence on spermatogenesis. However, further studies are needed”.
Comment 4. Please consider adding one paragraph stating the limitation of the study in relation to the number of the subject, limitation related to the types of spermiograms as well as methodologies.
Answer to comment 4. Dear Reviewer, we agree to include a paragraph on the study limitations. This has already been done in the previous version of the manuscript. That section is in the end of the discussion, and reads as follows:
“Study limitations include the relatively small cohort, although power analysis supports that the sample size is large enough to allow statistically significant conclusions. In addition, IGF1 isoforms could not be measured, thus adding another limitation to the interpretation of our data. Lastly, we did not assess IGF1 levels in the seminal fluid. Although this may be considered a limitation, IGF1 may influence the sperm counts likely acting on SCs. The latter are not exposed to the IGF1 levels in the seminal fluid due to the testis-blood-barrier, but to the levels in the bloodstream. Further studies on larger cohorts and intervention studies should be encouraged to confirm our findings. In addition, our results may not be generalizable to men from general population. Cross-sectional study design limits causal inference”.
We did not get what you mean with “types of spermiograms”. In case there should be anything you believe is needed to be considered for inclusion in the limitations, please let us know. Best regards.
