Main Research Findings

1) The use of clomiphene citrate, the parent compound of enclomiphene, leads to an increase in follicular oocytes reaching maturity.

2) After 6 weeks of continuous use, subjects using enclomiphene citrate exhibited a dramatic increase in testosterone levels in comparison to transdermal testosterone treatment .

3) Enclomiphene citrate increases levels of serum testosterone and luteinizing hormone (LH) to a level similar to topical testosterone treatments without the associated adverse side effects.

 

Selected Data

1) Researchers Yoshimura et. Al utilized 24 New Zealand White female rabbits for their experiment examining the effects of clomiphene citrate on the ovaries. The rabbits were housed individually and isolated in a controlled temperature for 3 weeks with free access to food and water. Prior to treatment the rats were put under anesthesia using 32 mg/kg of sodium pentobarbital. The ovarian artery was cannulated in preparation for the ovarian perfusion system. Following surgery, the test subjects were split into 3 experimental groups [2].

The first experimental group consisted of 6 rabbits. The ovary that was previously treated with gonadotropin experienced a perfusion of a 10^-5 M concentration of clomiphene citrate. The other ovary remained as the control variable as it was not treated with gonadotropin or perfused with clomiphene citrate. 30 minutes after perfusion began in the experimental ovary, 50 IUs of human chorionic gonadotropin (hCG) was added to the perfusion system in both the control and experimental ovary.

The second experimental group consisted of 12 rabbits. The purpose of the experiment was to examine the effects of clomiphene citrate in ovaries that were not previously treated with gonadotropin. Clomiphene citrate was perfused into 6 rabbits at a concentration of 10^-5 M while the other 6 rabbits received a concentration 10^-7 M into the experimental ovary. The other ovary was perfused with a medium in order to act as a control variable.

The final experimental group consisted of six rabbits. The purpose was to examine the effects elicited on follicular oocytes by a combination treatment of estradiol and clomiphene citrate. Both ovaries were perfused with a concentration of 10^-5 M of clomiphene citrate. The experimental ovary was additionally perfused with estradiol while the control ovary received a medium [2].

In all experiments the ovaries were frequently checked for follicle growth and rupture. The mature follicular cells that were obtained were perfused for 12 hours. All cells larger than 1.5 mM were punctured in aspiration in order to recover follicular oocytes for observation purposes. The ova were further classified into stages of maturity in order to compare the results of each experiment [2].

2) 48 men experiencing hypogonadism participated in a randomized, single-blind phase II study in order to determine the effects of three different doses of enclomiphene citrate on LH, follicle-stimulating hormone (FSH) and total testosterone levels. A fourth experimental group also transdermal testosterone in order to compare the results between the two treatments. The men ranged in age from 18-65, were considered to be in good health, and presented with testosterone levels greater than 350 ng/mL at baseline [3].

After the subjects were placed into three groups: the first group received 6.25 mg of enclomiphene citrate, the second group received 12 mg, and the third group of men received 25 mg of enclomiphene citrate, all on a daily basis. 44 men ended up completing the protocol method as 3 men from the 12.5 mg group and 1 from the 25 mg group had testosterone levels measuring greater than 350 ng/dL at baseline, disqualifying them from the study [3].

An enclomiphene citrate capsule was taken by the subjects between 7:00 h and 10:00 h every day. Transdermal testosterone gel was applied per package instructions. The gel was applied at the same time every day over a 6 week period and the subjects avoided bathing or swimming for 3-4 hours, following application. At visit 1, week 2 subjects underwent all necessary screening procedures. Eligible participants consented to a 24 hour assessment of LH, FSH, and total testosterone.

After all subjects consented to treatment and completed screening they were given a 2-week medication kit with instructions and supplies. The subjects periodically went in for visits in order to measure testosterone, FSH, and LH. Additional blood tests were conducted for a pharmacokinetic assessment of serum enclomiphene citrate. After 6 weeks of treatment all subjects underwent a 24 hour test measuring total testosterone, LH, and serum enclomiphene. At week 7 the men returned for a final measurement following a 1-week washout period [3].

Statistical analysis comparing changes in mean testosterone and LH levels between day 1 and day 42 were performed for all four experimental groups using ANOVA or the Kruskal-Wallis test. Changes in total testosterone and FSH levels were considered secondary variables and were checked throughout the stude. Blood tests assessing changes in lipids and hormones were performed during the baseline and week 6, while the pharmacokinetic analysis for serum enclomiphene citrate levels were taken over a 24 hour period during week 6. Levels of enclomiphene were recorded at hours 0, 1, 2, 3, 4, 8, 12, 16, and 24h [3].

3) 124 male subjects enrolled for this randomized, double-blind study and were split into 4 experimental treatment groups: enclomiphene citrate, 12.5 mg and 25 mg dose, a placebo, and a topical testosterone gel. The study took place over a 12 month period, starting in January 2011 and ending in December 2011. During the first visit hormone levels were recorded first thing in the morning as well as hemoglobin, hematocrit, glycosylated hemoglobin, and prostate-specific antigen.

The subjects had to have previous history or a current diagnosis of hypogonadism; if their testosterone levels were less than 250 ng/dL treatment was started immediately. Subjects with serum testosterone levels of greater than 300 ng/dL were disqualified from the study. A second measurement of testosterone levels were recorded right before treatment started in order to ensure proper procedures were followed. At the first visit the patients were provided with either a topical testosterone gel or enclomiphene citrate capsules, however subjects were unaware of which treatment they received to rule out bias [4].

The primary variables Wiehle et. Al were examining was the change in serum testosterone from baseline to month 3 of treatment. The comparison between the four treatment groups were analyzed using ANOVA. Secondary variables being observed were changes in FSH and LH levels from baseline to month three. Again, the comparison between the four groups were analyzed using ANOVA.

The test subjects frequently underwent various laboratory tests and physical examinations in order to record changes in various different tolerability variables. Semen samples were also obtained from the subjects in order to see how sperm concentration, semen volume, and motility changes in response to enclomiphene citrate versus topical testosterone [4].

 

Discussion

1) Results of the first experiment found that ovulation occurred in all ovaries including the control ovary that was treated with hCG alone. In this study the researchers refer to the percentages of mature follicles that rupture as the ovulatory efficiency. In group one, the ovulatory efficiency of the ovaries treated with both hCG and clomiphene citrate was similar to the control ovaries that received only hCG. There was also no observed difference in occurrences of ovulation between the experimental and control ovaries [2].

Table 1: Time and ovulation efficiency in the ovaries of group one

Additionally, perfusion with clomiphene citrate increased the percentage of follicular oocytes that reached the last stage of cell maturation by achieving germinal vesicle breakdown (GVBD). Results found that 84.6% of the oocytes treated with clomiphene citrate reached GVBD. This is in comparison to the control ovaries where 61.1% of the follicular oocytes reached GVBD.

Ovulation did not occur with either concentration administered of clomiphene citrate. However, perfusion with clomiphene citrate increased the percentage of follicular oocytes that reached the last stage of cell maturation by achieving germinal vesicle breakdown (GVBD). The 10^-5 M concentration led to a 59.3% increase in GVBD while the 10^-7 M concentration led to a 50% increase. The control ovary experienced far lower levels of GVBD, only increasing 10.3% and 15.2%, respectively [2].

Figure 1: Percentage of follicular oocytes racing GVBD in group 1 versus group 2

The subjects in the third experimental group did not experience ovulation in the ovary perfused with estradiol and clomiphene citrate or in the control ovary perfused with clomiphene citrate alone. Additionally, there was no difference between either ovary in the percentage of oocytes that reached GVBD. However, the results report that degeneration occurred in 79.3% of the follicular oocytes treated with clomiphene citrate alone. This is in comparison to the follicular oocytes treated with clomiphene and estradiol that exhibited only a 25% degeneration [2].

Figure 2: Percentage of oocytes reaching GVBD versus the oocytes that degenerated.

2) Researchers Wiehle et. Al determined that all four experimental groups experienced an increase in total testosterone in comparison to baseline levels. The improvements were most significant in the 6.25 mg enclomiphene citrate group, 25 mg enclomiphene citrate group, and the transdermal testosterone group. In regards to LH levels, the 6.25 mg and 25 mg of enclomiphene citrate groups resulted in an increase in serum LH from baseline levels, however, transdermal testosterone led to a decrease in serum LH levels. In the enclomiphene citrate groups the researchers observed that there were parallel increases in total testosterone and LH levels [3].

The results of the 24 hour pharmacodynamic studies were useful in comparing treatment with enclomiphene citrate versus transdermal testosterone. All treatment groups experienced a significant increase in testosterone after 6 weeks of following this treatment protocol. In terms of total testosterone concentration there was no variation in the baseline, mean, minimum, maximum, or range values between the experimental groups. On day 1 the transdermal testosterone group exhibited an increase in the mean and maximum values of total testosterone concentration, however, the increase was not considered significant.

Mean testosterone levels increased in all four experimental groups after 6 weeks of following treatment protocol. The researchers noted that transdermal testosterone treatment resulted in a large range of total testosterone value compared to enclomiphene citrate. The mean total testosterone range for transdermal testosterone was 590 +/- 465 ng/dL while the mean range for enclomiphene citrate was 314 +/- 65 ng/dL. The researchers noted that the wide variation in total testosterone could potentially be due to application issues [3].

Figure 2: Effects of enclomiphene versus transdermal testosterone in serum total testosterone levels. (A) transdermal testosterone, (B) 6.25 mg enclomiphene citrate, (C) 12.5 mg enclomiphene citrate, (D) 25 mg of enclomiphene citrate.

After 6 weeks of treatment, results reported that LH levels increased with enclomiphene citrate treatment and decreased with transdermal testosterone treatment. The increase in LH levels was observed for all doses of enclomiphene citrate. There was no significant difference between the serum LH levels of the 12.5 mg group and the 25 mg group. Mean LH concentration in all three enclomiphene citrate experimental groups were significantly higher than baseline levels. After treatment was completed and the subjects went back for the week 7 assessment, the group receiving 25 mg of enclomiphene citrate showed sustained increases in LH levels compared to the 6.25 mg group [3].

Figure 3: Changes in serum LH levels over the treatment period. 6.25 mg: open squares, 12.5 mg: green squares, 25 mg: orange triangles.

Similar results were found in the levels of FSH. Serum FSH levels decreased with transdermal testosterone treatment and increased with all three doses of enclomiphene citrate. Both the 12 mg and 25 mg groups increased FSH levels within two weeks of treatment. Transdermal testosterone reduced FSH levels throughout the entire 6 week period. The 12.5 mg and 25 mg doses were increasing at the same rate until the 4th week of treatment where serum FSH levels were measured as significantly higher in the 25 mg group than in the 12.5 or 6.25 mg groups. With the 6.25 and 12.5 mg doses of enclomiphene citrate, the rising FSH levels plateaued at week 4. The researchers noted that FSH serum levels seemed to increase gradually over the 6 week treatment period as there were no significant changes observed between weeks 2 and 4 or weeks 4 and 6, but an overall increase between week 1 and week 6 [3].

Figure 4: Changes in serum FSH levels over the treatment period 6.25 mg: open squares, 12.5 mg: green squares, 25 mg: pink squares, transdermal testosterone: orange triangles.

3) Results of the study reported that when comparing testosterone from control and baseline levels to the end of the 3-month dosing period, all three active treatment groups saw a significant increase in total testosterone. The researchers reported no statistical differences in testosterone levels between the two doses of enclomiphene citrate and topical testosterone. The topical testosterone treatment led to a decrease in the two hormones.

Additionally, results found that both doses of enclomiphene citrate increased levels of LH and FSH after 6 weeks of treatment. FSH levels in the 25 mg group increased 9.5 +/- 5.4 mIU/mL while the levels in the 12.5 mg group increased to 6.6 +/- 4.4 mIU/mL. LH levels of the 25 mg group increased 6.7 +/- 5.4 mIU/mL and the 12.5 mg group increased levels by 4.8 +/- 2.8 mIU/mL. These measurements were taken 1 month after treatment began [4].

The semen analysis reported that after 3 months of treatment, men in the topical testosterone treatment group had lowest sperm concentration out of all 4 experimental groups. The 12.5 mg and 25 mg groups did not significantly differ in their results, nor did either dose of enclomiphene citrate when compared to the placebo group. Throughout the course of the study 54% of the men using topical testosterone began to experience oligospermia. Only 14.6% of the enclomiphene citrate treatment groups became oligospermic. In comparison to the placebo group, there was no significant difference between sperm concentration of the placebo versus enclomiphene citrate. The men using topical testosterone had sperm concentration lower than those in the placebo group [4].

Figure 5: Effect of the different treatments on sperm concentration.

Furthermore, use of topical testosterone resulted in a decrease in median total sperm count measured by the ejaculatory emission. After 3 months of treatment the researchers reported significant differences in the sperm count in all 4 experimental groups. Similar to sperm concentration there was no significant difference between the two enclomiphene citrate groups and the placebo group. Sperm count was measured 1-month after stopping treatment; the 12.5 mg group was higher than the placebo group as was the 25 mg group but not to a significant level. The topical testosterone level was the only group significantly different from the placebo group [4].

 

Conclusion

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] “Enclomiphene, A New Treatment for Hypogonadism: Eric K. Seaman, MD: Urologist.” Male Fertility Doc, https://www.malefertilitydoc.com/blog/enclomiphene-a-new-treatment-for-hypogonadism. Accessed 25 April 2023.

[2] Yoshimura Y, Kitai H, Santulli R, Wright K, Wallach EE. Direct ovarian effect of clomiphene citrate in the rabbit. Fertil Steril. 1985 Mar;43(3):471-6. doi: 10.1016/s0015-0282(16)48451-7. PMID: 3979586.

[3] Wiehle R, Cunningham GR, Pitteloud N, Wike J, Hsu K, Fontenot GK, Rosner M, Dwyer A, Podolski J. Testosterone Restoration by Enclomiphene Citrate in Men with Secondary Hypogonadism: Pharmacodynamics and Pharmacokinetics. BJU Int. 2013 Jul 12;112(8):1188–200. doi: 10.1111/bju.12363. Epub ahead of print. PMID: 23875626; PMCID: PMC4155868.

[4] Wiehle RD, Fontenot GK, Wike J, Hsu K, Nydell J, Lipshultz L; ZA-203 Clinical Study Group. Enclomiphene citrate stimulates testosterone production while preventing oligospermia: a randomized phase II clinical trial comparing topical testosterone. Fertil Steril. 2014 Sep;102(3):720-7. doi: 10.1016/j.fertnstert.2014.06.004. Epub 2014 Jul 17. PMID: 25044085.

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