Ceiling effect of clomiphene citrate on the testosterone to estradiol ratio in eugonadal infertile men.

INTRODUCTION: The testosterone to estradiol ratio (T/E2 ratio) reportedly exerts a stronger effect on semen quality and sexual desire than does testosterone alone. Clomiphene citrate is a selective estrogen receptor modulator that has long been used as an empirical treatment option in the management of idiopathic oligozoospermia. Clomiphene may change the hypothalamus-pituitary-gonad axis and result in the alteration of the T/E2 ratio. No reliable data are available regarding the change in the T/E2 ratio after clomiphene use in eugonadism.
METHODS: This study included 24 male patients who were diagnosed with idiopathic infertility with eugonadism. They all received clomiphene citrate (25 mg/day) as empirical treatment. Blood tests for serum testosterone, estradiol, prolactin, luteinizing hormone, and follicle stimulating hormone were performed before and after 4 weeks of clomiphene use. Paired t-tests were used to evaluate the significance of the hormone level change.
RESULTS: Overall, the patients' T/E2 ratio did not increase significantly after clomiphene use. In the subgroup analysis, the T/E2 ratio of patients with a baseline ratio of <200 increased significantly after clomiphene use.
CONCLUSIONS: Clomiphene citrate may significantly increase the T/E2 ratio in eugonadal men under the premise of its ceiling effect (T/E2 ratio < 200), providing practitioners with guidance on the use of clomiphene in this demographic.

Introduction

Selective estrogen receptor modulators (SERMs) are drugs that act on estrogen receptors [1]. Such drugs can be divided into receptor agonists and antagonists. These drugs selectively act on specific organs, with effects differing by organs [1]. Clomiphene citrate is both a nonsteroidal antiestrogen drug and an SERM that competes with estradiol for estrogen receptors in the hypothalamus and blocks the normal negative feedback of circulating estradiol on the hypothalamus [2]. The production of gonadotroptin-releasing hormone (GnRH) may not be limited by estrogen; instead, the pituitary gland releases more follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and causes an increase in sperm and testosterone production by the testes [2]. High levels of intratesticular testosterone is a key factor in the nonconventional pathway of testosterone for spermatogenesis [3].

Abhyankar [4] revealed that the testosterone/estradiol (T/E2) ratio exerts a stronger effect on semen quality and sexual desire than did testosterone alone; the author concluded that a larger increase in the sperm concentration and total motility count was correlated with a larger increase in the posttreatment T/E2 ratio. A small population study by Shabsigh [5] also reported an increased T/E2 ratio after clomiphene use in patients with hypogonadism; the author concluded that low dose clomiphene citrate may improve the T/E2 ratio in men with hypogonadism. However, to our knowledge, no large study has been conducted on the T/E2 ratio change after clomiphene use in infertile patients with eugonadism. The aim of this preliminary study was to determine whether clomiphene citrate exerts an effect on the T/E2 ratio in infertile patients with eugonadism.

Materials and methods

This single-center, retrospective study was conducted in the Urology Department of #### from May 1, 2018, to May 31, 2019. Inclusion criteria were as follows: (1) being a man diagnosed with infertility; (2) taking clomiphene as testosterone restoration therapy; (3) accepting blood tests including those for prolactin, LH, FSH, testosterone, and estrogen; and (4) having an initial testosterone level within the normal range. Those who had undergone surgery of the testis or had received hormonal drugs that would affect the testosterone level (including dutasteride and leuprorelin) were excluded. Infertility was defined as failure to establish a clinical pregnancy after 12 months of regular and unprotected sexual intercourse. The normal range of the total testosterone level was defined as 240 to 870 ng/dL.

The study protocol was approved by the Institutional Review Board of #### with relevant jurisdiction (IRB No. 201903111RIN). The necessity of informed consent was confirmed by the institutional review board.

Before receiving clomiphene citrate treatment, blood samples were examined to record baseline serum total testosterone, estradiol, LH, FSH, and prolactin levels. All patients received clomiphene citrate 25 mg once a day for at least 1 month. Patients were followed up at 4 weeks after the treatment initiation to evaluate the treatment response. At the follow-up visit, the aforementioned serum levels were recorded to compare with those before clomiphene use. All hormone test(testosterone, estradiol, LH, FSH and prolactin) included in our study are sent to the central lab in “Department of Laboratory Medicine of National Taiwan University Hospital”. FSH, LH, E2 and prolactin were measured by using IMMULITE® 2000 immunoassay system and Testosterone was measured by the ARCHITECT® 2nd Generation Testosterone assay which is a chemiluminescent microparticle immunoassay for the quantitative determination of testosterone. Simple statistical analysis (paired t-tests) was used to analyze hormone level changes resulting from clomiphene therapy. The primary outcome of our study was changes in the T/E2 ratio after the administration of clomiphene citrate in eugonadal men with infertility. Secondary outcomes included changes in other hormone levels (FSH, LH, and prolactin).

Results

In total, 22 men were retrospectively reviewed in this study. The average age of these patients was 40.9 (range: 31–48) years, with 12 being older than 40 years. All patients were followed up in an outpatient clinic by one urologist. Most of these patients were healthy individuals seeking treatment for infertility; however, one had hypertension and ulcerative colitis, and another had liver cirrhosis, diabetes mellitus, and polyneuropathy.

The mean total testosterone level before treatment (n = 22) was 469 ± 204 (range: 212–947) ng/dL, and the mean estradiol level (n = 21) was 3.819 ± 1.312 (range: 1.3–7.8) ng/dL. The mean T/E2 ratio at the first visit (n = 21) was 160 ± 90 (Table 1). The mean LH (n = 21) and FSH (n = 22) levels were 4.89 ± 3.05 (range: 1.54–14.3) mIU/mL and 5.68 ± 3.17 (range: 1.59–14.4) mIU/mL, respectively. The mean prolactin level (n = 21) was 6.80 ± 3.32 (range: 0.9–12.7) ng/mL (Table 1).

Table 1

Endocrinology data at baseline and 4 weeks after initiating clomiphene treatment.

Hormone (Conc.)	Number of cases	Baselinea	Post treatmentb	Clomiphene effect	P value
Testosterone(ng/dL)	22	469±204	935±349*	↑99.2%	<0.001
Estradiol (ng/dL)	21	3.819±1.312	6.605±3.091*	↑80.8%	<0.001
T/E2 ratio	21	160±90	180±60	↑ 12.5%	0.302
LH (mIU/mL)	21	4.89±3.05	10.90±5.92*	↑ 123.1%	<0.001
FSH (mIU/mL)	22	5.68±3.17	11.43±7.86*	↑101.3%	0.002
Prolactin (ng/mL)	21	6.80±3.32	5.77±2.49	↓15.1%	0.297

Values are presented as the mean±standard deviation.

*Versus baseline data (p<0.05).

aBaseline hormone level before Clomiphene use.

bHormone level after Clomiphene use.

The patients were re‐evaluated approximately 4 weeks after initiating therapy with 25 mg of oral clomiphene citrate once a day. A significant increase was noted in the mean total testosterone level (n = 22) to 935 ± 349 (range: 285–1916) ng/dL, representing an increase of 99.2% (P < 0.001). The mean estradiol level (n = 21) increased significantly to 6.605 ± 3.091 (range: 2.57–1.30) ng/dL, representing a rise of 80.8% (P < 0.001). The T/E2 ratio also increased from 160 ± 90 to 180 ± 60, a rise of 12.5%; however, the difference was not significant (P = 0.302; Table 1). The posttreatment mean LH (n = 21) and FSH (n = 22) levels increased significantly to 10.90 ± 5.92 (range: 2.93–22.2) mIU/mL (P < 0.001) and 11.43 ± 7.86 (range: 4.25–36.9) mIU/mL (P = 0.002), representing an increase of 123.1% and 101.3%, respectively. The posttreatment mean prolactin level (n = 21) decreased to 5.77 ± 2.49 (range: 2.84–12.20) ng/mL; this 15.1% decrease, however, did not reach significance (P = 0.297; Table 1).

The 21 patients included were further analyzed according to the baseline T/E2 ratio. Four patients had a baseline T/E2 ratio of <100. Their mean T/E2 ratio increased significantly from 070 ± 10 to 110 ± 20 after clomiphene use, representing an increase of 57.1% (P = 0.006). Twelve patients had a baseline T/E2 ratio of < 150. Their mean T/E2 ratio significantly increased from 090 ± 30 to 130 ± 40 after clomiphene use, which is an increase of 44.4% (P = 0.006). Fifteen patients had a baseline T/E2 ratio of <200. Their mean T/E2 ratio significantly increased from 120 ± 40 to 150 ± 50 after clomiphene use, representing an increase of 25.0% (P = 0.0017). The T/E2 ratio of the five patients with a baseline T/E2 ratio < 250 increased from 120 ± 50 to 160 ± 50; however, the difference was not significant (P = 0.09), which is similar to the result in the group with all patients included (n = 21, P = 0.302; Table 2).

Table 2

T/E2 ratio of men with a lower baseline ratio (<0.20) increased significantly after clomiphene treatment.

Upper limit of T/E2	Number of cases	Baselinea	Post treatmentb	Clomiphene effect	P value
Any	21	160±90	0.18±0.06	↑12.5%	0.302
<0.25	17	120±50	160±50	↑33.3%	0.09
<0.20	15	120±40	150±50*	↑25.0%	0.017
<0.15	12	90±30	130±40*	↑44.4%	0.006
<0.10	4	70±10	110±20*	↑57.1%	0.006

Values are presented as the mean±standard deviation.

*Versus baseline data (p<0.05).

a Baseline T/E2 level before clomiphene use.

b T/E2 after clomiphene use.

Discussion

Clomiphene citrate, an SERM, is a nonsteroidal antiestrogen drug that has empirically been used in the management of idiopathic oligospermia. The drug competes with estradiol for estrogen receptors in the hypothalamus and blocks the normal negative feedback of circulating estradiol on the hypothalamus [6, 7]. Under clomiphene therapy, the amplitude of GnRH pulses increases, stimulating the pituitary gland to produce more FSH and LH. As a result, testicular total testosterone also increases [6, 8–10]. When used to treat male infertility, clomiphene is well tolerated with no identified serious adverse effects [11-13].

Few studies have investigated the relationship between clomiphene citrate use and the T/E ratio. Shabsigh [4] recruited 36 Caucasian men with hypogonadism, defined as having a serum total testosterone level of <300 ng/dL. Each patient was treated with a daily dose of 25 mg clomiphene citrate. The serum levels of total testosterone and estradiol were recorded at baseline and follow-up visits. By the first follow-up visit (at 4–6 weeks), the mean total testosterone level had increased significantly (P < 0.00001). Moreover, the T/E ratio improved from 8.7 to 14.2 (P < 0.001). Thus, the T/E2 ratio of patients with hypogonadism increased after clomiphene use. No further research on clomiphene use and the testosterone to estradiaol ratio in eugonadal patients has been conducted, prompting the current study.

In our study, we observed a significant increase in the mean total testosterone level, with an increase of 99.2% (P < 0.001). The mean estradiol level also increased significantly, with a rise of 80.8% (P < 0.001). These results are similar to those of patients with hypogonadism in the aforementioned study [4] who took clomiphene citrate. As for other hormone levels, the mean FSH and LH levels increased significantly after clomiphene treatment, with a rise of 101.3% (P = 0.002) and 123.1% (P < 0.001), respectively. Because clomiphene citrate competes with estradiol for estrogen receptors in the hypothalamus, it blocks the normal negative feedback of circulating estradiol in the hypothalamus. As a result, LH and FSH levels would increase, which is consistent with our results.

Regarding the T/E2 ratio, compared with the T/E2 ratio in the general male population, that of patients enrolled in our study was relatively low. Gong [14] enrolled 337 patients in a single center as the control group to study the correlation between the T/E2 ratio and cardiovascular events, revealing the normal range of the T/E2 ratio to be 190 ± 60. In our study, the mean baseline T/E2 ratio was 160 ± 90. When including all patients, we noted an increase of 6.25% in the ratio, but the difference was not significant. We obtained different results when setting an upper limit of the T/E2 ratio. The mean T/E2 ratio increased significantly (57.1%) after clomiphene use if only patients with a T/E2 ratio of <100 were included. A significant increase in the T/E2 ratio after clomiphene use was also noted in those with a T/E2 ratio of <200. After slightly increasing the upper limit to a T/E2 ratio of <250, we still noted an increase of 33.3% in the T/E2 ratio; however, the difference was not significant. Therefore, we suggest ceiling effects for increasing the T/E2 ratio; in the current study, the ceiling effect was set at a T/E2 ratio of <200. This result suggests that using clomiphene in infertile men with eugonadism whose T/E ratio is >200 is not advisable.

This study has some limitations. Only 24 patients were included; thus, the sample was relatively small. Moreover, because this was a retrospective study, the exposure method could not be controlled, potentially resulting in variable clomiphene citrate dosage and frequency. Possible factors that may affect total testosterone and estradiol level, such as BMI level, waistline and testicular volume, were also not included. Future studies should prospectively include these parameters for analysis to confirm the T/E2 ratio changeafter clomiphene citrate use in infertile men with eugonadism.

Conclusion

Clomiphene citrate may significantly increase the T/E2 ratio in eugonadal infertile men under the premise of its ceiling effect (T/E2 ratio < 200), providing useful guidance for the use of clomiphene in this group of patients. Further investigations, especially those with a larger sample size, are required to confirm the results.

17 Nov 2021

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Reviewer #1: I appreciate the manuscript and the work that went into preparing it. Just a few suggestions to strengthen the product:

1. As aromatase breaks down T to E2, would report on the effect of BMI on results/outcomes.

2. Should the Cirrhosis patient be excluded since liver disease effects sex hormone binding globulin production (effects total Testosterone)?

3. Is the standard to use ng/mL for T? I'm more used to ng/dL...with the normal T:E ratio being 10:1.

Best regards.

Reviewer #2: Dear Dr. Hong-Chiang Chang

Based on my own reading of your paper, I have some issues I want to address. I would like to say right away that the theme “ceiling effect” is quite interesting as a predictor for clomiphene citrate (CC) use.

I emphasize that the PDF of the article I received does not have line:numbers, which made the review a bit difficult.

1) Introduction: I did not observe references for all the information contained in the paragraph below. Despite being general domain information, it is important to always add reference sources, even if it is in the introduction.

“Selective estrogen receptor modulators (SERMs) are drugs that act on estrogen receptors. Such drugs can be divided into receptor agonists and antagonists (Ref?). These drugs selectively act on specific organs, with effects differing by organs (Ref?). Clomiphene citrate is both a nonsteroidal antiestrogen drug and an SERM that competes with estradiol for estrogen receptors in the hypothalamus and blocks the normal negative feedback of circulating estradiol on the hypothalamus (Ref?). The production of gonadotroptin-releasing hormone (GnRH) may not be limited by estrogen; instead, the pituitary gland releases more follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and causes an increase in sperm and testosterone production by the testes (Ref?). High levels of intratesticular testosterone is a key factor in the nonconventional pathway of testosterone for spermatogenesis (Ref?).

2) Materials and Methods

I would like the authors to specify which reference was used to define the diagnosis of eugonadism (total testosterone 2.4 to 8.7 ng/mL).

The laboratory diagnosis of hypogonadism is based on the measurement of total testosterone. The American Society of Endocrinology recommends the value of 264 ng/dL (9.2 nmol/L) as the lower limit to define the hypogonadal state; however, the cutoff levels for diagnosis are not a consensus. So I would like the authors to specify the Guideline used.

(Testosterone Therapy in Men With Hypogonadism: An Endocrine Society* Clinical Practice Guideline)

I would like the authors to write “range of the total testosterone level” instead of just “range of the testosterone level” – here it can be confusing if total or free testosterone was used as a parameter.

I would like more detail in the table of 22 patients at baseline with data about BMI, waistline and testicular volume if possible

3) Discussion

An important point to be considered is the ability of the CC to induce a response in patients with central or mixed hypogonadism. In the different studies in which its use was evaluated, it was possible to define response (elevation of testosterone) in patients with Leydig cells capable of reacting to the central stimulus restored by CC (that's why I would like to know about testicular volume).

It is known that the unbalance of the T:E2 ratio is directly implicated in the pathophysiology of hypogonadism. It is important to emphasize that the increase in estrogen with the use of CC results from the aromatization of testosterone in the adipose tissue, especially the visceral tissue. I would like to see the details of the abdominal fat of these patients in order to understand whether the ceiling effect proposed by the authors is related to patients with larger abdominal fat/waistline and therefore greater visceral fat.

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Reviewer #2: Yes: Andressa Heimbecher Soares

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30 Nov 2021

*To editor:

We really appreciate for reminding us all additional journal requirements. The article has now met PLOS ONE's style requirements. As for the Data Availability statement, we stated that there are no supporting information in the main body of our article.

*To reviewer 1:

Q: 1. As aromatase breaks down T to E2, would report on the effect of BMI on results/outcomes.

Answer: Different BMI levels may probably affect T/E2 ratio. However, we did not include patients’ BMI in our data base. Your suggestion is good for further study of this topic and data of BMI would make a more precise analysis.

Q: 2. Should the Cirrhosis patient be excluded since liver disease effects sex hormone binding globulin production (effects total Testosterone)?

Answer: Cirrhosis surely affect sex hormone binding globulin production, indirectly influencing the bioavailability of testosterone. As for the patient included in our study with liver cirrhosis, his Child-Pugh score is only grade A with normal bilirubin, PT/APTT and albumin level, which may hardly have an impact on sex hormone binding globulin production.

Q: 3. Is the standard to use ng/mL for T? I'm more used to ng/dL...with the normal T:E ratio being 10:1.

Answer: There may not be a standard unit of testosterone level. According to EAU(European Association of Urology) guideline, ng/mL and nmol/L have both been used as the unit of testosterone level.

*To reviewer 2:

1) Introduction:

Q: I did not observe references for all the information contained in the paragraph below. Despite being general domain information, it is important to always add reference sources, even if it is in the introduction.

“Selective estrogen receptor modulators (SERMs) are drugs that act on estrogen receptors. Such drugs can be divided into receptor agonists and antagonists (Ref?). These drugs selectively act on specific organs, with effects differing by organs (Ref?). Clomiphene citrate is both a nonsteroidal antiestrogen drug and an SERM that competes with estradiol for estrogen receptors in the hypothalamus and blocks the normal negative feedback of circulating estradiol on the hypothalamus (Ref?). The production of gonadotroptin-releasing hormone (GnRH) may not be limited by estrogen; instead, the pituitary gland releases more follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and causes an increase in sperm and testosterone production by the testes (Ref?). High levels of intratesticular testosterone is a key factor in the nonconventional pathway of testosterone for spermatogenesis (Ref?).

Answer: We really appreciate the advice of adding references. Three references have been added to the introduction. The following paragraph is the revised introduction.

“Selective estrogen receptor modulators (SERMs) are drugs that act on estrogen receptors and such drugs can be divided into receptor agonists and antagonists [1]. These drugs selectively act on specific organs, with effects differing by organs [1]. Clomiphene citrate is both a nonsteroidal antiestrogen drug and an SERM that competes with estradiol for estrogen receptors in the hypothalamus and blocks the normal negative feedback of circulating estradiol on the hypothalamus [2]. The production of gonadotroptin-releasing hormone (GnRH) may not be limited by estrogen; instead, the pituitary gland releases more follicle-stimulating hormone (FSH) and luteinizing hormone (LH) and causes an increase in sperm and testosterone production by the testes [2]. High levels of intratesticular testosterone is a key factor in the nonconventional pathway of testosterone for spermatogenesis [3].”

Reference:

1. Chua, M.E., et al. Revisiting oestrogen antagonists (clomiphene or tamoxifen) as medical empiric therapy for idiopathic male infertility: a meta-analysis. Andrology, 2013. 1: 749.

2. Ribeiro, R.S., et al. Clomiphene fails to revert hypogonadism in most male patients with conventionally treated nonfunctioning pituitary adenomas. Arq Bras Endocrinol Metabol, 2011. 55: 266.

3. Hussein, A., et al. Clomiphene administration for cases of nonobstructive azoospermia: a multicenter study. J Androl, 2005. 26: 787.

2) Materials and Methods

Q: I would like the authors to specify which reference was used to define the diagnosis of eugonadism (total testosterone 2.4 to 8.7 ng/mL).

The laboratory diagnosis of hypogonadism is based on the measurement of total testosterone. The American Society of Endocrinology recommends the value of 264 ng/dL (9.2 nmol/L) as the lower limit to define the hypogonadal state; however, the cutoff levels for diagnosis are not a consensus. So I would like the authors to specify the Guideline used.

(Testosterone Therapy in Men With Hypogonadism: An Endocrine Society* Clinical Practice Guideline)

Answer: Exactly I agree that the cutoff level of hypogonadism and eugonadism has not reached a consensus. Different hospital may have different lab reference level with regard to their laboratory department. All patient included in our study have done blood examination in National Taiwan University Hospital. The reference range of total testosterone(2.4 to 8.7 ng/mL) is defined by Department of Laboratory Medicine of National Taiwan University Hospital.

Q: I would like the authors to write “range of the total testosterone level” instead of just “range of the testosterone level” – here it can be confusing if total or free testosterone was used as a parameter.

Answer: We really appreciate the advice of modifying “testosterone” to “total testosterone” not to be confusing. Adjustment have been made throughout the whole manuscript(changing the testosterone level to the total testosterone level)

Q: I would like more detail in the table of 22 patients at baseline with data about BMI, waistline and testicular volume if possible

Answer: BMI level, waistline and testicular volume are surely the possible factor that may affect total testosterone and estradiol level, indirectly influencing the T/E2 ratio. Unfortunately, these parameters were not included in our study. Due to the retrospective nature of our study, these factors were not recorded in our data base. Further prospective study including these factors may be needed.

3) Discussion

Q: An important point to be considered is the ability of the CC to induce a response in patients with central or mixed hypogonadism. In the different studies in which its use was evaluated, it was possible to define response (elevation of testosterone) in patients with Leydig cells capable of reacting to the central stimulus restored by CC (that's why I would like to know about testicular volume).

It is known that the unbalance of the T:E2 ratio is directly implicated in the pathophysiology of hypogonadism. It is important to emphasize that the increase in estrogen with the use of CC results from the aromatization of testosterone in the adipose tissue, especially the visceral tissue. I would like to see the details of the abdominal fat of these patients in order to understand whether the ceiling effect proposed by the authors is related to patients with larger abdominal fat/waistline and therefore greater visceral fat.

Answer: According to EAU (European Association of Urology) guideline, larger abdominal fat and greater visceral fat has now been established to be related to hypogonadism. Increase in estrogen by aromatase in visceral fat has also been established in several studies. Unfortunately, these factors were also not included in our study and may not be achieved due to the retrospective nature of our study. We will work our best on the further prospective study of relevant topics.

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

30 Dec 2021

3 Jan 2022

Reviewer #2:

Q: 1) In this part of the manuscript, I would like to correct “A small population study by Shabsigh [5] also reported an increased T/E ratio after clomiphene use in patients with hypogonadism” to “A small population study by Shabsigh [5] also reported an increased T/E2 ratio after clomiphene use in patients with hypogonadism”. The number 2 on E was missing.

A: We really appreciate for checking through the manuscript for any missing word or number. “Number 2” has now been added to T/E ratio.

Q: 2) I would like the description of the laboratory kits used for the analysis of total testosterone level, estradiol level, LH, FSH and prolactin level to be detailed.

A: We add the following description about the measurement methods in my document. All hormone test(testosterone, estradiol, LH, FSH and prolactin) included in our study are sent to the central lab in “Department of Laboratory Medicine of National Taiwan University Hospital”. FSH, LH, E2 and prolactin were measured by using IMMULITE® 2000 immunoassay system and Testosterone was measured by the ARCHITECT® 2nd Generation Testosterone assay which is a chemiluminescent microparticle immunoassay for the quantitative determination of testosterone.

Q:3) I would like to know if the dosage of sex hormone binding globulin (SHBG) levels was measured via morning lab draws.

A: Sex hormone binding globulin (SHBG) levels is surely a possible factor that may affect bioavailable testosterone, indirectly influencing the T/E2 ratio. Unfortunately, this parameter was not included in our study. Due to the retrospective nature of our study, this factor was not recorded in our data base. Further prospective study including this factor may be needed.

Q:4) To make it easy in terms of standardization, I would like to put the total testosterone level in ng/dL and estradiol levels in ng/dL (https://doi.org/10.1016/j.juro.2017.02.2652)

A: We really appreciate for this suggestion. All testosterone level and estradiol level have been changed to ng/dL. T/E2 ratio has also been adjusted due to the unit change of testosterone and estradiol.

Q:5) In the “limitations” part of the study, I would like it to be detailed that there wasn't possible to get BMI level, waistline and testicular volume data. I suggest that the authors should proceed with the study to evaluate obese men versus men with normal weight and analyse if there are differences in the ceiling effect.

A: We appreciate for the advice of adding this part to out limitation. We surely will proceed with the study to include these parameters for analysis to see if there are differences in the ceiling effect.

Q:6) If I may suggest, a greater number of participants would improve the strength of the results

A: Thanks for reviewer’s nice suggestion. Small study population is surely a limitation of our study. Further studies will include more participants to strengthen our result.

Q:7) The study generates an interesting hypothesis about which subgroup of patients may benefit most from the use of clomiphene citrate

A: We are glad that this study leads to an interesting result of “Clomiphene citrate significantly increase the T/E2 ratio in eugonadal infertile men under the premise of its ceiling effect (T/E2 ratio < 200)”. We would work hard on increasing the sample size and including more important parameters for further analysis.

Submitted filename: Response to Reviewers.docx

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10 Jan 2022

Ceiling Effect of Clomiphene Citrate on the Testosterone to Estradiol Ratio in Eugonadal Infertile Men

PONE-D-21-24474R2

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21 Jan 2022

PONE-D-21-24474R2

Ceiling Effect of Clomiphene Citrate on the Testosterone to Estradiol Ratio in Eugonadal Infertile Men

Dear Dr. Chang:

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