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F S Sci. Author manuscript; available in PMC 2020 Sep 9.
Published in final edited form as:
Published online 2020 Apr 14. doi:10.1016/j.xfss.2020.03.002
PMCID: PMC7480784
NIHMSID: NIHMS1583679
PMID: 32914138
Kevin Y. Chu, M.D.,a Shathiyah Kulandavelu, Ph.D.,b Thomas A. Masterson, M.D.,a Emad Ibrahim, M.D.,a Himanshu Arora, Ph.D.,a and Ranjith Ramasamy, M.D.a
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Abstract
Objective
To compare the effect of exogenous short-acting and long-acting testosterone on male reproductive potential in mice.
Design
In vivo mouse model.
Setting
University-based basic science research laboratory
Animals
A total of 30 wild-type C57BL/6 male and female mice were used for this experimentation. The male mice were used for control group and testosterone supplementation, while both male and female mice were used for the breeding portion of the study.
Intervention(s)
Exogenous testosterone was administered either in short-acting formulation (Monday–Wednesday–Friday dosing schedule, testosterone propionate 0.2 mg/kg), or long-acting formulation (3-month dosing schedule - testosterone pellets 150 mg) to male mice.
Main Outcome Measure(s)
Time to pregnancy, Luteinizing hormone (LH) levels, and testicular weight.
Results
Mice treated with long-acting testosterone appear to have longer time to pregnancy when compared to wild-type (33 ± 11 vs 23 ± 2.6 days, p ≤ 0.05) and mice that received short-acting testosterone propionate (26 ± 5.9 days). Mice treated with long-acting testosterone had smaller testes weight when compared to control (0.08 ± 0.01 vs 0.11 ± 0.01g; p ≤ 0.01), while the short-acting testosterone treated mice had similar testis weight when compared to control (0.09 ± 0.02 vs 0.11 ± 0.01g; ns). The serum testosterone level was elevated in mice that received testosterone pellets (285.78 ng/dL) and testosterone propionate (122.16 ng/dL) versus control (68.4 ng/dL). In mice that received long-acting testosterone pellets, LH levels at 3 months were almost undetectable while those that received short-acting testosterone remained similar to control (0.017 ± 0.058 vs 0.348 ± 0.232 IU/L; p ≤ 0.01). Female reproductive potential parameters including litter size and pup weight were collected and observed to have no difference between groups.
Conclusion
Through a mouse breeding study, mice that received short-acting testosterone were shown to have fertility potential similar to wild-type male mice. Long-acting exogenous testosterone appeared to impair male reproductive capacity and LH levels when compared to short-acting testosterone. Short-acting testosterone appeared to cause less LH suppression. Identifying strategies to increase testosterone while simultaneously preserving male fertility is important for treating young men with hypogonadism.
Keywords: testosterone, male infertility, reproductive potential, mouse model
Capsule
Mice that received short-acting testosterone were shown to have fertility potential similar to wild-type male mice, whereas mice that received long-acting testosterone appeared to have impaired fertility potential.
Introduction
Male hypogonadism is defined as a clinical state of low serum testosterone in combination with symptoms such as decreased libido, erectile dysfunction, poor energy, and depression (1). Various large-scale population studies have estimated the prevalence to be from 2.1 to 5.6%, with increasing incidence in aging men (2)(3)(4). Current direct testosterone replacement therapy (TRT) delivery systems include transdermal application (gels, creams, and patches), intranasal gel treatment, injectable therapy, and subcutaneous implantable pellets (5). While these therapies are usually able to successfully raise serum testosterone levels, they include a side effect profile of decreased testicular volume, waning gonadotropin levels (luteinizing hormone (LH) and follicle stimulating hormone (FSH)) and worsening sem*n parameters. In particular, current TRTs can be considered as contraceptives and may inadvertently cause male infertility in hypogonadal men seeking to conceive (6).
Injectable and subcutaneous testosterone therapy have differing pharmaco*kinetics and pharmacodynamic properties. Injectable testosterone therapy used in humans is commonly in the form of either testosterone cypionate, enanthate, and undecanoate. In this experiment, we utilized testosterone propionate, re-constituted in grape seed oil. The terminal half-life of testosterone propionate is approximately 19 hours, and thus suggested injection intervals are every 2–3 days to achieve maintained therapeutic response (7). Subcutaneous testosterone pellets have a terminal half-life of approximately 70.8 days, with an apparent mean residence time of 87 days. From previous research studies, serum testosterone returned to baseline after approximately 300 days (8).
TRT is postulated to suppress the hypothalamic-pituitary-gonadal (HPG) axis through negative feedback, which results in decreased gonadotropin levels and impaired spermatogenesis (9). Early clinical data using a short-acting intranasal formulation (Natesto™), has shown increased serum testosterone levels with preservation of gonadotropin levels and spermatogenesis after six months of use (10). We hypothesized that the short-acting property of nasal testosterone gel provides less suppression of the HPG axis and GnRH pulsatility. GnRH pulsatility is critical for the release of LH and FSH and detectable levels of LH and FSH are important for spermatogenesis (11) (12). In this study, we utilized a murine model to determine if males that received exogenous short-acting testosterone will have lesser effect on reproductive potential as compared males that received long-acting testosterone.
Materials and Methods
Mice
All procedures and mice experiments were performed in accordance with the National Institute of Health (NIH) guidelines and approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Miami Miller School of Medicine (Protocol # 17–113-adm01). Male and female C57BL/6 mice were purchased from The Jackson Laboratory (Bal Harbor, ME). All mice were housed in a temperature and humidity-controlled environment outfitted with standard 12:12 hour light-darkness cycle. Each mouse cage had free access to pellet concentrate and water. All mice were evaluated on a daily basis throughout the study period to ensure no animal pain or impairment.
Testosterone Supplementation Phase
Fifteen C57Bl/6 male mice at six-weeks or older received: wild-type (WT) placebo injections with saline, short-acting testosterone, and long-acting testosterone. Each group of five mice were placed into the same cage. The short-acting testosterone group consisted of five mice that received 0.2 mg/kg testosterone propionate injections administered with BD Veo insulin syringes with BD Ultra-Fine 6mm x 31G needle, intraperitoneally, on a Monday-Wednesday-Friday dosing schedule. The injected testosterone propionate was prepared by diluting stock testosterone propionate (Empower Pharmacy, Houston, TX) in grape seed oil. The group consisted of five mice implanted sub-dermally with two testosterone pellets (Testopel, Endo Pharmaceuticals Inc., Malvern, PA). The testosterone supplementation portion of the study proceeded for three months to ensure at least two cycles of spermatogenesis, 34.5 days in mice, were completed (13). Interim testosterone and gonadotropin levels were measured at the halfway point of the supplementation phase to ensure appropriate hormonal increase. At the end of these three months, blood was obtained from the tail vein of each male mouse for measurement of gonadotropin levels. Blood was drawn in the morning, prior to 11 a.m.
Breeding Phase
The breeding phase of the study commenced upon completion of the testosterone supplementation phase. Each male mouse was placed into a separate cage with one C57BL/6 female mice, for a total of fifteen breeding cages. The short-acting testosterone group continued to receive 0.2 mg/kg testosterone propionate injections on the same dosing schedule. The female mice were evaluated on a daily basis for plugging and signs of pregnancy. Time to pregnancy and delivery were recorded. Litter size and neonate weights was evaluated the day after birth. The breeding phase was conducted over a three-month period. Per the Jackson Laboratory Breeding Manual, C57BL/6 mice have a gestation period of approximately 18.5 days and average 5.5 litters. A three-month breeding phase was decided upon in maximizing potential litters, while also allowing for evaluation of non-productive breeders at the 60-day period as recommended (14).
Tissue Collection
At the end of the three-month breeding phase, the male mice were anesthetized with isoflurane and blood was collected through cardiac puncture. Blood was allowed to clot at room temperature for 15 minutes, and was centrifuged at 2,000g for 10 minutes at 4°C, and the serum was stored at −80°C for future use. Additionally, testicular tissue was removed, weighed, and then flash frozen in liquid nitrogen and stored at −80°C for future use. Tissue collection was performed in the morning, prior to 11 a.m.
Testosterone, LH, FSH Assay
Total testosterone, LH, and FSH levels were measured according to provided protocols using the Ligand Assay & Analysis Core of the Center for Research in Reproduction at the University of Virginia (Charlottesville, VA, USA). Protocol details are delineated at https://med.virginia.edu/research-in-reproduction/ligand-assay-analysis-core/assaymethods/. LH levels were measured with ultrasensitive enzyme-linked immunosorbent assay, and samples were prepared by diluting whole blood 6 μL in assay buffer 54 μL. Testosterone and FSH were measured using a multiplex assay from serum.
Statistical Analysis
Data were analyzed for significance utilizing One-way analysis of variance with the post hoc Tukey-Kramer or/and Bonferroni’s multiple comparisons test as indicated. A group size of n = 5 males provided 90% power to detect a 33% reduction of time to pregnancy (ANOVA, α set at 0.05). To establish rigor, the treatment assignment was blinded to investigators who participated in drug administration and endpoint analyses. All analyses were performed using GraphPad prism 8.30 (GraphPad, South San Francisco, CA, USA), and a p value less than 0.05 was considered significant. All data are presented as mean ± standard deviation.
Results
Testicular weight was decreased in mice that received exogenous long-acting testosterone as compared to mice that received short-acting testosterone
Testis weights of mice that received exogenous long-acting testosterone (n = 5) were decreased when compared to testis weight from WT mice (n = 3; 0.083 ± 0.01 vs 0.115 ± 0.012 g; p ≤ 0.01). Testis weights of mice that received exogenous short-acting testosterone (n = 5, 0.097 ± 0.024) were only slightly decreased when compared to WT mice (ns) (Figure 2). This suggests that spermatogenesis was preserved to levels comparable to WT mice in male mice that received exogenous short-acting testosterone and impaired in male mice that received exogenous long-acting testosterone.
Figure 2.
Testicular weight was observed to be lower in the long-acting testosterone group, when compared to the control group and short-acting testosterone group.
Serum hormone levels of mice that received long-acting testosterone were more affected then mice that received short-acting testosterone.
LH levels in mice that received short-acting testosterone were preserved at control levels, while suppressed in mice receiving long-acting testosterone. We compared the levels of FSH, LH, and testosterone in WT mice and mice that received short-acting testosterone or long-acting testosterone. LH levels of mice that received exogenous long-acting testosterone (n = 5) were noted to be significantly suppressed when compared to both LH levels from WT mice (n = 3; 0.017 ± 0.058 vs 0.403 ± 0.123 IU/L; p ≤ 0.001) and short-acting exogenous testosterone mice (n = 5; 0.017 ± 0.058 vs 0.348 ± 0.232 IU/L; p ≤ 0.01; Fig 3B). This suggests less effect by short-acting testosterone on the hypothalamus-pituitary-gonadal axis when compared to long-acting testosterone. Serum testosterone levels were noted to be increased in both mice receiving short-acting testosterone (n = 5; mean = 122.16 ± 119.95; median = 73.9, IQR 55.9 – 108.5 ng/dL) and long-acting testosterone (n = 3; mean = 168 ± 0; median = 168.0, IQR 168.0 – 168.0 ng/dL) in comparison to WT mice (n = 5; mean = 68.4 ± 47.7; median = 51.7, IQR 33 – 80.2 ng/dL; Fig 3A). This represents successful testosterone supplementation throughout the experiment.
Figure 3.
(a) Testosterone supplementation was evident with increased serum testosterone levels in both experimental groups. (b) LH levels were grossly suppressed in mice that received long-acting testosterone in comparison to both WT and mice that received short-acting testosterone.
Male mice that received exogenous long-acting testosterone appeared to have longer time to pregnancy when compared to both WT mice and mice that received short-acting testosterone.
To study the effect of short-acting and long-acting testosterone on male reproductive potential, time to pregnancy was the primary outcome recorded during the three-month breeding phase. We observed the mice that received long-acting testosterone (n = 4) had an average time to pregnancy longer than WT mice (n = 5; 33 ± 11 vs 23 ± 2.6 days; p ≤ 0.05; Fig 4). When the mice that received short-acting testosterone (n = 6) were compared to WT mice, there was only a slight increase in time to pregnancy that was not statistically significant (26 ± 5.9 vs 23 ± 2.6 days; ns). This represents greater effect on male fertility potential by long-acting testosterone versus short-acting testosterone.
Figure 4.
Exogenous long-acting testosterone supplemented mice appeared to have longer time to pregnancy when compared to control mice and mice that received short-acting testosterone.
Female reproductive potential showed no significant difference between breeding groups
Variance in female reproductive potential may play a confounding factor on time to pregnancy data, and as such total pups per litter and pup weight data were collected as secondary outcomes. These measurements are better representative of female reproductive potential and can identify if there is significant variance. The total number of deliveries during the breeding period were recorded and compared amongst the test groups. WT male x WT female mice delivered a total of 9 litters while long-acting testosterone male x WT female yielded 4 litters, and short-acting testosterone male x WT female yielded 6 litters (Supplemental Figure 1). There was no significant difference when comparing the number of pups per litter between groups (WT, average pups per litter (ppl) = 5.75 ± 1.71; long-acting exogenous testosterone, 7.50 ± 1.73; short-acting testosterone, 5.50 ± 2.59, ns) (WT, median pups per litter = 6 (IQR 5 – 8); long-acting exogenous testosterone, 7.5 (IQR 6 – 9); sho rt-acting testosterone, 6 (IQR 3.75 – 6.75)). The average pup weight only showed very slight variance (WT, 1.33 ± 0.22g; long-acting exogenous testosterone, 1.40 ± 0.40g; short-acting testosterone, 1.27 ± 0.28 g, ns) (Supplemental Figure 2).
Discussion
Male hypogonadism prevalence has reached an estimated prevalence of approximately 13.8 million men over the age of 45 in the United States (15). With the rising age of fatherhood and incidence of 3–8% hypogonadism in men between 20–45 years old, strategies to treat hypogonadism while preserving fecundity is of paramount importance (16) (17). There currently are four official guidelines from academic societies regarding testosterone replacement therapy, including the American Urological Association, Endocrine Society, International Society for the Study of the Aging Man, and International Society for Sexual Medicine. Each of these guidelines has some variance in regard to diagnosis and treatment of the hypogonadal male (18). Exogenous testosterone replacement therapy cannot be utilized in men seeking to maintain fertility, as it acts as an unintended contraceptive by lowering sperm concentration (19). Recently, short-acting intranasal testosterone has been shown to preserve spermatogenesis while treating low testosterone in clinical trials (10). We postulated that this preservation is due in part to the maintenance of pulsatile GnRH release, a consequence of the short-acting property of the testosterone preparation. While this clinical data is promising, research regarding conception outcomes on short-acting testosterone have been limited due to size and duration of present clinical trials. We sought to address this shortcoming by studying the effects of short-acting and long-acting testosterone on fecundity in mice, as they have reliable breeding patterns and short gestational periods. Additionally, mouse spermatogenesis and oogenesis have been observed to be comparable to humans (20). We observed that mice treated with short-acting testosterone had lesser effects on male reproductive potential when compared to long-acting testosterone. These findings suggest that short-acting testosterone preparations, such as intranasal testosterone gel, may maintain fecundity in male hypogonadal patients seeking treatment.
Upon completion of the breeding study, we observed mice that received long-acting testosterone had smaller testes than both mice that received short-acting testosterone and WT mice. This implies stronger spermatogenesis impairment and possible HPG suppression in the mice that received long-acting testosterone when compared to the mice that received-short acting testosterone. Sperm production has been strongly correlated with testicular weight in rats, who have similar physiology to mice (21). Effect on HPG axis was further supported with the LH ultrasensitive assay showing significant LH suppression in mice that received long-acting testosterone, while levels were preserved in mice that received short-acting testosterone comparable to WT. This parallels the reported findings in humans by Connors et al., in which gonadotropin levels were maintained within the normal range in hypogonadal men being treated with Natesto™ at b.i.d. or t.i.d. dosing (22). To ensure that the LH suppression in mice was due to the exogenous testosterone administration, our measurements of serum testosterone levels were shown to be increased in both mice that received short-acting or long-acting testosterone when compared to WT. Rogol et al. performed a randomized, open-label, dose-ranging study that showed short-acting testosterone administered two to three times daily resulted in restoration of testosterone levels within normal limits in hypogonadal men (23).
The most significant outcome we aimed to follow in this experiment was the time to pregnancy, as this is inferred to be a reflection of male fertility potential (24). As we observed, there was significantly increased time to pregnancy for the mice that received long-acting testosterone. The WT and mice that received short-acting testosterone groups had times to pregnancy that were similar in comparison. Further strengthening the primary outcome finding was the lack of difference in our secondary outcomes amongst the control and test groups, inferring little variance in female reproductive potential. The current perception towards most TRT are that they act as contraceptives, impairing spermatogenesis (19). With this study, in conjunction with early data from the Natesto™ clinical trial, it is tempting to speculate that exogenous short-acting testosterone preparations may be used in hypogonadal men who desire maintain fertility potential.
We postulate that exogenous short-acting testosterone is able to achieve this preservation of the HPG axis by its pulsatile release pattern that mimics that of GnRH. Release of LH respond to the varying pulsatile patterns of GnRH (11) (12). Exogenous long-acting testosterone acts in a non-pulsatile manner, resulting in a steady stream of negative feedback that results in HPG axis suppression. As we further research the effects of short-acting testosterone preparations on the HPG axis and male fertility, we can better guide hypogonadal patients in the treatment therapy they should undergo.
Limitations of this study included the small sample size that were supplemented with short-acting or long-acting testosterone, as well as for inclusion in breeding study. Additionally, reproductive behavior such as mounting to further support male fertility parameters were unable to be observed consistently due to limited personnel resources. Our data did not show significant difference amongst the three test groups in regard to testosterone level differences after supplementation, and this was due to a limitation of our small research population size. Presence of outliers (variability in serum testosterone levels), can be addressed in future studies with a larger group of animals. Another challenge was promoting consistent receptivity for mating among the female mice. Our data as presented in the supplemental figures indicated that there was no variability in reproductive potential in our female mice breeding group, but the narrow size of the female mice population limited an effort to increase sample size with dependable births. Other limitations of our study included the use of male mice that were not hypogonadal to begin with. In the real world, it is mostly hypogonadal men whom undergo TRT, with baseline decreased spermatogenesis. Additionally, there was an issue with the orchiectomy procedure for two wild-type mice, and as a result adequate and appropriate weight measurements were unable to be obtained, and as a result omitted in our study analysis. Furthermore, testosterone levels have been shown to possibly be affected by group-housing in male mice, with an observed increase from baseline levels (25). We maintained group-housing for our male mice during the supplementation phase in a systematic fashion, in order to minimize bias and heterogeneity.
In future directions, additional measures to improve mating will allow for increased validity of the study. Additionally, more various dosing schedules for the exogenous short-acting and long-acting testosterone and more frequent hormone level evaluations can be undertaken to better delineate the effects on male fertility potential. While our data suggests observable differences between short-acting and long-acting testosterone, extrapolation of our data for clinical application requires increased mice study population, inclusion of hypogonadal male mice, as well as a longer breeding phase to six-months to better assess fertility potential.
Conclusions
Through a mouse breeding study, mice that received short-acting testosterone were shown to have fertility potential similar to wild-type male mice. Male mice that received exogenous long-acting testosterone appeared to have impaired reproductive potential as the time to pregnancy was measurably longer. Short-acting testosterone seemed to only slightly impair the HPG axis in comparison to long-acting testosterone in mice.
Figure 1.
Experimental Design.
Supplementary Material
Supplemental Figure 1
Supplementary Figure 1. Total number of deliveries per breeding group.
Click here to view.(297K, pdf)
Supplemental Figure 2
Supplementary Figure 2. (a) Average pups per litter per breeding group (b) Average pup weight per breeding group.
Click here to view.(673K, pdf)
Acknowledgements / Disclosure
Investigator - initiated grant from Aytu Bioscience, Inc.
Footnotes
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