madman
Super Moderator
An update on male infertility and intratesticular testosterone— insight into novel serum biomarkers(2021)
Karim Sidhom , Kapilan Panchendrabose , Uday Mann and Premal Patel
Intratesticular testosterone is vital for spermatogenesis, male fertility, and virility. Currently, the only method to assess levels of intratesticular testosterone is to perform testicular biopsy which is invasive and can lead to several complications. Approaches to assessing intratesticular testosterone have been understudied but hold promise as future male contraceptive agents and may grant the ability to monitor patients undergoing hormonal changes from therapeutic and diagnostic perspectives. Previous studies have sought to assess the utility of 17-hydroxyprogesterone (17-OHP) and insulin-like factor 3 (INSL3) as accurate surrogate biomarkers of intratesticular testosterone. The aim of this review is thus to highlight the importance of intratesticular testosterone and the consequent advances that have been made to elucidate the potential of biomarkers for intratesticular testosterone in the context of male infertility.
INTRODUCTION
Infertility has become a ubiquitous challenge for many couples and is estimated to affect more than 185 million people globally [1, 2]. Male infertility specifically has been found to contribute to up to 50% of reported cases within North America and is defined by the World Health Organization (WHO) as an inability to conceive after 1 year of unprotected intercourse [3–5]. The prevalence of male infertility has caused semen analyses to become routine practice in the evaluation of male spermatogenesis and quality assessment. Nevertheless, up to 60% of male infertility cases are due to indeterminable causes, leaving many men stratified into the oligoasthenoteratozospermic spectrum with little more than a descriptive diagnosis [5]. Following a medical history and physical exam, sperm morphology testing is typically accompanied by serum analyses of luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone, and prolactin to discover the clinical cause of male infertility [6]
Testosterone is the principal hormone responsible for both spermatogenesis and fertility, as well as the development of primary male sexual characteristics [7]. Serum testosterone acts as a reflection of the hypothalamic-pituitary-gonadal (HPG) axis, self-regulating gonadal function via negative feedback. Starting first with a pulsatile patterned secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus to signal LH and FSH secretion from the anterior pituitary that then act on the gonads as illustrated in Fig. 1. FSH and LH are released from gonadotrophic cells compromising about 15% of the anterior pituitary mass to then bind G protein-coupled receptors on the gonads to regulate function [8]. In males, FSH acts to support spermatogonia differentiation and maturation via membrane-bound receptors on Sertoli cells and LH is required for testosterone secretion using Leydig cells. Testis, therefore, have the dual function of virility via testosterone and fertility via spermatozoa respectively, the two being naturally intertwined [9]. Once cholesterol is converted into testosterone by Leydig cells, it then acts to promote spermatogenesis by acting on androgen receptors (ARs) found on Leydig, Sertoli, and peritubular cells. Still, GnRH is the precursor of both virility and fertility, and gonadotropin withdrawal naturally leads to the failure of spermatogenesis. This last statement is mainly due to the loss of testosterone production, as FSH is dispensable for male fertility, but LH is needed [10].
*THE INTRATESTICULAR ENVIRONMENT
Testosterone, and more specifically, intratesticular testosterone (ITT) levels are essential to male fertility [11–14]. ITT and FSH are required to remain at consistently high levels to maintain normal spermatogenesis. In mice models, FSH-receptor knockout mice were found to remain fertile, albeit reduced and with delayed puberty [15–17]. Moreover, in human males with homozygous inactivation of the FSH-receptor spermatogenic inadequacy was shown, though no cases demonstrated complete azoospermia or infertility. Transgenic mouse models have shown via cell-selective ablation of the androgen receptor found in Sertoli cells a complete block of meiosis as it pertains to spermatogenesis [18]. In contrast, LH-receptor knockout mice were found to be completely infertile with reduced testes size and significantly reduced ITT levels (beyond 95%) [10]. Note that a normal male’s ITT levels range between 400-600 ng/g while their serum levels are usually almost 100x lower [9]. All in all, the Leydig cell acts as a vital mediator between the HPG axis and the effect of androgens on fertility, hence leading to the intratesticular environment necessary to conserve both fertility and virility. Although significant, FSH alone cannot complete the necessary cycle of spermatogenesis. Similarly, even though an absence of FSH may lead to spermatogenic impairment, decreased levels of ITT would completely abolish spermatogenesis.
That said, the volume of smooth endoplasmic reticulum correlates to Leydig cells’ capacity to secrete testosterone [19]. It is hypothesized that testosterone is crucial to the seventh stage of the spermatogenic cycle (in the rat model) within seminiferous tubules and that the corresponding Leydig cells (peritubular cells) secreting testosterone (in the presence of LH) to these tubules act in harmony with this maturation process [20]. This is supported by work demonstrating cyclical AR upregulation leading to a reciprocal progressive increase from stage 2 to 7, which then drop to barely detectable levels in subsequent stages [21–23]. It is important to realize that a typical cross-section of testis shows a heterogeneous presentation of these tubules at various points in their respective 14 stage seminiferous epithelial tubule cycle (in mice) [24, 25]. Given this radial distribution of seminiferous tubule maturation with human testis, and their mixed development, these findings suggest a local, and thus paracrine variable involving ITT [26]. This variable thus must pass by an endocrine fashioned mechanism involving testicular blood flow. Note that the entry of all nutrients, hormones, etc. required for spermatogenesis is controlled by the testicular vasculature. Meaning that all chemicals must pass across the vascular endothelium and into the testicular interstitial fluid to their respective targets to achieve any level of bioactive relevance.
The blood-testis barrier (BTB) represents one of the most unique, and accordingly, specialized barriers within the mammalian body. As opposed to other barriers which rely on mostly tight junctions such as the blood-retinal and blood-brain barriers, the BTB is more intricate. It relies on a unique mix of tight junctions, basal ectoplasmic specializations, gap junctions, and desmosomes that may undergo restructuring during the 8th stage of the seminiferous tubule cycle [27]. Moreover, the BTB shows a unique dependency on ITT in that it is needed for Sertoli-germ cell junction assembly and restructuring, as well as spermatid release from the seminiferous epithelium and tight junction remodeling. ITT thus allows for the maintenance of a dynamic BTB [28–31].
Monitoring ITT would be a great addition to the andrology toolkit in understanding male fertility cases. Testing ITT however requires invasive percutaneous aspiration normally done via a 19- gauge 10 mL needle into seminiferous tubules [26]. Though this method has been adapted to be minimally invasive, biopsy is still an invasive procedure with substantial risk compared to serum analysis, and aspirates may be contaminated by interstitial fluid leading to a potentially misleading assessment. Nevertheless, the need for ITT assessment is apparent as mentioned earlier that a large proportion of men are diagnosed with idiopathic infertility. It is possible that these cases may be the result of subnormal ITT levels, a very different problem from subnormal serum testosterone levels, hence why using serum biomarkers for ITT levels has become an attractive topic of recent research. Not only would such a marker allow for serial monitoring in a non-invasive manner, but it may also allow us to better understand otherwise idiopathic causes of infertility, and with that, aid in the development of male contraceptives [32].
*SERUM BIOMARKERS OF INTRATESTICULAR TESTOSTERONE
Serum 17-hydroxyprogesterone (17-OHP)
17-OHP is an intermediate steroid synthesized within the adrenal gland zona fasciculata and the testes. 70% of the circulating 17-OHP is derived from the testes and the rest is thought to originate from the adrenal gland [33]. Adrenal steroidogenesis has been highly documented in the literature, however, the gonadal pathway remains to be fully elucidated though, high concentrations of 17-OHP have been documented in testicular biopsy as well as ovarian follicular biopsy [34]. 17-OHP has already shown its potential as a biomarker since it is used to screen for congenital adrenal hyperplasia [35].
Figure 2 illustrates the precursor biomolecules of 17-OHP and the subsequent products. Previous studies have shown serum 17-OHP to be correlated with high levels of ITT [33, 36]. However, there may be a sensitivity threshold between the association of 17-OHP and ITT. Roth et al. studied the effects of human chorionic gonadotropin (hCG) by administering dose-dependent injections subcutaneously consisting of GnRH, acyline, and dose-dependent hCG ranging from 0, 15, 60, or 125 IU or testosterone gel in 37 healthy males for 10 days. They measured serum 17-OHP using chromatography and mass spectrometry and found no significant association between 17-OHP and ITT [37]. The authors suggest the low doses of hCG administered as the main factor, otherwise, an association may have been observed. The same group was able to verify this by assessing 17- OHP directly from testicular biopsies using an immunofluorometric assay in 29 healthy males who received dose-dependent hCG ranging from 0, 125 IU, 250 IU, or 500 IU for 3 weeks. They found 17-OHP was strongly associated with ITT during treatment and increased by 70% when undergoing the highest dose.
To better study the relationship between ITT and 17-OHP, Lima et al. assessed serum 17-OHP and total testosterone levels in 93 men: 42 healthy men, 30 men who received 1500 IU hCG weekly or clomiphene citrate (CC) 25 mg every other day for at least 3 months, and 21 men who received exogenous testosterone for at least 3 months. The authors found that all men had normal ranges of serum testosterone [34] however, men receiving exogenous testosterone had significantly reduced levels of serum 17-OHP compared to men receiving either hCG/CC therapy or no medications [38]. Men receiving exogenous testosterone exhibited an almost 4-fold decrease of 17-OHP compared to the hCG/CC group and also a significantly lower level when compared to the control group after 3 months. Whereas men receiving hCG/CC therapy had similar levels to controls but increased by approximately twofold after treatment. The authors state that such a decrease may be attributed to the exogenous testosterone via a negative feedback loop affecting the HPG axis. Similar to testosterone, 17-OHP is also regulated by the HPG axis and undergoes diurnal variation mirroring testosterone, with peaks in the morning and falls by the evening to 60% of its original high point [39, 40].
In hopes of further elucidating how 17-OHP and ITT levels may be impacted, the same group sought to assess lifestyle factors and aging. They assessed serum samples in 340 men and found males with >25 BMI had higher rates of statistically significant decreases in serum testosterone and 17-OHP compared to normal BMI males [41]. Furthermore, their data shows aging by 1 year and 1 unit increase in BMI decreased 17-OHP by 0.84 ng/dl and 1.73 ng/dl, respectively. There was also a positive correlation between 17-OHP and serum testosterone, though this was not observed when serum testosterone levels were low. A similar study conducted by Martínez-Montoro studied 266 non-diabetic, ≥ 30 BMI men using high-performance liquid chromatography-mass spectrometry and found significant associations between 17-OHP, BMI, waist circumference, insulin levels, total and free testosterone levels [42]. However, in contrast to Lima et al.’s results, they found no association between age and 17-OHP but did so with LH. The two groups used different methods to assess measurements which may explain the differing results. These contrasting findings make it unclear whether increasing BMI causes decreased 17-OHP via negative feedback through the HPG axis and a subsequent decrease in hormones such as LH, or if it may rather have a more direct impact on 17-OHP and therefore intratesticular levels. How volumes of ITT and thus 17-OHP originating from the adrenal gland relate also remains unclear.
To further corroborate a direct effect on Leydig cells, Lima et al. found no changes between serum testosterone, 17-OHP, LH, FSH before and after varicocelectomy [43]. 17-OHP’s utility as a biomarker is also observed to predict when semen parameters may be upgraded when men are undergoing hCG/CC therapy. A previous study showed men with ≤55 ng/dL 17-OHP at baseline had upgraded semen quality as per assisted reproduction eligibility [44].
INSULIN-LIKE FACTOR 3 (INSL3)
INSL3 is a peptide hormone synthesized in adult Leydig cells and has been previously shown to be able to act as a biomarker for Leydig cell functionality [45]. Additionally, since INSL3 is constitutively produced by adult Leydig cells, it is temporally reflective when Leydig cells have differentiated. INSL3 levels are therefore capable to represent the population of differentiated Leydig cells and are more of an accurate predictor than serum testosterone. Serum testosterone is regulated by the HPG axis and undergoes large diurnal fluctuations [45, 46]. The large variability throughout the day and between patients with the same diseases makes it less of a candidate to represent Leydig cell function. INSL3 has also been shown to likely be solely produced from Leydig cells and its regulation is unaffected by LH. Bay et al. assessed the levels of serum INSL3 in 135 healthy males and 21 anorchid men using time-resolved fluorescence immunoassays and found median INSL3 levels were 0.99 ng/ml and 0.05 ng/ml respectively [47]. They also found INSL3 levels to be 15 times higher in blood samples drawn from vena spermatic compared to peripheral blood of two infertile males. This is suggestive that INSL3 may be solely localized in the testes.
INSL3 can be an insightful biomarker for patients undergoing hCG monotherapy. Although INSL3 levels have been observed to not significantly differ in hypogonadal patients within 72 and 96 h, hCG was highly associated with higher levels of INSL3. Hence, it is thought that hCG acts to differentiate Leydig cells in hypogonadal men. The Roth et al. study previously mentioned further corroborates the effects of hCG by administering dose-dependent injections subcutaneously consisting of gonadotropin-releasing hormone antagonist, acyline, and hCG or testosterone gel for 10 days in healthy males. Patients receiving only the antagonist had significantly lower INSL3 levels than the baseline and higher doses of hCG normalized INSL3 again [48]. These levels are directly correlated with serum testosterone levels.
INHIBIN B
Inhibin B is a dimeric glycoprotein whose bioactivity is dependent on its dimerization of its respective α and βB subunits. Its function is representative of Sertoli cells, acting as a negative regulator to pituitary FSH [50]. Inhibin B has been shown to be a valid marker of Sertoli cell function; inhibin B levels positively correlate with sperm concentration and negatively correlate with serum FSH levels. Inhibin B was even found to be an objectively better marker of spermatogenesis when compared to FSH as it more strongly correlated with testicular volume and semen parameters. As Roth et.al were studying INSL3 [43], they searched for reciprocal changes in other potential biomarkers, inhibin B being one of them, as well as 17-OHP and anti-Mullerian hormone – all of which showed no statistical significance in their study [48, 51]. Hence, although important to serum testosterone testing, further study is needed to understand inhibin B’s possible relationship with ITT. Figure 1 displays an overall review of the biomarkers emphasized here in the context of the intratesticular environment.
*CLINICAL IMPLICATIONS AND FUTURE DIRECTIONS
The intratesticular milieu is essential to spermatogenesis, though it is still far from being well understood [27]. More work is needed to better elucidate its role and consequently, its pathophysiologic potential. Although surgical testicular biopsies are minimally invasive, there are many common risks including pain, bleeding, infection, and testicular injury [52, 53]. Therefore, these biopsies are rarely used in the clinical setting.
The use of surrogate ITT biomarkers would not only allow more frequent and less risky monitoring but could also improve physiologic understanding of this environment. This is especially critical given the poor correlation between serum testosterone and ITT levels. Standard ITT was found to be around 170 times higher than serum testosterone and did not correlate with serum LH, FSH, testosterone, DHT (dihydrotestosterone), or estradiol [37]. DHT was found to be only 11 times higher within the testes than in the serum, likely due to the fact that most of it are made by the conversion of 5-alpha reductase in peripheral tissue [49] (most of which is concentrated to the prostate with minimal expression in genital skin), as further shown in Fig. 2 [54]. Nevertheless, when it comes to ITT, a quantitative relationship with spermatogenesis has yet to be clearly derived in men. Studies in rats have found that a reduction of ITT by up to 80% did not affect spermatogenesis, though, below this, spermatogenesis was severely impacted [55]. This study implies that there is far more testosterone in the testicles than required to maintain spermatogenesis, and moreover, a quantitative relationship with spermatogenesis does exist below a certain ITT threshold. Work towards establishing a lower limit of normal would likely prove to be a future therapeutic target for the clinical use of potential serum biomarkers.
A clinical study by Coviello et al. worked to address this gap by further examining the relationship between ITT and serum testosterone via a male contraceptive regimen using 100 mg weekly injections of testosterone enanthate and oral levonorgestrel 62.5 or 31.25 μg daily [56]. When comparing the results before and after this 6-month trial in seven men assessed via serum and intratesticular biopsy, they found the following: mean ITT decreased by 98%, no significant difference in serum T, and mean sperm count was suppressed by 98% (six of seven men reached a count of zero). Hence it is clear from a diagnostic viewpoint that a lack of ITT assessment may lead to the erroneous conclusion of idiopathic infertility, as is all too common.
Therapeutics
If patients are found to have low ITT levels, they are treated with hCG as well as recombinant FSH and CC [56–59]. The European consensus statement on the treatment of congenital hypogonadotropic hypogonadism states that testosterone replacement therapy is the mainstay of therapy for virilization purposes (so as to avoid infantile infantilism) [60]. However, this does not address the issue of fertility in these patients. The induction of male fertility mandates the induction of testosterone production and consequently spermatogenesis, hence relying on combined gonadotropin therapy via the guidance of an experienced urologist or endocrinologist. Moreover, AUA guidelines also refer to the use of aromatase inhibitors, HCG, and selective estrogen receptor modulators when treating men with testosterone deficiency who desire to maintain fertility (guideline statement 27) [61]. This allows patients suffering from hypogonadotropic hypogonadism or anabolic steroid abuse shutdown to initiate spermatogenesis, as well as improve spermatogenesis in men with idiopathic oligospermia. hCG acts as an LH mimic, thus stimulating Leydig testosterone production and secretion to raise ITT and serum testosterone [62]. CC is a racemic mixture of enclomiphene and zuclomiphene acting as a selective estrogen receptor modulator to block the negative feedback from testosterone [59, 63]. This indirectly increases LH and FSH secretion from the anterior pituitary to allow for more testosterone production and improved spermatogenesis [64]. In doing so, it is well-tolerated, showing minimal side effects such as hair loss, gastrointestinal distress, gynecomastia, and in a few cases visual disturbances, overall leading to restoration rather than replacement as traditional testosterone therapy would entail [64]. In addition to this routine trio, multivitamins and antioxidants are commonly used and have shown to be effective, though the evidence present is of lower quality [65]. Given the importance of ITT and its discrepancy from serum testosterone, the use of a biomarker would allow for routine monitoring. This would provide evidence that the above-mentioned therapeutics are acting appropriately.
Male contraception
Conversely, this monitoring process could act to support a male contraceptive regimen’s effectiveness. Currently, men are limited to condoms and vasectomies, but novel hormonal contraceptives are under development [66]. Their mechanism relies on the suppression of LH and FSH, presupposing completely reversible inhibition. Both the initiation and the reversal process would benefit from monitoring of both ITT and serum testosterone levels. Most of these regimens rely on exogenous testosterone, which via negative feedback shuts down endogenous FSH and LH. Meaning that although testosterone is still present exogenously, spermatogenesis is suppressed. Hence virility is maintained while fertility is repressed via azoospermia. However, side effects such as acne, weight gain, and fluctuating mood as well as a 5% failed reversibility rate over 52 weeks are barriers to the popularization of such a regimen [67]. Additionally, chronic testosterone administration does not reliably induce azoospermia, hence why monitoring is imperative, yet if this monitoring meant monthly testicular biopsy, the acceptability of such a regimen would undoubtedly decline [68].
CONCLUSION
Assessing 17-OHP and INSL3 as surrogate biomarkers for ITT is a promising way to address male infertility and assist in the creation of novel hormonal male contraceptives. There are many factors ranging from lifestyle to bioavailable concentrations that may affect the utility of these biomarkers and different factors are being investigated to determine whether they are efficacious. Although 17-OHP and INSL3 have potential, further investigations are required to elucidate their efficacy as biomarkers of ITT. An ongoing investigation is required to develop novel techniques to quantify levels of ITT biomarkers - techniques that are both effortless for the physician and safe for the patient. All in all, a better understanding of ITT via accurate serum biomarkers will deepen our understanding of the intratesticular environment and in so doing, conceivably transform the future of male fertility diagnostics and therapeutics.
Karim Sidhom , Kapilan Panchendrabose , Uday Mann and Premal Patel
Intratesticular testosterone is vital for spermatogenesis, male fertility, and virility. Currently, the only method to assess levels of intratesticular testosterone is to perform testicular biopsy which is invasive and can lead to several complications. Approaches to assessing intratesticular testosterone have been understudied but hold promise as future male contraceptive agents and may grant the ability to monitor patients undergoing hormonal changes from therapeutic and diagnostic perspectives. Previous studies have sought to assess the utility of 17-hydroxyprogesterone (17-OHP) and insulin-like factor 3 (INSL3) as accurate surrogate biomarkers of intratesticular testosterone. The aim of this review is thus to highlight the importance of intratesticular testosterone and the consequent advances that have been made to elucidate the potential of biomarkers for intratesticular testosterone in the context of male infertility.
INTRODUCTION
Infertility has become a ubiquitous challenge for many couples and is estimated to affect more than 185 million people globally [1, 2]. Male infertility specifically has been found to contribute to up to 50% of reported cases within North America and is defined by the World Health Organization (WHO) as an inability to conceive after 1 year of unprotected intercourse [3–5]. The prevalence of male infertility has caused semen analyses to become routine practice in the evaluation of male spermatogenesis and quality assessment. Nevertheless, up to 60% of male infertility cases are due to indeterminable causes, leaving many men stratified into the oligoasthenoteratozospermic spectrum with little more than a descriptive diagnosis [5]. Following a medical history and physical exam, sperm morphology testing is typically accompanied by serum analyses of luteinizing hormone (LH), follicle-stimulating hormone (FSH), testosterone, and prolactin to discover the clinical cause of male infertility [6]
Testosterone is the principal hormone responsible for both spermatogenesis and fertility, as well as the development of primary male sexual characteristics [7]. Serum testosterone acts as a reflection of the hypothalamic-pituitary-gonadal (HPG) axis, self-regulating gonadal function via negative feedback. Starting first with a pulsatile patterned secretion of gonadotropin-releasing hormone (GnRH) from the hypothalamus to signal LH and FSH secretion from the anterior pituitary that then act on the gonads as illustrated in Fig. 1. FSH and LH are released from gonadotrophic cells compromising about 15% of the anterior pituitary mass to then bind G protein-coupled receptors on the gonads to regulate function [8]. In males, FSH acts to support spermatogonia differentiation and maturation via membrane-bound receptors on Sertoli cells and LH is required for testosterone secretion using Leydig cells. Testis, therefore, have the dual function of virility via testosterone and fertility via spermatozoa respectively, the two being naturally intertwined [9]. Once cholesterol is converted into testosterone by Leydig cells, it then acts to promote spermatogenesis by acting on androgen receptors (ARs) found on Leydig, Sertoli, and peritubular cells. Still, GnRH is the precursor of both virility and fertility, and gonadotropin withdrawal naturally leads to the failure of spermatogenesis. This last statement is mainly due to the loss of testosterone production, as FSH is dispensable for male fertility, but LH is needed [10].
*THE INTRATESTICULAR ENVIRONMENT
Testosterone, and more specifically, intratesticular testosterone (ITT) levels are essential to male fertility [11–14]. ITT and FSH are required to remain at consistently high levels to maintain normal spermatogenesis. In mice models, FSH-receptor knockout mice were found to remain fertile, albeit reduced and with delayed puberty [15–17]. Moreover, in human males with homozygous inactivation of the FSH-receptor spermatogenic inadequacy was shown, though no cases demonstrated complete azoospermia or infertility. Transgenic mouse models have shown via cell-selective ablation of the androgen receptor found in Sertoli cells a complete block of meiosis as it pertains to spermatogenesis [18]. In contrast, LH-receptor knockout mice were found to be completely infertile with reduced testes size and significantly reduced ITT levels (beyond 95%) [10]. Note that a normal male’s ITT levels range between 400-600 ng/g while their serum levels are usually almost 100x lower [9]. All in all, the Leydig cell acts as a vital mediator between the HPG axis and the effect of androgens on fertility, hence leading to the intratesticular environment necessary to conserve both fertility and virility. Although significant, FSH alone cannot complete the necessary cycle of spermatogenesis. Similarly, even though an absence of FSH may lead to spermatogenic impairment, decreased levels of ITT would completely abolish spermatogenesis.
That said, the volume of smooth endoplasmic reticulum correlates to Leydig cells’ capacity to secrete testosterone [19]. It is hypothesized that testosterone is crucial to the seventh stage of the spermatogenic cycle (in the rat model) within seminiferous tubules and that the corresponding Leydig cells (peritubular cells) secreting testosterone (in the presence of LH) to these tubules act in harmony with this maturation process [20]. This is supported by work demonstrating cyclical AR upregulation leading to a reciprocal progressive increase from stage 2 to 7, which then drop to barely detectable levels in subsequent stages [21–23]. It is important to realize that a typical cross-section of testis shows a heterogeneous presentation of these tubules at various points in their respective 14 stage seminiferous epithelial tubule cycle (in mice) [24, 25]. Given this radial distribution of seminiferous tubule maturation with human testis, and their mixed development, these findings suggest a local, and thus paracrine variable involving ITT [26]. This variable thus must pass by an endocrine fashioned mechanism involving testicular blood flow. Note that the entry of all nutrients, hormones, etc. required for spermatogenesis is controlled by the testicular vasculature. Meaning that all chemicals must pass across the vascular endothelium and into the testicular interstitial fluid to their respective targets to achieve any level of bioactive relevance.
The blood-testis barrier (BTB) represents one of the most unique, and accordingly, specialized barriers within the mammalian body. As opposed to other barriers which rely on mostly tight junctions such as the blood-retinal and blood-brain barriers, the BTB is more intricate. It relies on a unique mix of tight junctions, basal ectoplasmic specializations, gap junctions, and desmosomes that may undergo restructuring during the 8th stage of the seminiferous tubule cycle [27]. Moreover, the BTB shows a unique dependency on ITT in that it is needed for Sertoli-germ cell junction assembly and restructuring, as well as spermatid release from the seminiferous epithelium and tight junction remodeling. ITT thus allows for the maintenance of a dynamic BTB [28–31].
Monitoring ITT would be a great addition to the andrology toolkit in understanding male fertility cases. Testing ITT however requires invasive percutaneous aspiration normally done via a 19- gauge 10 mL needle into seminiferous tubules [26]. Though this method has been adapted to be minimally invasive, biopsy is still an invasive procedure with substantial risk compared to serum analysis, and aspirates may be contaminated by interstitial fluid leading to a potentially misleading assessment. Nevertheless, the need for ITT assessment is apparent as mentioned earlier that a large proportion of men are diagnosed with idiopathic infertility. It is possible that these cases may be the result of subnormal ITT levels, a very different problem from subnormal serum testosterone levels, hence why using serum biomarkers for ITT levels has become an attractive topic of recent research. Not only would such a marker allow for serial monitoring in a non-invasive manner, but it may also allow us to better understand otherwise idiopathic causes of infertility, and with that, aid in the development of male contraceptives [32].
*SERUM BIOMARKERS OF INTRATESTICULAR TESTOSTERONE
Serum 17-hydroxyprogesterone (17-OHP)
17-OHP is an intermediate steroid synthesized within the adrenal gland zona fasciculata and the testes. 70% of the circulating 17-OHP is derived from the testes and the rest is thought to originate from the adrenal gland [33]. Adrenal steroidogenesis has been highly documented in the literature, however, the gonadal pathway remains to be fully elucidated though, high concentrations of 17-OHP have been documented in testicular biopsy as well as ovarian follicular biopsy [34]. 17-OHP has already shown its potential as a biomarker since it is used to screen for congenital adrenal hyperplasia [35].
Figure 2 illustrates the precursor biomolecules of 17-OHP and the subsequent products. Previous studies have shown serum 17-OHP to be correlated with high levels of ITT [33, 36]. However, there may be a sensitivity threshold between the association of 17-OHP and ITT. Roth et al. studied the effects of human chorionic gonadotropin (hCG) by administering dose-dependent injections subcutaneously consisting of GnRH, acyline, and dose-dependent hCG ranging from 0, 15, 60, or 125 IU or testosterone gel in 37 healthy males for 10 days. They measured serum 17-OHP using chromatography and mass spectrometry and found no significant association between 17-OHP and ITT [37]. The authors suggest the low doses of hCG administered as the main factor, otherwise, an association may have been observed. The same group was able to verify this by assessing 17- OHP directly from testicular biopsies using an immunofluorometric assay in 29 healthy males who received dose-dependent hCG ranging from 0, 125 IU, 250 IU, or 500 IU for 3 weeks. They found 17-OHP was strongly associated with ITT during treatment and increased by 70% when undergoing the highest dose.
To better study the relationship between ITT and 17-OHP, Lima et al. assessed serum 17-OHP and total testosterone levels in 93 men: 42 healthy men, 30 men who received 1500 IU hCG weekly or clomiphene citrate (CC) 25 mg every other day for at least 3 months, and 21 men who received exogenous testosterone for at least 3 months. The authors found that all men had normal ranges of serum testosterone [34] however, men receiving exogenous testosterone had significantly reduced levels of serum 17-OHP compared to men receiving either hCG/CC therapy or no medications [38]. Men receiving exogenous testosterone exhibited an almost 4-fold decrease of 17-OHP compared to the hCG/CC group and also a significantly lower level when compared to the control group after 3 months. Whereas men receiving hCG/CC therapy had similar levels to controls but increased by approximately twofold after treatment. The authors state that such a decrease may be attributed to the exogenous testosterone via a negative feedback loop affecting the HPG axis. Similar to testosterone, 17-OHP is also regulated by the HPG axis and undergoes diurnal variation mirroring testosterone, with peaks in the morning and falls by the evening to 60% of its original high point [39, 40].
In hopes of further elucidating how 17-OHP and ITT levels may be impacted, the same group sought to assess lifestyle factors and aging. They assessed serum samples in 340 men and found males with >25 BMI had higher rates of statistically significant decreases in serum testosterone and 17-OHP compared to normal BMI males [41]. Furthermore, their data shows aging by 1 year and 1 unit increase in BMI decreased 17-OHP by 0.84 ng/dl and 1.73 ng/dl, respectively. There was also a positive correlation between 17-OHP and serum testosterone, though this was not observed when serum testosterone levels were low. A similar study conducted by Martínez-Montoro studied 266 non-diabetic, ≥ 30 BMI men using high-performance liquid chromatography-mass spectrometry and found significant associations between 17-OHP, BMI, waist circumference, insulin levels, total and free testosterone levels [42]. However, in contrast to Lima et al.’s results, they found no association between age and 17-OHP but did so with LH. The two groups used different methods to assess measurements which may explain the differing results. These contrasting findings make it unclear whether increasing BMI causes decreased 17-OHP via negative feedback through the HPG axis and a subsequent decrease in hormones such as LH, or if it may rather have a more direct impact on 17-OHP and therefore intratesticular levels. How volumes of ITT and thus 17-OHP originating from the adrenal gland relate also remains unclear.
To further corroborate a direct effect on Leydig cells, Lima et al. found no changes between serum testosterone, 17-OHP, LH, FSH before and after varicocelectomy [43]. 17-OHP’s utility as a biomarker is also observed to predict when semen parameters may be upgraded when men are undergoing hCG/CC therapy. A previous study showed men with ≤55 ng/dL 17-OHP at baseline had upgraded semen quality as per assisted reproduction eligibility [44].
INSULIN-LIKE FACTOR 3 (INSL3)
INSL3 is a peptide hormone synthesized in adult Leydig cells and has been previously shown to be able to act as a biomarker for Leydig cell functionality [45]. Additionally, since INSL3 is constitutively produced by adult Leydig cells, it is temporally reflective when Leydig cells have differentiated. INSL3 levels are therefore capable to represent the population of differentiated Leydig cells and are more of an accurate predictor than serum testosterone. Serum testosterone is regulated by the HPG axis and undergoes large diurnal fluctuations [45, 46]. The large variability throughout the day and between patients with the same diseases makes it less of a candidate to represent Leydig cell function. INSL3 has also been shown to likely be solely produced from Leydig cells and its regulation is unaffected by LH. Bay et al. assessed the levels of serum INSL3 in 135 healthy males and 21 anorchid men using time-resolved fluorescence immunoassays and found median INSL3 levels were 0.99 ng/ml and 0.05 ng/ml respectively [47]. They also found INSL3 levels to be 15 times higher in blood samples drawn from vena spermatic compared to peripheral blood of two infertile males. This is suggestive that INSL3 may be solely localized in the testes.
INSL3 can be an insightful biomarker for patients undergoing hCG monotherapy. Although INSL3 levels have been observed to not significantly differ in hypogonadal patients within 72 and 96 h, hCG was highly associated with higher levels of INSL3. Hence, it is thought that hCG acts to differentiate Leydig cells in hypogonadal men. The Roth et al. study previously mentioned further corroborates the effects of hCG by administering dose-dependent injections subcutaneously consisting of gonadotropin-releasing hormone antagonist, acyline, and hCG or testosterone gel for 10 days in healthy males. Patients receiving only the antagonist had significantly lower INSL3 levels than the baseline and higher doses of hCG normalized INSL3 again [48]. These levels are directly correlated with serum testosterone levels.
INHIBIN B
Inhibin B is a dimeric glycoprotein whose bioactivity is dependent on its dimerization of its respective α and βB subunits. Its function is representative of Sertoli cells, acting as a negative regulator to pituitary FSH [50]. Inhibin B has been shown to be a valid marker of Sertoli cell function; inhibin B levels positively correlate with sperm concentration and negatively correlate with serum FSH levels. Inhibin B was even found to be an objectively better marker of spermatogenesis when compared to FSH as it more strongly correlated with testicular volume and semen parameters. As Roth et.al were studying INSL3 [43], they searched for reciprocal changes in other potential biomarkers, inhibin B being one of them, as well as 17-OHP and anti-Mullerian hormone – all of which showed no statistical significance in their study [48, 51]. Hence, although important to serum testosterone testing, further study is needed to understand inhibin B’s possible relationship with ITT. Figure 1 displays an overall review of the biomarkers emphasized here in the context of the intratesticular environment.
*CLINICAL IMPLICATIONS AND FUTURE DIRECTIONS
The intratesticular milieu is essential to spermatogenesis, though it is still far from being well understood [27]. More work is needed to better elucidate its role and consequently, its pathophysiologic potential. Although surgical testicular biopsies are minimally invasive, there are many common risks including pain, bleeding, infection, and testicular injury [52, 53]. Therefore, these biopsies are rarely used in the clinical setting.
The use of surrogate ITT biomarkers would not only allow more frequent and less risky monitoring but could also improve physiologic understanding of this environment. This is especially critical given the poor correlation between serum testosterone and ITT levels. Standard ITT was found to be around 170 times higher than serum testosterone and did not correlate with serum LH, FSH, testosterone, DHT (dihydrotestosterone), or estradiol [37]. DHT was found to be only 11 times higher within the testes than in the serum, likely due to the fact that most of it are made by the conversion of 5-alpha reductase in peripheral tissue [49] (most of which is concentrated to the prostate with minimal expression in genital skin), as further shown in Fig. 2 [54]. Nevertheless, when it comes to ITT, a quantitative relationship with spermatogenesis has yet to be clearly derived in men. Studies in rats have found that a reduction of ITT by up to 80% did not affect spermatogenesis, though, below this, spermatogenesis was severely impacted [55]. This study implies that there is far more testosterone in the testicles than required to maintain spermatogenesis, and moreover, a quantitative relationship with spermatogenesis does exist below a certain ITT threshold. Work towards establishing a lower limit of normal would likely prove to be a future therapeutic target for the clinical use of potential serum biomarkers.
A clinical study by Coviello et al. worked to address this gap by further examining the relationship between ITT and serum testosterone via a male contraceptive regimen using 100 mg weekly injections of testosterone enanthate and oral levonorgestrel 62.5 or 31.25 μg daily [56]. When comparing the results before and after this 6-month trial in seven men assessed via serum and intratesticular biopsy, they found the following: mean ITT decreased by 98%, no significant difference in serum T, and mean sperm count was suppressed by 98% (six of seven men reached a count of zero). Hence it is clear from a diagnostic viewpoint that a lack of ITT assessment may lead to the erroneous conclusion of idiopathic infertility, as is all too common.
Therapeutics
If patients are found to have low ITT levels, they are treated with hCG as well as recombinant FSH and CC [56–59]. The European consensus statement on the treatment of congenital hypogonadotropic hypogonadism states that testosterone replacement therapy is the mainstay of therapy for virilization purposes (so as to avoid infantile infantilism) [60]. However, this does not address the issue of fertility in these patients. The induction of male fertility mandates the induction of testosterone production and consequently spermatogenesis, hence relying on combined gonadotropin therapy via the guidance of an experienced urologist or endocrinologist. Moreover, AUA guidelines also refer to the use of aromatase inhibitors, HCG, and selective estrogen receptor modulators when treating men with testosterone deficiency who desire to maintain fertility (guideline statement 27) [61]. This allows patients suffering from hypogonadotropic hypogonadism or anabolic steroid abuse shutdown to initiate spermatogenesis, as well as improve spermatogenesis in men with idiopathic oligospermia. hCG acts as an LH mimic, thus stimulating Leydig testosterone production and secretion to raise ITT and serum testosterone [62]. CC is a racemic mixture of enclomiphene and zuclomiphene acting as a selective estrogen receptor modulator to block the negative feedback from testosterone [59, 63]. This indirectly increases LH and FSH secretion from the anterior pituitary to allow for more testosterone production and improved spermatogenesis [64]. In doing so, it is well-tolerated, showing minimal side effects such as hair loss, gastrointestinal distress, gynecomastia, and in a few cases visual disturbances, overall leading to restoration rather than replacement as traditional testosterone therapy would entail [64]. In addition to this routine trio, multivitamins and antioxidants are commonly used and have shown to be effective, though the evidence present is of lower quality [65]. Given the importance of ITT and its discrepancy from serum testosterone, the use of a biomarker would allow for routine monitoring. This would provide evidence that the above-mentioned therapeutics are acting appropriately.
Male contraception
Conversely, this monitoring process could act to support a male contraceptive regimen’s effectiveness. Currently, men are limited to condoms and vasectomies, but novel hormonal contraceptives are under development [66]. Their mechanism relies on the suppression of LH and FSH, presupposing completely reversible inhibition. Both the initiation and the reversal process would benefit from monitoring of both ITT and serum testosterone levels. Most of these regimens rely on exogenous testosterone, which via negative feedback shuts down endogenous FSH and LH. Meaning that although testosterone is still present exogenously, spermatogenesis is suppressed. Hence virility is maintained while fertility is repressed via azoospermia. However, side effects such as acne, weight gain, and fluctuating mood as well as a 5% failed reversibility rate over 52 weeks are barriers to the popularization of such a regimen [67]. Additionally, chronic testosterone administration does not reliably induce azoospermia, hence why monitoring is imperative, yet if this monitoring meant monthly testicular biopsy, the acceptability of such a regimen would undoubtedly decline [68].
CONCLUSION
Assessing 17-OHP and INSL3 as surrogate biomarkers for ITT is a promising way to address male infertility and assist in the creation of novel hormonal male contraceptives. There are many factors ranging from lifestyle to bioavailable concentrations that may affect the utility of these biomarkers and different factors are being investigated to determine whether they are efficacious. Although 17-OHP and INSL3 have potential, further investigations are required to elucidate their efficacy as biomarkers of ITT. An ongoing investigation is required to develop novel techniques to quantify levels of ITT biomarkers - techniques that are both effortless for the physician and safe for the patient. All in all, a better understanding of ITT via accurate serum biomarkers will deepen our understanding of the intratesticular environment and in so doing, conceivably transform the future of male fertility diagnostics and therapeutics.
