Review Reports - Reduction of Prostate Cancer Risk: Role of Frequent Ejaculation-Associated Mechanisms

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Mechanisms Regulating the Reduction of Prostate Cancer Risk

According to my understanding from the title, the authors aim to talk about the general mechanisms regulating the reduction of prostate cancer risk. However, upon reading through, it becomes evident that the authors emphasize the effects of frequent ejaculation on reducing prostate cancer risk. More surprisingly, the majority of the content describes factors affecting ejaculation rather than the mechanism by which frequent ejaculation reduces prostate cancer risk. Therefore, I found this review lacks a clear direction regarding its main message. If the authors want to focus on how frequent ejaculation benefits prostate cancer risk reduction, they should change the title accordingly and focus on the mechanism by which frequent ejaculation reduces prostate cancer risk.

Major Issues:

As mentioned above, the authors need to determine their primary focus and modify the entire review accordingly, especially the title and overall structure.

Minor Issues:

Some sentences lack periods, such as those on line 80 and line 210. Please review the entire manuscript for similar errors.
Regarding the sentence "The more frequent ejaculation occurs without risky sexual behavior, the more the risk of prostate cancer decreases in men,” consider changing it to "The more frequent ejaculation occurs without risky sexual behavior, the less the risk of prostate cancer."
Several paragraphs are underlined. What does this signify?
There are repetitive sentences throughout the manuscript, such as lines 34-36 and lines 43-44. Please review the entire manuscript for similar issues.
For the description "The prostate gland is part of the male reproductive system located inside the body," could you please modify it to a more professional phrasing, such as the description on lines 54-55?
In the author contribution section, please omit the instructional content.

Author Response

Author response to Reviewer 2

Dear Editor,

Enclosed find please our response to the valuable comment of reviewer 01

Reviewer 1

Comments and Suggestions for Authors

Mechanisms Regulating the Reduction of Prostate Cancer Risk

Major Issues:

Comment 1: As mentioned above, the authors need to determine their primary focus and modify the entire review accordingly, especially the title and overall structure.

Authors’ response: Thank you very much for your valuable comment. Accordingly, we modified the title to be more suitable and we made significant modification and changes in the overall structure of the manuscript. Accordingly, the following modifications and changes have been made.

See lines: 2-3; The current title [Mechanisms Regulating the Reduction of Prostate Cancer Risk ] has been modified and replaced by the following title [Reduction of Prostate Cancer Risk: Role of Frequent Ejaculation-Associated Mechanisms]

See Lines:794-831; the following chapter [8. Mechanisms of frequent ejaculation-induced reduction of prostate cancer risk The regulation of PCa risk induced by frequent ejaculation appears to be mediated by several mechanisms that have been proposed to explain the association between frequent ejaculation and reduced PCa [372, 373]. The higher the frequency of ejaculation, the more inhibited the metabolic switch from citrate secretion to citrate oxidation in the epithelial cells in the peripheral zone. One of the possible mechanisms by which frequent ejaculation may reduce the risk of tumor development in the prostate [373]. Also, frequent ejaculation is expected to reduce PCa risk via mechanism mediated by restore the development of intraluminal eosinophilic structures in the prostate, which are considered a sign of higher risk for PCa. [374, 375]. Another mechanism by which frequent ejaculation can reduce PCA risk is expected to be mediated by reducing psychological tension and suppressing the central sympathetic nervous system, resulting in a significant attenuation of the stimulation of prostate epithelial cell division. [376].

As is generally accepted, cancer is one of the most common diseases caused by environmental and external influences [377, 378]. The development of around 90% of cancers diagnosed worldwide is attributed mainly to harmful environmental chemicals [379]. Among these substances, several active harmful chemicals that are recognized as endocrine disruptors [380, 381]. These endocrine disruptors have common characteristics that are mostly associated with disruption of hormone regulation and action [382]. Endocrine disruptors act primarily via nuclear hormone receptors, such as estrogen and progesterone receptors [383, 384]. The mechanisms of endocrine disruptors are much more comprehensive than commonly reported [385, 386].

Like other organs, the prostate is a target of numerous environmentally harmful chemicals including endocrine disruptors that can induce multi-organ disease through mechanisms based on interaction with the cellular receptors or components [387, 388]. Therefore, the interference of these harmful chemicals with the action of hormones, which are involved in the different cellular functions of the prostate gland is expected to develop hormone-dependent malignancies [389, 390]. Ultimately, many reports suggested that endocrine disruptors have the potential to significantly alter the epigenomic landscape in cancers, including prostate cancer [391, 392]. Thus, frequent ejaculation is expected to protect the prostate by flushing out these harmful chemicals. Consequently, men who ejaculate more frequently can have healthy lifestyle habits harmful chemicals that can, in turn, reduce the risk of prostate cancer.

The stimulation of endocannabinoid release in human by masturbation to orgasm suggests an essential role for the frequent ejaculation in the reduction of prostate cancer via endogenous 2-arachidonoylglycerol (2-AG) -dependent mechanism [393]. Also, several studies demonstrated the ability of 2-AG to inhibit the invasion of androgen-independent prostate cancer PC-3 and DU-145) cell lines in vitro [393]. ] has been added to the main text.

See lines:1447-1802;

Lozano-Lorca, M.; Olmedo-Requena, R.; Barrios-Rodríguez, R.; Jiménez-Pacheco, A.; Vázquez-Alonso, F.; Castillo-Bueno, H. M.; Rodríguez-Barranco, M.; Jiménez-Moleón, J. J. Ejaculation Frequency and Prostate Cancer: CAPLIFE Study. World J Mens Health 2023, 41 (3), 724-733. DOI: 10.5534/wjmh.220216

Costello, L. C.; Franklin, R. B. The clinical relevance of the metabolism of prostate cancer; zinc and tumor suppression: connecting the dots. Mol Cancer 2006, 5, 17. DOI: 10.1186/1476-4598-5-17.

Svatek, R. S.; Karam, J. A.; Rogers, T. E.; Shulman, M. J.; Margulis, V.; Benaim, E. A. Intraluminal crystalloids are highly associated with prostatic adenocarcinoma on concurrent biopsy specimens. Prostate Cancer Prostatic Dis 2007, 10 (3), 279-282. DOI: 10.1038/sj.pcan.4500954.

Del Rosario, A. D.; Bui, H. X.; Abdulla, M.; Ross, J. S. Sulfur-rich prostatic intraluminal crystalloids: a surgical pathologic and electron probe x-ray microanalytic study. Hum Pathol 1993, 24 (11), 1159-1167. DOI: 10.1016/0046-8177(93)90210-8.

Newman, H. F.; Reiss, H.; Northup, J. D. Physical basis of emission, ejaculation, and orgasm in the male. Urology 1982, 19 (4), 341-350. DOI: 10.1016/0090-4295(82)90186-8.

Parsa, N. Environmental factors inducing human cancers. Iran J Public Health 2012, 41 (11), 1-9.

Baillie-Hamilton, P. F. Chemical toxins: a hypothesis to explain the global obesity epidemic. J Altern Complement Med 2002, 8 (2), 185-192. DOI: 10.1089/107555302317371479.

Madia, F.; Worth, A.; Whelan, M.; Corvi, R. Carcinogenicity assessment: Addressing the challenges of cancer and chemicals in the environment. Environ Int 2019, 128, 417-429. DOI: 10.1016/j.envint.2019.04.067.

Jensen, T. K.; Andersson, A. M.; Jørgensen, N.; Andersen, A. G.; Carlsen, E.; Petersen, J. H.; Skakkebaek, N. E. Body mass index in relation to semen quality and reproductive hormones among 1,558 Danish men. Fertil Steril 2004, 82 (4), 863-870. DOI: 10.1016/j.fertnstert.2004.03.056.

Macdonald, A. A.; Stewart, A. W.; Farquhar, C. M. Body mass index in relation to semen quality and reproductive hormones in New Zealand men: a cross-sectional study in fertility clinics. Hum Reprod 2013, 28 (12), 3178-3187. DOI: 10.1093/humrep/det379.

[296] La Merrill, M. A.; Vandenberg, L. N.; Smith, M. T.; Goodson, W.; Browne, P.; Patisaul, H. B.; Guyton, K. Z.; Kortenkamp, A.; Cogliano, V. J.; Woodruff, T. J.; et al. Consensus on the key characteristics of endocrine-disrupting chemicals as a basis for hazard identification. Nat Rev Endocrinol 2020, 16 (1), 45-57. DOI: 10.1038/s41574-019-0273-8.

Lee, H. R.; Jeung, E. B.; Cho, M. H.; Kim, T. H.; Leung, P. C.; Choi, K. C. Molecular mechanism(s) of endocrine-disrupting chemicals and their potent oestrogenicity in diverse cells and tissues that express oestrogen receptors. J Cell Mol Med 2013, 17 (1), 1-11. DOI: 10.1111/j.1582-4934.2012.01649.x.

Tabb, M. M.; Blumberg, B. New modes of action for endocrine-disrupting chemicals. Mol Endocrinol 2006, 20 (3), 475-482. DOI: 10.1210/me.2004-0513..

Diamanti-Kandarakis, E.; Bourguignon, J. P.; Giudice, L. C.; Hauser, R.; Prins, G. S.; Soto, A. M.; Zoeller, R. T.; Gore, A. C. Endocrine-disrupting chemicals: an Endocrine Society scientific statement. Endocr Rev 2009, 30 (4), 293-342. DOI: 10.1210/er.2009-0002.

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Corti, M.; Lorenzetti, S.; Ubaldi, A.; Zilli, R.; Marcoccia, D. Endocrine Disruptors and Prostate Cancer. Int J Mol Sci 2022, 23 (3). DOI: 10.3390/ijms23031216.

Bleak, T. C.; Calaf, G. M. Breast and prostate glands affected by environmental substances (Review). Oncol Rep 2021, 45 (4). DOI: 10.3892/or.2021.7971

Lacouture, A.; Lafront, C.; Peillex, C.; Pelletier, M.; Audet-Walsh, É. Impacts of endocrine-disrupting chemicals on prostate function and cancer. Environ Res 2022, 204 (Pt B), 112085. DOI: 10.1016/j.envres.2021.112085.

Prins, G. S. Endocrine disruptors and prostate cancer risk. Endocr Relat Cancer 2008, 15 (3), 649-656. DOI: 10.1677/ERC-08-0043.

Lehle, J. D.; McCarrey, J. R. Differential susceptibility to endocrine disruptor-induced epimutagenesis. Environ Epigenet 2020, 6 (1), dvaa016. DOI: 10.1093/eep/dvaa016.

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Fuss, J.; Bindila, L.; Wiedemann, K.; Auer, M. K.; Briken, P.; Biedermann, S. V. Masturbation to Orgasm Stimulates the Release of the Endocannabinoid 2-Arachidonoylglycerol in Humans. J Sex Med 2017, 14 (11), 1372-1379. DOI: 10.1016/j.jsxm.2017.09.016.

Nithipatikom, K.; Endsley, M. P.; Isbell, M. A.; Falck, J. R.; Iwamoto, Y.; Hillard, C. J.; Campbell, W. B. 2-arachidonoylglycerol: a novel inhibitor of androgen-independent prostate cancer cell invasion. Cancer Res 2004, 64 (24), 8826-8830. DOI: 10.1158/0008-5472.CAN-04-3136. have been added to reference section

Minor Issues:

Comment1: Some sentences lack periods, such as those on line 80 and line 210. Please review the entire manuscript for similar errors.

Authors ‘response: Thank you very much for your valuable comment. Accordingly, we corrected the lack of periods overall in the manuscript.

Comment 2: Regarding the sentence "The more frequent ejaculation occurs without risky sexual behavior, the more the risk of prostate cancer decreases in men,” consider changing it to "The more frequent ejaculation occurs without risky sexual behavior, the less the risk of prostate cancer."

Authors’response: Thank you very much for your comment. Accordingly, we considered your suggestion replaced the sentence as required

See lines:27-28; the following sentence [The more frequent ejaculation occurs without risky sexual behavior, the more the risk of prostate cancer decreases in men] is replaced by the following sentence [The more frequent ejaculation occurs without risky sexual behavior, the less the risk of prostate cancer ]

Comment 3: Several paragraphs are underlined. What does this signify?

Authors’ response: Thank you very much for your comment. The underlined paragraphs were the authors ‘response to the comment of the editor. Accordingly, we underlined the changes that have been made in the manuscript.

Comment 4: There are repetitive sentences throughout the manuscript, such as lines 34-36 and lines 43-44. Please review the entire manuscript for similar issues.

Authors’ response: Thank you very much for your valuable comment. Accordingly, we made the required correction overall in the manuscript.

See lines:35-36;43-45

Comment 5: For the description "The prostate gland is part of the male reproductive system located inside the body," could you please modify it to a more professional phrasing, such as the description on lines 54-55?

Authors’response: Thank you very much for your comment. As required, we modified the sentence to be more professional.

See lines:37-38; the following sentence [The prostate gland is part of the male reproductive system located inside the body. ] has been modified and replaced by the following sentence [The prostate is a small gland that is anatomically located below the bladder and in front of the rectum]

Comment 6: In the author contribution section, please omit the instructional content.

Authors’response: Thank you very much for your comment. As requested, we omitted the instructural content.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This is an interesting paper dealing with the relationship between ejaculation frequency and prostate cancer risk and a well-done survey of different groups of neurotransmittors is given.My comments are the following:

page 10/30, third paragraph: The description of the prostatic secretion is too potted. Bivalent cations Mg2+ and Ca2+ should be mentioned together with Zn2+ and most importantly that the prostatic secretion also contains an abundance of subcellular, exosome-like extracellular vesicles denoted "prostasomes". Prostasomes play a crucial role in development of prostate cancer (Prostate Cancer and Prostatic Diseases 7 (2004) 21-31). Moreover, activation of the renin-angiotensin system (RAS) plays an important role in normal physiology. Angiotensin-converting enzyme (ACE) degrades vasodilator kinins and generates angiotensin II, being the major effector peptide in the RAS-system. ACE synthesis by the prostate gland has been reported (Int J Androl 12 (1989) 22-28; J Pathol 195 (2001) 571-579) and the activity of ACE is much higher in seminal plasma than in blood serum and most activity is membrane-bound to prostasomes. Angiotensin II stimulates neovascularization which is one of the requirements for tumor growth. Angiotensin II can also act as a tumor growth factor with stimulation of cell replication and is associated with genes that control cell growth. A cohort study with nested case-control analysis showed a lower risk of subsequent prostate cancer in hypertensive patients medicated with a well-established inhibitor of ACE (The Prostate 58(2004) 50-56). Similar results with reduced prostate cancer risk have been subseqently achieved with hypertensive patients, now medicated with antagonists towards the angiotensin II receptor. This aspect of reduction of prostate risk should be mentioned and briefly discussed by the authors. Minor comment: page 20/30: Reference 81 is incomplete

Author Response

Author response to Reviewer 2

Dear Editor,

Enclosed find please our response to the valuable comment of reviewer 02

Comments and Suggestions for Authors

Comment 1: page 10/30, third paragraph: The description of the prostatic secretion is too potted. Bivalent cations Mg2+ and Ca2+ should be mentioned together with Zn2+ and most importantly that the prostatic secretion also contains an abundance of subcellular, exosome-like extracellular vesicles denoted "prostasomes". Prostasomes play a crucial role in development of prostate cancer (Prostate Cancer and Prostatic Diseases 7 (2004) 21-31). Moreover, activation of the renin-angiotensin system (RAS) plays an important role in normal physiology. Angiotensin-converting enzyme (ACE) degrades vasodilator kinins and generates angiotensin II, being the major effector peptide in the RAS-system. ACE synthesis by the prostate gland has been reported (Int J Androl 12 (1989) 22-28; J Pathol 195 (2001) 571-579) and the activity of ACE is much higher in seminal plasma than in blood serum and most activity is membrane-bound to prostasomes. Angiotensin II stimulates neovascularization which is one of the requirements for tumor growth. Angiotensin II can also act as a tumor growth factor with stimulation of cell replication and is associated with genes that control cell growth. A cohort study with nested case-control analysis showed a lower risk of subsequent prostate cancer in hypertensive patients medicated with a well-established inhibitor of ACE (The Prostate 58(2004) 50-56). Similar results with reduced prostate cancer risk have been subseqently achieved with hypertensive patients, now medicated with antagonists towards the angiotensin II receptor. This aspect of reduction of prostate risk should be mentioned and briefly discussed by the authors.

Authors’response: Thank you very much for your valuable comment. Accordingly, we added several paragraphs as you suggested. The following changes in the manuscript have been made. The title of chapter 2 to be more suitable

See line: 101 The following Chapter title [2. Anatomy and Physiological Functions of Prostate ] has been replaced by the following one [2. Anatomy, Physiological Functions and secretion of Prostate ].

See lines:116-247; the following text [As an additional sexual organ, the prostate secretes fluid into the semen, which supports sperm release and fertility [8, 46]. The cells of the prostate can be roughly divided into two cell types: the parenchymal epithelial cells that form the acini and ducts of the gland and secrete prostatic fluid, and the stromal cells, which surround the acini and ducts and provide support and structure to the gland [46, 47].

The main function of the accessory sex tissues, which include the prostate, seminal vesicles and urethral glands, is the production of seminal plasma, the non-gametic portion of semen [48, 49]. The main function of the seminal plasma is to serve as a buffered, nutrient-rich transport medium for sperm when they are deposited in the female genital tract [50, 51]. Seminal plasma is characterized by its alkalinity that is essential to neutralize the acidic environment of the vagina [52, 53]. While the main function of the prostaglandins hat are present in seminal plasma is to stimulate the contraction of smooth muscles along the genital tract of the female to facilitate the transport of the sperm to the egg [54,55]. In addition to zinc and IgA that are characterized by their bacteriostatic properties, the seminal plasm many prostatic proteins that are essential to prevent autoagglutination of sperm [56, 57].

As is known, in normal men, seminal plasma production, including the prostatic component, is regulated by circulating androgens via a hormonal control dependence [19, 58]. Once circulating testosterone is transported into the prostate cells, it is converted by 5α-reductase into dihydrotestosterone (DHT), which in turn binds the androgen receptor (AR), dimerizes and translates into the cell nucleus. The nuclear AR then binds the response elements of androgen-regulated genes to trigger gene expression of secretory proteins or enzymes to be secreted into the semen [19, 39].

In addition to hormonal control, the prostatic secretions also underlay neural control [19, 59]. It is noteworthy that the components of the prostate including both epithelial and stromal derived tissues are innervated by the autonomic nervous system [19, 60].

As has been widely reported and documented, prostate fluid is rich in enzymes, proteins and minerals that are essential for the protection and nutrition of sperm [60, 61]. Due to sexual arousal, the prostate can push prostate fluid through the ducts into the urethra, where it mixes with sperm and other fluids and is then ejaculated as semen [62, 63]. The most common prostatic fluid components secretions include Zinc, citric acid, Sodium, potassium, calcium, magnesium and chloride, spermine, prostaglandins, Cholesterol and lipids, immunoglobulins, acid phosphatase, and prostate specific antigen (PSA) as well as extracellular vesicles [64, 65].

The highest concentration of zinc has been reported in prostate gland, when compared he other organs within human body [64, 65]. Therefore, the prostatic secretions are the main source for higher concentration of zinc in the seminal plasma that is estimated to be nearly 100-fold higher than those in serum [66, 67] [66]. In addition to its function as an antibacterial factor both in seminal plasma and in non-ejaculatory prostate secretions as well, zinc is also thought to play an important role in the development of normal testes, spermatogenesis and sperm motility. [68]. Furthermore, the zinc in seminal plasma has been reported to function as a cell membrane and nuclear chromatin stabilizer in spermatozoa [69].

The binding of zinc to semenogelin I and II in seminal plasma is also essential for the regulation of semen liquefaction [70, 71]. Semenogelin is a protein reported to be involved in the formation of a gel matrix that coats ejaculated sperm to prevent capacitation [72, 73]. Citric acid is also one of the secretory products of the prostate, which is characterized by its ability to bind metal ions. It is also one of the main anions in human seminal plasma [74, 75]. Although higher concentrations of calcium and magnesium have been found in seminal plasma compared to other body fluids, the contribution of the prostate to the increase in these anions is not reported. [78]. However, variations in sodium, potassium, calcium, magnesium and chloride concentrations in prostate fluid have been reported to be closely associated with health or disease status [65, 78]. This is partly a passive response to varying amounts of citrate secretion in the prostate fluid in the disease state

Spermine is one of the polyamines that is discovered in semen [79, 80]. Spermine is one of the components of semen and its concentration in the prostate gland is the highest spermine concentration in the human body [81, 82]. It is also the main source of polyamine in seminal plasma. [83, 84].. The enzyme ornithine decarboxylase (ODC) is characterized by its ability to serve as a rate-limiting enzyme in the synthesis of polyamines and spermine [85]. ODC expression is increased in benign prostatic hyperplasia (BPH) tissue, suggesting a possible role of polyamines in the pathogenesis of this disease [86, 87].

Although prostaglandins are present throughout the body, the highest concentrations of prostaglandins in the human body have been found in the seminal vesicles [88, 89]. Prostaglandins are characterized by their potent physiological effects on men, particularly those involved in erection, ejaculation, sperm motility, and contractions of the testes and penis [90, 91]. While the deposition of prostaglandins in seminal fluid in the vagina has been reported to affect cervical mucus, vaginal secretions and uterine contractions [92].

The main source of the lipid fraction in seminal plasma comes from the prostate [93, 94]. The most common components of the lipid fraction in semen include cholesterol and phospholipids [95]. These phospholipids are composed of 44% sphingomyelin, 12.3% ethanolamine plasmalogen and 11.2% phosphatidylserine [96, 97]. While the ratio of cholesterol to phospholipids is considered important to protect sperm from temperature and environmental shocks, [98, 99].

The presence of immunoglobulins including IgG and IgA as well as IgM in seminal plasma at lower concentrations than those examined in blood [100, 101].

While the IgM concentration was lower in seminal plasma, when compared to those of IgG and IgA [102, 103].

The acid phosphatase activity is one of the components of the seminal fluid higher in the prostate than those reported in any other tissue [105, 106]. Prostatic acid phosphatase is a glycoprotein dimer, whose substrate in the seminal plasma is the phosphorylcholine phosphate that can rapidly be hydrolyzed by the acid phosphatase [106, 107]. Overexpression of acid phosphatase is observed in cancerous prostate tissues. For example, about 95% of patients with prostate cancer are characterized by high expression of acid phosphatase, especially in patients with metastases in the bones [107, 108]. Accordingly, acid phosphatase is considered an important serum marker for screening and staging of prostate cancer, even if it is replaced by the more specific prostate-specific antigen. (PSA).

PSA is a glycoprotein that acts as a serine protease and is produced almost exclusively in epithelial cells of the prostate [109, 110]. PSA is involved in the dissolution of the seminal clot and uses semenogelin as a substrate [111]. The clinical utilization of PSA as an important serum marker for prostate cancer has been established [106, 112]. PSA is an organ-specific rather than cancer-specific marker that is present in the serum of patients with BPH or prostatitis [113, 114].

As part of the components of seminal fluid, extracellular vehicles (EVs) are lipid bilayer nanovesicles, typically ranging in size from 30 to 1000 nm in diameter [115]. EVs serve as transporter of a wide range of molecules, including proteins, nucleic acids, metabolites and lipids [116,117]. Seminal plasma contains comparatively more EVs compared to those circulated in blood or cerebrospinal fluid [115, 116].

The main source of EVs in semen are the cells of the testes, epididymis and vas deferens, as well as the cells of the male accessory glands, including the prostate and seminal vesicles. [116]. Like EVs identified in other body fluids and containing a heterogeneous population characterized by their variable size, shape and composition [117, 118]. Semen EVs have the potential to bind to and exchange active molecules with mature sperm and endometrial epithelial cells and are thereby involved in the regulation of several reproductive processes. [116]. Furthermore, it has been reported that the cargo load of sEVs differs between normozoospermic and non-normozoospermic men. [119]. Seminal EVs would also facilitate the safe transit of spermatozoa in the female genital tract by regulating the uterine immune response [49, 116]. Taken together, these findings suggest that semen EVs may be involved in fertility and that some biomolecules encapsulated in EVs could be considered as biomarkers of male fertility. However, among the molecules encapsulated in sEVs, none are currently recognized as biomarkers for male fertility. To determine how far science has advanced in this field, the present systematic review aimed to summarize existing published evidence on the association of EVs with fertility and to identify candidate EV molecules that could function as biomarkers of fertility or infertility.

Prostasomes are EVs that is known as macrovesicles that can be secreted the acinar epithelial cells of prostate gland [57, 120]. Prosteosomes are an intracellular vesicles that are mostly found inside large storage vesicles Intracellular macrovesicles are similar to the multivesicular bodies of the late endosomal origin [57]. Therefore, it was assumed that prostasomes play an important role in intercellular communication, especially by directly interacting between the immotile acinar cells of the prostate and the motile sperm. In addition to the transfer membrane components Prostasomes can also transfer genetic material to sperm [121]. Once the proteases are released from the secretory epithelial cells of the prostate gland and become part of the sperm, they can interact with the sperm membrane and eventually fuse with it. [57, 120], thus transferring molecules from prostatic secretory cell to the spermatozoon has been reported to be mediated by prosteosomes [57, 120].

Accumulated evidence suggests activation of the renin-angiotensin system (RAS) in the human prostate [122-124]. The expression and cellular localization of angiotensin II (Ang II) and AT1 receptor proteins were analyzed in normal human prostate and benign prostatic hyperplasia (BPH). As has been reported, increased expression of Ang II and significant reduction of the AT1 receptor are common in prostate hyperplasia are common [122-124]. While in the normal prostate the immunoreactivity was restricted to the basal layer of the epithelium, the AT1 receptor was predominantly localized in both the stroma and the smooth muscle of the prostatic vessels [122, 123]. The downregulation of the AT1 receptor together with increased tissue levels of Ang II as well as increased angiotensin-converting enzyme (ACE) at both mRNA and protein levels in BPH [125, 126], suggesting that RAS activation in BPH depends on its cellular localization and function. The role of Ang II and AT1 receptors in the regulation of prostate cancer has been demonstrated in several studies [127, 128]. The observation suggests clinical relevance to inhibition of RAS activation in the treatment and prevention of PCa [129-131]. Several studies have also shown that angiotensin II receptor blockers (ARBs) have the potential to reduce elevated PSA levels in patients without PCa and significantly reduce the risk of PCa. [131, 132]. .]has been added to chapter 2

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Comment 2: Minor comment: page 20/30: Reference 81 is incomplete

Authors’response: Thank you very much for comment. We apologize for the mistake. Accordingly, we replaced the incomplete reference with the corrected one

See lines: 1263-164; the incomplete reference [, B. The role of oxytocin in male and female reproductive behavior. Eur J Pharmacol 2015, 753, 209-228. DOI: 10.1016/j.ejphar.2014.07.045.] by the corrected one [ Veening, J. G.; de Jong, T. R.; Waldinger, M. D.; Korte, S. M.; Olivier, B. The role of oxytocin in male and female reproductive behavior. Eur J Pharmacol 2015, 753, 209-228. DOI: 10.1016/j.ejphar.2014.07.045.]

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Editor

The title of the review is misleading. The appropriate title should be: How frequent ejaculations reduce the risk of prostate cancer?
The abstract basically says nothing. Pressure space is taken by anatomical and physiological explanations that are not relevant to the subject.
Then comes long and out of the subject endocrinological description.
The connection between frequent ejaculation and reduced PC risk is not a general knowledge and one would expect a paragraph describing this.
Some of the text is underlined without any obvious reason.
It is only on page 14 that the authors finally get to the point. Chapter 7 is well written, and its summary should appear in the abstract.

Author Response

Author response to Reviewer 2

Dear Editor,

Enclosed find please our response to the valuable comment of reviewer 02

Reviewer 3

Comments and Suggestions for Authors

Commen 1: The title of the review is misleading. The appropriate title should be: How frequent ejaculations reduce the risk of prostate cancer?

Authors’response: Thank you very much for your comment, As required, we changed the title to be more suitable.

Comment 2: The abstract basically says nothing. Pressure space is taken by anatomical and physiological explanations that are not relevant to the subject.

Authors’response:

Comment 3: Then comes long and out of the subject endocrinological description.

Authors’response: Thank you very much for your comment

Comment 4: The connection between frequent ejaculation and reduced PC risk is not general knowledge and one would expect a paragraph describing this.

Authors’response: Thank you very much for your valuable comment. Accordingly, we modified the title to be more suitable and also, we made significant modification and changes in the overall structure of the manuscript. Accordingly, the following modifications and changes have been made.

See lines: 1748-1802; the following references [

Newman, H. F.; Reiss, H.; Northup, J. D. Physical basis of emission, ejaculation, and orgasm in the male. Urology 1982, 19 (4), 341-350. DOI: 10.1016/0090-4295(82)90186-8.

Parsa, N. Environmental factors inducing human cancers. Iran J Public Health 2012, 41 (11), 1-9.

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La Merrill, M. A.; Vandenberg, L. N.; Smith, M. T.; Goodson, W.; Browne, P.; Patisaul, H. B.; Guyton, K. Z.; Kortenkamp, A.; Cogliano, V. J.; Woodruff, T. J.; et al. Consensus on the key characteristics of endocrine-disrupting chemicals as a basis for hazard identification. Nat Rev Endocrinol 2020, 16 (1), 45-57. DOI: 10.1038/s41574-019-0273-8.

Tabb, M. M.; Blumberg, B. New modes of action for endocrine-disrupting chemicals. Mol Endocrinol 2006, 20 (3), 475-482. DOI: 10.1210/me.2004-0513..

Ankolkar, M.; Balasinor, N. H. Endocrine control of epigenetic mechanisms in male reproduction. Horm Mol Biol Clin Investig 2016, 25 (1), 65-70. DOI: 10.1515/hmbci-2016-0007

Corti, M.; Lorenzetti, S.; Ubaldi, A.; Zilli, R.; Marcoccia, D. Endocrine Disruptors and Prostate Cancer. Int J Mol Sci 2022, 23 (3). DOI: 10.3390/ijms23031216.

Bleak, T. C.; Calaf, G. M. Breast and prostate glands affected by environmental substances (Review). Oncol Rep 2021, 45 (4). DOI: 10.3892/or.2021.7971

Prins, G. S. Endocrine disruptors and prostate cancer risk. Endocr Relat Cancer 2008, 15 (3), 649-656. DOI: 10.1677/ERC-08-0043.

Lehle, J. D.; McCarrey, J. R. Differential susceptibility to endocrine disruptor-induced epimutagenesis. Environ Epigenet 2020, 6 (1), dvaa016. DOI: 10.1093/eep/dvaa016.

Comment 5: Some of the text is underlined without any obvious reason.

Authors’response: Authors’ response: Thank you very much for your comment. The underlined paragraphs were the authors ‘response to the comment of the editor. Accordingly, we underlined the changes that have been made in the manuscript.

Comment 6: It is only on page 14 that the authors finally get to the point. Chapter 7 is well written, and its summary should appear in the abstract.

Authors’response: Thank you very much for your valuable comment. As you suggested, we modified the abstract

See lines:34-45; the following text [Abstract: Prostate cancer (PCa) accounts for roughly 15% of diagnosed cancer among men, with disease incidence increasing worldwide. Age, family history and ethnicity, diet, physical activity, and chemoprevention all play a role in reducing PCa risk. Although frequent ejaculation has been reported to play a significant role in the reduction of PCa risk, the mechanisms regulating frequent ejaculation- mediated reduction of PCa risk are not well described. The prostate gland is part of the male reproductive system located inside the body. As an exocrine gland, the prostate is a multi-functional gland involved in reproductive aspects such as male ejaculation and orgasmic ecstasy, as well as plays key roles in the regulation of local and systemic concentrations of 5α-dihydrotestosterone. 5α-dihydrotestosterone, expressed in the prostate, is an important potent androgen which results from the conversion of the precursor testosterone via mechanism mediated by isoenzyme 5α- reductase-2. Recent evidence indicates that frequent ejaculation plays a potential role in reducing PCa risk. Accordingly, more frequent ejaculation in the absence of risky sexual behavior likewise improves PCa risk. In this review, we provide an insight into possible mechanisms regulating the impact of frequent ejaculation on reducing PCa risk. ] has been replaced by a new one [Abstract: Prostate cancer (PCa) accounts for roughly 15% of diagnosed cancer among men, with disease incidence increasing worldwide. Age, family history and ethnicity, diet, physical activity, and chemoprevention all play a role in reducing PCa risk. The prostate is an exocrine gland that is characterized by its multi-function involved in reproductive aspects such as male ejaculation and orgasmic ecstasy, as well as plays key roles in the regulation of local and systemic concentrations of 5α-dihydrotestosterone. The increase in androgen receptors at the ventral prostate is the first elevated response induced by copulation. The regulation of prostate growth and function is mediated by androgens-dependent mechanism. Binding 5-DHT binds to androgen receptors (AR) results in the formation of a 5α-DHT:AR complex. The interaction of 5α-DHT:AR complex with the specific DNA enhancer element of androgen-regulated genes leading to the regulation of androgen-specific target genes to maintain prostate homeostasis. Consequently, ejaculation may play a significant role in the reduction of PCa risk. Thus, the frequent ejaculation in the absence of risky sexual behavior likewise is a possible approach or the prevention of PCa. In this review, we provide an insight into possible mechanisms regulating the impact of frequent ejaculation on reducing PCa risk.]

Author Response File: Author Response.pdf

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have significantly improved the manuscript!

Author Response

Authors‘response to Reviewer 1

Comments and Suggestions for Authors: The authors have significantly improved the manuscript!

Authors ‘response: Thank you very much for your comment

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

This revised manuscript is sufficiently improved.

Please observe a small notice:

An error has slipped into the text in next last paragraph of Discussion (before References):

Prostasomes have quite accidentally been denoted "prosteosomes"

Author Response

Authors ‘response to Reviewer 2

Comments and Suggestions for Authors: This revised manuscript is sufficiently improved. Please observe a small notice: An error has slipped into the text in next last paragraph of Discussion (before References): Prostasomes have quite accidentally been denoted "prosteosomes

Auhors’response: Thank you very much for your valuable comment. We apologize for this mistake. We have made the required corrections.

See line:226, 228, 231, 234; The word “ prosteosomes” has been corrected and replaced by the corrected word “ Prostasomes “

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

I did not find the response to comment 2

Author Response

Authors’response to Reviewer 3

Comment 2: The abstract basically says nothing. Pressure space is taken by anatomical and physiological explanations that are not relevant to the subject.

Authors ‘response: Thank you very much for your comment, We apologize that we forget to mention the changes performed and modification of the abstract.

See lines:37-52; the following abstract [ Prostate cancer (PCa) accounts for roughly 15% of diagnosed cancer among men, with disease incidence increasing worldwide. Age, family history and ethnicity, diet, physical activity, and chemoprevention all play a role in reducing PCa risk. Although frequent ejaculation has been reported to play a significant role in the reduction of PCa risk, the mechanisms regulating frequent ejaculation- mediated reduction of PCa risk are not well described. The prostate gland is part of the male reproductive system located inside the body. As an exocrine gland, the prostate is a multi-functional gland involved in reproductive aspects such as male ejaculation and orgasmic ecstasy, as well as plays key roles in the regulation of local and systemic concentrations of 5α-dihydrotestosterone. 5α-dihydrotestosterone, expressed in the prostate, is an important potent androgen which results from the conversion of the precursor testosterone via mechanism mediated by isoenzyme 5α- reductase-2. Recent evidence indicates that frequent ejaculation plays a potential role in reducing PCa risk. Accordingly, more frequent ejaculation in the absence of risky sexual behavior likewise improves PCa risk. In this review, we provide an insight into possible mechanisms regulating the impact of frequent ejaculation on reducing PCa risk. ] has been modified and replaced by the following abstract[Abstract: Prostate cancer (PCa) accounts for roughly 15% of diagnosed cancer among men, with disease incidence increasing worldwide. Age, family history and ethnicity, diet, physical activity, and chemoprevention all play a role in reducing PCa risk. The prostate is an exocrine gland that is characterized by its multi-function involved in reproductive aspects such as male ejaculation and orgasmic ecstasy, as well as plays key roles in the regulation of local and systemic concentrations of 5α-dihydrotestosterone. The increase in androgen receptors at the ventral prostate is the first elevated response induced by copulation. The regulation of prostate growth and function is mediated by androgens-dependent mechanism. Binding 5-DHT binds to androgen receptors (AR) results in the formation of a 5α-DHT:AR complex. The interaction of 5α-DHT:AR complex with the specific DNA enhancer element of androgen-regulated genes leading to the regulation of androgen-specific target genes to maintain prostate homeostasis. Consequently, ejaculation may play a significant role in the reduction of PCa risk. Thus, the frequent ejaculation in the absence of risky sexual behavior likewise is a possible approach or the prevention of PCa. In this review, we provide an insight into possible mechanisms regulating the impact of frequent ejaculation on reducing PCa risk. ]

Author Response File: Author Response.pdf