This work is licensed under a Abstract
Objective
Thyroid dysfunction modulates the hypothalamic–pituitary–gonadal axis in men, potentially altering sex hormone bioavailability. Prior studies relied on immunoassays for the measurement of sex steroids. We investigated the effect of primary thyroid dysfunction on testosterone (T) and estradiol (E2) in men, using high-specificity liquid chromatography–tandem mass spectrometry (LC–MS/MS).
Methods
In this prospective cohort study, adult men with newly diagnosed hyperthyroidism or hypothyroidism were evaluated at diagnosis and at the first follow-up visit. Changes in serum markers of gonadal function were assessed: follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin, sex hormone-binding globulin (SHBG), total and calculated free testosterone, total estradiol, and total testosterone:estradiol (T:E2) ratio.
Results
Sixteen men with hyperthyroidism and eleven with hypothyroidism were included. In the hyperthyroid cohort, total T, total E2, and SHBG levels decreased during treatment (P = 0.0131, P = 0.0063, and P = 0.0003), while free T, total T:E2 ratio, FSH, LH, and prolactin remained unchanged. In the hypothyroid cohort, increased SHBG, LH, and FSH levels were observed during levothyroxine treatment (P = 0.0014, P = 0.0117, and P = 0.0039), while total and free T, total E2, and prolactin remained unchanged. In the subgroup with overt hypothyroidism, the total T:E2 ratio increased during levothyroxine treatment.
Conclusion
In hyperthyroid males during treatment, short-term changes in the gonadal axis are mainly mediated by reduced SHBG, without changes in free T, gonadotropins, or T:E2 ratio. In hypothyroid males, SHBG and gonadotropins increased under levothyroxine supplementation. These findings highlight the importance of assessing SHBG when evaluating the male gonadal axis during thyroid dysfunction.
Keywords: thyroid dysfunction, hypothalamic–pituitary–gonadal axis, sex steroids, sex hormone-binding globulin
Introduction
Thyroid dysfunction may profoundly affect multiple physiological systems, including the hypothalamic–pituitary–gonadal (HPG) axis (1, 2). The HPG axis plays a central role in regulating the production of sex steroids, such as testosterone (T) and estradiol (E2), which are essential for reproductive health and sexual function (3, 4). In circulation, these sex steroids are mainly bound in steroid-specific proportions to sex hormone-binding globulin (SHBG), produced by the liver. As a result, only a small fraction of T and E2 circulate in an unbound or free form. According to the free hormone hypothesis, it is the free, unbound fraction of sex steroids that drive biological activity, highlighting the physiological relevance of changes in SHBG levels (5, 6). Thyroid hormones act on this delicate equilibrium by enhancing the expression of hepatic nuclear factor 4α (HNF-4α), a key regulator of SHBG transcription.
Hyperthyroidism leads to increased hepatic SHBG production, resulting in decreased sex steroid bioavailability (7). To maintain stable free T levels, luteinizing hormone (LH) secretion increases, enhancing testicular steroidogenesis and the aromatization of T to E2 (8, 9, 10). As a result, hyperthyroidism has been shown to give rise to elevated total T and E2 concentrations (11, 12). Although rare, gynecomastia can be the first clinical manifestation of hyperthyroidism and is believed to be the consequence of reduced free T availability caused by elevated SHBG levels (13). Notably, the measurement of T and E2 in previous studies was performed by immunoassay (9, 10, 11, 12). This is particularly relevant for E2, as circulating concentrations in men are low and immunoassays are less reliable in this range, which may have contributed to uncertainty in previous reports on estrogen status in thyroid dysfunction (14, 15, 16). Although this may be less problematic for T in adult men, assay-related variation may still limit precise assessment of subtle differences in sex steroid homeostasis (16, 17). To our knowledge, no studies to date have employed mass spectrometry (MS), the gold standard for steroid hormone quantification, to evaluate changes in both T and E2 levels in men with thyroid dysfunction.
Hypothyroidism, on the other hand, is associated with reduced SHBG levels, potentially leading to an increase in the free T and E2 fraction (18, 19). In addition, hypothyroidism can also lead to increased prolactin levels, resulting in hypogonadotropic hypogonadism, which can potentially also result in the development of gynecomastia (1, 13). The underlying mechanism linking primary hypothyroidism and hyperprolactinemia is thought to involve elevated thyrotropin-releasing hormone (TRH), which stimulates prolactin secretion. Hyperprolactinemia, in its turn, inhibits the pulsatile release of gonadotropin-releasing hormone (GnRH), potentially leading to central hypogonadism (3). Apart from gynecomastia, thyroid dysfunction-related sex steroid imbalances in males may also contribute to other clinical manifestations, such as infertility and erectile dysfunction, highlighting the importance of assessing sexual desire and erectile function in men with hypo- and hyper-thyroidism (20, 21).
In patients with differentiated thyroid cancer (DTC), thyroid hormone withdrawal (THW) is used prior to radioiodine (RAI) administration to achieve elevated endogenous TSH levels and thereby stimulate iodine uptake by residual thyroid tissue or tumor foci (22). As such, THW induces a transient but profound state of primary hypothyroidism with a predictable onset and duration, a clinical context that enables to study short-term changes in the gonadal axis.
The objective of this study was to prospectively evaluate changes in HPG axis parameters in adult men treated for thyroid dysfunction (primary hyperthyroidism and hypothyroidism), with a specific focus on alterations in SHBG, T, E2, and their ratio (T:E2 ratio). In doing so, we also aimed to address an important methodological gap by using liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based steroid measurements, particularly relevant for E2 assessment in men.
Methods
Participants and study design
A prospective clinical cohort study was conducted at the University Hospitals Leuven in Belgium between June 2012 and June 2016. Male patients referred to the thyroid outpatient clinic with a diagnosis of primary hyper- or hypothyroidism and patients with DTC prior to THW in preparation for RAI administration causing transient profound primary hypothyroidism were invited to participate. Patients with any cause of primary hyper- and hypothyroidism, as well as any type of treatment, were included. Patients using medication interfering with the gonadal axis (e.g. T supplements, beta-HCG, opioids, neuroleptics) and patients with acute illness around the timing of blood sampling were excluded.
The Ethical Committee of the University Hospitals Leuven approved the study (s54034, ClinicalTrials.gov No. NCT02434562). Written informed consent was obtained from each participant.
Biochemical analysis
Two blood samples were collected for each patient: the first sample at the visit where hyper- or hypothyroidism was diagnosed (visit 1) and the second sample at the follow-up visit (visit 2). All samples were collected in the morning to early afternoon hours, during standard-of-care visits. For the hypothyroid subgroup under THW, visit 1 corresponds to blood sampling at 6 weeks after levothyroxine (LT4) cessation and 2 weeks after liothyronine cessation, and visit 2 corresponds to blood sample collection after RAI when LT4 was recommenced for at least 6 weeks.
Thyroid-stimulating hormone (TSH), total thyroxine (TT4), free thyroxine (fT4), total triiodothyronine (TT3), LH, follicle-stimulating hormone (FSH), and prolactin were measured by electrochemiluminescence immunoassay on the Roche Cobas® 8,000 analyzer (Roche Diagnostics, Germany). Free thyroxine index (FTI) was calculated as the product of total T4 and T3 uptake ratio (23). SHBG was measured using the Modular E170 platform electrochemiluminescence immunoassay (Roche Diagnostics). E2 concentrations were measured by LC–MS/MS as described previously (15). The lower limit of quantification for E2 was 4.8 pmol/L, and total imprecision was 8.8% at 82.6 pmol/L. Serum T levels were quantified by LC–MS/MS using the Xevo TQ-XS IVD mass spectrometer coupled to an Acquity UPLC I-class (Waters Corp, USA) equipped with an Acquity UPLC HSS T3 column (1.8 μm, 2.1 mm × 50 mm). Mobile phases consisted of MilliQ water and acetonitrile with post-column infusion of 2.5 mM ammonium fluoride solution at 20 μL/min. Samples (50 μL serum diluted in 250 μL internal standard solution) were incubated for 30 min at −20°C to facilitate protein precipitation. Following centrifugation (10 min at 21,130 g), 250 μL of the supernatant was transferred to a 96-deepwell plate and subsequently dried under nitrogen flow. Evaporated samples were redissolved in 30% acetonitrile and incubated on a shaker for 5 min at 1,000 r.p.m. After dissolving, 10 μL of sample was injected into the LC–MS/MS system. Measurements were acquired in multiple reaction monitoring mode by exploiting transitions m/z 289.3 > 97.1 and 289.3 > 109.1 for T and 292.2 > 100.0 for the internal standard (T-13C3). Data processing was performed using TargetLynx software (Waters Corp). The lower limit of quantification for T was 0.03 nmol/L and long-term (one year) precision was 3.9% at 0.38 nmol/L. Free T was calculated using the Vermeulen formula (24). The total T:E2 ratio was calculated using total T (nmol/L) and total E2 (nmol/L) (25).
Statistical analysis
Statistical analyses were conducted using GraphPad Prism (version 10.3.1, USA). Descriptive statistics were used to summarize the baseline characteristics. Normality was tested using the Shapiro–Wilk test. All data were visualized as median (range). Paired t-test or Wilcoxon matched-pairs signed-rank test was used where appropriate. Subgroup analysis was performed in the hyperthyroid subgroup with overt hyperthyroidism defined as fT4 or FTI above the upper limit of normal and in the overt hypothyroid subgroup defined as THW post-thyroidectomy. Spearman’s rank correlation was used to evaluate associations between changes in hormonal parameters. Two-tailed P-value < 0.05 was considered statistically significant.
Results
Primary hyperthyroidism and changes in the pituitary–gonadal axis and SHBG in adult males
A total of 16 men with hyperthyroidism were included (8 patients with overt hyperthyroidism and 8 patients with mild hyperthyroidism). The patients' age, etiology of hyperthyroidism, time between first and second blood sampling, and treatment are presented in Table 1. Thyroid hormones at diagnosis (visit 1) and at follow-up (visit 2) are shown in Table 2. As intended, TSH increased, while TT4, FTI, fT4, and fT3 decreased after treatment initiation.
Table 1.
Study population. Data are presented as n or as median (range).
| Patient characteristics | Hyperthyroid group (n = 16) | Hypothyroid group (n = 11) |
|---|---|---|
| Age (years) | 57.9 (39.7–85.6) | 55.2 (25.3–76.1) |
| Etiology of thyroid dysfunction, n | ||
| Autoimmune | 6 | 3 |
| Toxic nodule | 6 | |
| Iodine-induced | 2 | |
| Thyroiditis | 1 | |
| Amiodarone | 1 | |
| THW | 8 | |
| Severity of thyroid dysfunction, n | ||
| Overt | 8 | 8 |
| Mild | 8 | 3 |
| Time between first and second sample (days) | 54.0 (20.1–364.8) | 111.3 (35.1–256.5) |
| Overt subgroup* | 31.6 (20.1–238.5) | 117.2 (97.2–256.5) |
| Mild subgroup | 66.6 (21.0–364.8) | 56.1 (35.1–63.1) |
| Treatment, n | ||
| Thiamazole | 9 | |
| RAI | 5 | |
| LT4 | 11 | |
| None | 2 |
RAI, radioiodine; THW, thyroid hormone withdrawal; LT4, levothyroxine.
Overt subgroup defined as free T4 or free thyroxine index above the upper limit of normal for the hyperthyroid group, and THW (6 weeks after LT4 cessation and 2 weeks after liothyronine cessation) post-thyroidectomy for the hypothyroid group.
Table 2.
Thyroid function tests at diagnosis of hyperthyroidism (visit 1) and during treatment (visit 2). Data are presented as median (range). Statistically significant values are in bold. Normality was tested using the Shapiro–Wilk test. Overt subgroup defined as free T4 or free thyroxine index above the upper limit of normal at visit 1.
| Visit 1 | Visit 2* | Ref. interval | n | P value | |
|---|---|---|---|---|---|
| TSH (mIU/L)‡ | 0.00 (0.00–0.21) | 0.07 (0.00–16.58) | 0.27–4.20 | 16 | 0.0010 |
| Overt subgroup‡ | 0.00 (0.00–0.01) | 0.01 (0.00–16.58) | 8 | 0.2500 | |
| Total T4 (μg/dL)‡ | 8.80 (6.30–47.80) | 7.75 (4.30–11.70) | 5.10–14.10 | 16 | <0.0001 |
| Overt subgroup‡ | 13.05 (8.50–47.80) | 8.75 (5.70–11.70) | 8 | 0.0078 | |
| Free thyroxine index‡ | 9.60 (5.60–158.20) | 7.30 (3.80–53.90) | 1.30–4.20 | 15 | 0.0001 |
| Overt subgroup‡ | 22.00 (9.90–158.20) | 9.00 (5.50–53.90) | 7 | 0.0156 | |
| Free T4 (ng/dL)‡ | 1.68 (0.91–4.13) | 1.10 (0.74–3.43) | 0.80–1.80 | 13 | 0.0017 |
| Overt subgroup† | 2.61 (1.96–4.13) | 1.63 (0.95–3.43) | 5 | 0.0029 | |
| Total T3 (ng/dL)‡ | 148.00 (112.00–351.00) | 109.00 (70.00–189.00) | 80.00–200.00 | 15 | <0.0001 |
| Overt subgroup† | 215.00 (138.00–351.00) | 108.00 (70.00–189.00) | 7 | 0.0118 |
In 2 patients (1 with thyroiditis +1 with autonomous nodule), a watchful waiting approach was adopted and no specific treatment was initiated.
Paired t-test.
Wilcoxon matched-pairs signed-rank test was used where appropriate.
Biochemical parameters of the HPG axis at diagnosis and follow-up are shown in Table 3 and Supplementary Fig. 1 (see section on Supplementary materials given at the end of the article). At visit 2, total T, total E2, and SHBG levels decreased, while all other parameters (LH, FSH, prolactin, free T, and total T:E2 ratio) showed no statistically significant differences between diagnosis and during treatment in either the total group or the overt hyperthyroidism subgroup. At visit 1, of patients with hyperthyroidism, one had total T levels below and three patients had total T levels above the reference range; the latter patients were in the overt hyperthyroidism subgroup. At visit 2, three patients had total T below the reference range and none were above it. For free T, three patients were below the reference range at visit 1 and none were above; at visit 2, four patients were below the reference range, again with no patients above it.
Table 3.
Changes in the pituitary–gonadal axis and SHBG in male hyperthyroid patients*. Data are presented as median (range). Significant results in bold. Normality was tested using the Shapiro–Wilk test. Overt subgroup defined as free T4 or free thyroxine index above the upper limit of normal.
| Parameters | Visit 1 | Visit 2 | Ref. interval | n | P value |
|---|---|---|---|---|---|
| LH (IU/L)‡ | 6.50 (1.60–34.80) | 6.30 (2.30–30.20) | 1.70–8.60 | 16 | 0.2363 |
| Overt subgroup† | 6.50 (4.20–12.20) | 5.75 (3.40–10.10) | 8 | 0.2941 | |
| FSH (IU/L)‡ | 6.10 (2.00–37.80) | 5.95 (1.60–35.90) | 1.20–7.70 | 16 | 0.8099 |
| Overt subgroup‡ | 6.10 (2.00–18.00) | 5.45 (2.20–19.40) | 8 | >0.9999 | |
| Prolactin (μg/L)‡ | 4.55 (2.20–35.00) | 4.75 (2.20–36.00) | 2.00–18.00 | 16 | 0.6096 |
| Overt subgroup‡ | 4.05 (2.70–14.50) | 4.15 (2.20–11.40) | 8 | 0.3125 | |
| Total T (nmol/L)‡ | 22.43 (8.38–56.35) | 17.39 (5.16–32.13) | 10.40–34.67 | 16 | 0.0131 |
| Overt subgroup† | 26.72 (8.38–56.35) | 18.69 (5.16–32.13) | 8 | 0.0897 | |
| Free T (nmol/L)‡ | 0.31 (0.11–0.40) | 0.29 (0.10–0.63) | 0.17–0.69 | 16 | 0.1591 |
| Overt subgroup† | 0.23 (0.11–0.38) | 0.25 (0.10–0.63) | 8 | 0.7062 | |
| Total E2 (pmol/L)‡ | 124.27 (59.11–243.41) | 79.85 (42.59–181.73) | 36.71–146.85 | 16 | 0.0063 |
| Overt subgroup† | 124.27 (59.11–239.74) | 79.85 (42.59–181.73) | 8 | 0.1881 | |
| Total T:E2 ratio† | 188.59 (66.27–323.30) | 195.70 (83.31–392.28) | 16 | 0.8104 | |
| Overt subgroup† | 223.11 (87.31–323.30) | 182.92 (112.25–392.28) | 8 | 0.4394 | |
| SHBG (nmol/L)‡ | 68.50 (33.00–180.00) | 43.00 (17.00–102.00) | 24.00–55.00 | 16 | 0.0003 |
| Overt subgroup† | 114.50 (33.00–180.00) | 63.00 (28.00–101.00) | 8 | 0.0053 |
In 2 patients (1 with thyroiditis +1 with autonomous nodule), a watchful waiting approach was adopted and no specific treatment was initiated.
E2, estradiol; FSH, follicle-stimulating hormone; LH, luteinizing hormone; T, testosterone; SHBG, sex hormone-binding globulin.
Paired t-test.
Wilcoxon matched-pairs signed-rank test was used when appropriate.
Correlation analyses are shown in Table 4. Changes in TT4 were positively correlated with changes in SHBG (r = 0.61, P = 0.014). No significant correlations were observed between changes in the other thyroid hormone parameters (FTI, TT3, and fT4) and sex steroid parameters. Changes in total T were positively correlated with changes in free T (r = 0.65, P = 0.007), total E2 (r = 0.69, P = 0.004), and SHBG (r = 0.65, P = 0.007). Changes in free T were also positively correlated with changes in total E2 (r = 0.60, P = 0.015).
Table 4.
Correlation matrix of changes (Δ) in thyroid and sex hormone parameters between visit 1 (diagnosis of hyperthyroidism) and visit 2 (treatment) measurements. Values within the matrix represent Spearman correlation coefficients. Significant results in bold.
| Δ Total T | Δ Free T | Δ Total E2 | Δ Total T:E2 ratio | Δ SHBG | |
|---|---|---|---|---|---|
| Δ Total T4 | 0.22 | −0.05 | −0.01 | 0.49 | 0.61 * |
| Δ Free T4 | 0.29 | 0.00 | 0.23 | −0.12 | 0.36 |
| Δ Free thyroxine index | −0.24 | −0.26 | −0.29 | 0.27 | 0.08 |
| Δ Total T3 | 0.08 | −0.34 | −0.13 | 0.30 | 0.48 |
T, testosterone; E2, estradiol; SHBG, sex hormone-binding globulin.
P < 0.05.
Primary hypothyroidism and changes in the pituitary–gonadal axis and SHBG in adult males
A total of 11 men with hypothyroidism were included, with 8 patients undergoing THW post-thyroidectomy for DTC. The patients’ age, etiology of hypothyroidism, time between first and second serum sampling, and treatment are presented in Table 1. Thyroid hormones at diagnosis (visit 1) and at follow-up (visit 2) are shown in Table 5. All patients were treated with LT4. As intended, TSH decreased while TT4, FTI, and fT4 increased after LT4 initiation.
Table 5.
Thyroid function tests at diagnosis of hypothyroidism (visit 1) and during LT4 treatment (visit 2). Data are presented as median (range). Significant results in bold. Normality was tested using the Shapiro–Wilk test. Overt subgroup defined by patients undergoing thyroid hormone withdrawal (6 weeks after LT4 cessation and 2 weeks after liothyronine cessation) post-thyroidectomy.
| Visit 1 | Visit 2 | Ref. interval | n | P value | |
|---|---|---|---|---|---|
| TSH (mIU/L)† | 56.66 (4.30–255.10) | 0.29 (0.02–1.40) | 0.27–4.20 | 11 | 0.0010 |
| Overt subgroup† | 79.75 (32.09–255.10) | 0.09 (0.02–1.39) | 8 | 0.0078 | |
| Total T4 (μg/dL)† | 0.90 (0.50–6.10) | 9.70 (7.00–11.00) | 5.10–14.10 | 7 | 0.0156 |
| Overt subgroup† | 0.55 (0.50–0.90) | 10.00 (8.80–11.00) | 4 | 0.1250 | |
| Free thyroxine index† | 0.70 (0.30–5.70) | 10.20 (6.40–13.10) | 1.30–4.20 | 7 | 0.0156 |
| Overt subgroup† | 0.40 (0.30–0.70) | 10.00 (8.80–11.00) | 4 | 0.1250 | |
| Free T4 (ng/dL)† | 0.06 (0.03–0.92) | 1.86 (1.47–2.56) | 0.80–1.80 | 8 | 0.0078 |
| Overt subgroup† | 0.05 (0.03–0.13) | 2.02 (1.70–2.56) | 6 | 0.0312 | |
| Total T3 (ng/dL) | NA | NA | NA | NA |
NA, not available.
Paired t-test.
Wilcoxon matched-pairs signed-rank test was used where appropriate.
Biochemical parameters of the HPG axis at diagnosis and during treatment for hypothyroidism are shown in Table 6 and Supplementary Fig. 2. Under LT4 treatment, LH and FSH levels increased, and prolactin decreased, although this change was not significant. Despite increased SHBG, total T was not significantly different. Furthermore, E2 did not change significantly. In the overt subgroup, the T:E2 ratio increased under LT4 treatment.
Table 6.
Changes in the pituitary–gonadal axis and SHBG in male hypothyroid patients. Data are presented as median (range). Significant results in bold. Normality was tested using the Shapiro–Wilk test. Overt subgroup defined by patients undergoing thyroid hormone withdrawal post-thyroidectomy.
| Parameters | Visit 1 | Visit 2 | Ref. interval | n | P value |
|---|---|---|---|---|---|
| LH (IU/L)† | 4.80 (4.40–7.30) | 6.55 (4.20–17.20) | 1.70–8.60 | 10 | 0.0117 |
| Overt subgroup† | 5.00 (4.50–7.30) | 8.40 (4.20–17.20) | 7 | 0.0312 | |
| FSH (IU/L)† | 6.70 (2.50–26.40) | 10.85 (2.60–49.80) | 1.20–7.70 | 10 | 0.0039 |
| Overt subgroup† | 8.80 (4.10–26.40) | 16.70 (7.60–49.80) | 7 | 0.0487 | |
| Prolactin (μg/L)† | 6.95 (2.50–11.50) | 4.40 (2.40–6.10) | 2.00–18.00 | 6 | 0.1250 |
| Overt subgroup* | 9.90 (4.00–11.50) | 5.70 (3.10–6.10) | 3 | 0.1214 | |
| Total T (nmol/L)* | 11.68 (4.02–18.85) | 14.61 (8.51–18.44) | 10.40–34.67 | 11 | 0.1011 |
| Overt subgroup* | 9.75 (4.02–17.63) | 12.75 (8.51–18.32) | 8 | 0.1145 | |
| Free T (nmol/L)* | 0.22 (0.09–0.38) | 0.24 (0.18–0.33) | 0.17–0.69 | 10 | 0.5374 |
| Overt subgroup* | 0.21 (0.09–0.30) | 0.21 (0.18–0.31) | 7 | 0.5879 | |
| Total E2 (pmol/L)* | 80.77 (38.92–124.09) | 73.79 (38.92–119.32) | 36.71–146.85 | 11 | 0.8417 |
| Overt subgroup* | 76.73 (38.92–124.09) | 73.06 (38.92–119.32) | 8 | 0.7992 | |
| Total T:E2 ratio* | 144.57 (55.23–393.69) | 169.89 (106.84–388.79) | 10 | 0.2098 | |
| Overt subgroup* | 133.22 (55.23–393.69) | 177.25 (106.84–388.79) | 7 | 0.0358 | |
| SHBG (nmol/L)* | 31.00 (18.00–55.00) | 43.00 (21.00–57.00) | 24.00–55.00 | 11 | 0.0014 |
| Overt subgroup† | 31.00 (27.00–55.00) | 43.00 (28.00–57.00) | 8 | 0.0078 |
E2, estradiol; FSH, follicle-stimulating hormone; LH, luteinizing hormone; T, testosterone; SHBG, sex hormone-binding globulin.
Paired t-test.
Wilcoxon matched-pairs signed-rank test was used when appropriate.
At visit 1, five hypothyroid patients had total T levels below the reference range (four in the overt subgroup and one in the autoimmune subgroup); at visit 2, two of these patients (both in the overt subgroup) remained below the reference range. At visit 1, three patients (all in the overt subgroup) had free T levels below the reference range; at visit 2, none were below the reference range.
No significant correlations were observed between changes in thyroid hormone parameters and sex steroid parameters (data not shown).
Discussion
This study investigated the relationship between primary thyroid dysfunction and male gonadal hormone homeostasis, focusing on SHBG, total and free T, total E2, and the T:E2 ratio. Our findings provide valuable insights into how thyroid dysfunction alters T and E2 in the circulation, using LC–MS/MS, a highly accurate method to assess sex steroid hormone concentrations (26). The added value of LC–MS/MS in this context is particularly relevant for E2, as circulating E2 concentrations in men are low and immunoassays are less reliable in this range (14, 15).
In hyperthyroid patients between the diagnosis and the follow-up visit, we observed a significant decrease in total T, total E2, and SHBG levels. Free T levels and the total T:E2 ratio remained unchanged. However, these patterns should be interpreted in the context of the small and clinically heterogeneous hyperthyroid group, in which the observed changes may reflect both treatment effects and changes in thyroid status over time. Our findings are consistent with previous findings showing that hyperthyroidism is associated with increased SHBG and total T levels (27, 28). It has been suggested that to maintain stable free T levels, LH secretion may increase (8, 11, 28). In our study, LH levels appeared higher at diagnosis, particularly in the overt hyperthyroid subgroup, and declined during treatment, although this change was not statistically significant. This is in line with the observation reported by Abalovich et al. that hyperthyroid patients had significantly higher basal LH levels compared with healthy controls and that the LH response to GnRH stimulation was significantly greater in hyperthyroid patients (11).
Previous studies examining E2 levels in men with hyperthyroidism have reported elevated E2 concentrations, using both cross-sectional comparisons between hyperthyroid and euthyroid individuals and longitudinal designs (11, 20, 29). In line with these findings, we observed significantly higher total E2 concentrations at diagnosis compared with levels measured during treatment after a median follow-up of 1–3 months. To our knowledge, this is the first study to assess E2 in adult men with thyroid dysfunction using LC–MS/MS, a method with greater analytical specificity than the immunoassays employed in earlier research (14, 15, 26). This methodological aspect is particularly relevant for E2, as circulating concentrations in men are low and immunoassay-based measurements show limited agreement with LC–MS/MS in this range, whereas agreement is generally better for T (30). The main added value of LC–MS/MS in this study, therefore, lies in the more reliable assessment of E2 concentrations. Our findings strengthen the evidence for elevation of E2 concentration in men with hyperthyroidism.
In men, circulating E2 is predominantly derived from peripheral aromatization of T (31). Consequently, decreased T levels, such as those observed in our study during treatment of hyperthyroidism, result in parallel changes in E2 concentrations (32). Changes in SHBG showed a strong association with total T, whereas no significant association was observed with total E2. This discrepancy is likely explained by the markedly higher affinity of SHBG for androgens than for estrogens (33). The decrease in fT4, TT4, or TT3 was not associated with changes in the T:E2 ratio, suggesting that hyperthyroidism may not be associated with altered aromatization.
Hyperthyroidism has several potential clinical implications for the male gonadal axis. When hyperthyroidism is suspected or diagnosed and assessment of male gonadal function is indicated based on symptoms (e.g. loss of libido, gynecomastia), it is important to measure both total T and SHBG to assess free T (5, 6). Conversely, when total T levels above the reference range are found (after exclusion of exogenous origin of androgens), SHBG should be measured, and the patient should be screened for hyperthyroidism. Some hyperthyroid patients in our cohort presented with total T levels above the upper normal limit, while the free T fraction remained within the reference range. No patients presented with gynecomastia in our study. In cases of hyperthyroidism-induced gynecomastia, it could be speculated that T levels were already borderline or reduced prior to the onset of hyperthyroidism with inadequate testicular compensation, or that an exaggerated hepatic increase in SHBG occurred in hyperthyroid conditions. In the case of newly diagnosed gynecomastia, it remains advisable to also measure E2, prolactin, LH, and FSH along with T and SHBG to rule out other underlying pathologies (13).
In the hypothyroid group under LT4 treatment, we observed a significant increase in SHBG levels, which is in line with earlier studies (20, 34). Furthermore, we observed a significant increase in gonadotropin levels (both LH and FSH), suggesting increased stimulation of the HPG axis. Velásquez et al. showed that hypothyroid patients exhibited a significantly reduced LH in response to GnRH compared with controls, suggesting an impaired gonadotropic response in a hypothyroid state (12). A study with healthy older subjects showed that LH levels significantly increased following a T3 challenge over a period of 4–5 days (28). We observed a trend toward decreased prolactin levels during LT4 treatment; however, this reduction did not reach statistical significance, likely due to the limited sample size, as prolactin measurements were only available for six patients (three in the overt subgroup and three in the autoimmune subgroup). Donnelly et al. found that while hyperprolactinemia is a recognized cause of hypogonadotropic hypogonadism in both men and women, prolactin levels in hypothyroid men were not significantly different from those in euthyroid controls. Nonetheless, they observed a mild but significant decrease in prolactin following LT4 replacement (35). Hekimsoy et al. reported elevated prolactin levels in 36% of patients with overt hypothyroidism and 22% with subclinical hypothyroidism, with normalization upon LT4 treatment (36). Van der Spoel et al. showed that prolactin levels significantly decreased in both men and women following a T3 challenge (28). These data support the concept of hypothyroidism-induced central suppression of the HPG axis, mediated by hyperprolactinemia.
As expected, alterations in the HPG axis were more pronounced in the overtly hypothyroid subgroup, likely due to the severely iatrogenic hypothyroid state, which is schematically illustrated in Fig. 1. Previous studies in women undergoing THW have shown a similar increase in SHBG (37, 38).
Figure 1.
Schematic representation of the effects of primary hypothyroidism on the hypothalamic–pituitary–thyroid axis (HPT axis) and the hypothalamic–pituitary–gonadal axis (HPG axis). TRH, thyrotropin-releasing hormone; TSH, thyroid-stimulating hormone; PRL, prolactin; GnRH, gonadotropin-releasing hormone; LH, luteinizing hormone; FSH, follicle-stimulating hormone; T, testosterone; E2, estradiol; SHBG, sex hormone-binding globulin. Adapted with permission from: Gabrielson et al. (3).
Hypothyroidism likewise has important clinical implications for the evaluation of the male gonadal axis. Both total T and SHBG should be measured to accurately assess free T in hypothyroid men (5, 6). This is particularly relevant in patients presenting with low total T in the context of reduced SHBG levels, as also seen in the context of male obesity, which could otherwise lead to misclassification of hypogonadism if free T is not considered.
In contrast to the hyperthyroid group, in which treatment was associated with a decrease in total T and E2 levels but not in the total T:E2 ratio, we observed no significant changes in total T or E2 levels in the hypothyroid group. However, the overt subgroup demonstrated a significant increase in the total T:E2 ratio during LT4 treatment. This finding is likely attributable to the concurrent rise in SHBG levels observed under LT4 replacement. Given the higher binding affinity of SHBG for androgens compared with estrogens, total T may increase disproportionately relative to E2, leading to an elevated total T:E2 ratio despite stable absolute hormone levels.
This study is the first, to our knowledge, to comprehensively map changes in sex steroids (both T and E2) measured by LC–MS/MS in male adults in the context of both hyper- and hypo-thyroidism. Its main strength lies in the use of LC–MS/MS for the measurement of T and especially E2, for which accurate measurement is challenging in adult men. Our study has limitations. The sample size is small and the cohort is heterogeneous, with different underlying causes of thyroid dysfunction potentially influencing the observed results. The interval between the two study visits was not standardized. The degree of hyperthyroidism in our cohort was relatively mild. Timing during the day of the serum sampling is not standardized, which may have underestimated serum T concentrations. Sexual symptoms were not recorded. Future studies in larger cohorts with longer follow-up should investigate the long-term effects of thyroid dysfunction and its treatment on the HPG axis and SHBG, as well as the clinical implications.
Conclusion
In this cohort, the main short-term finding during treatment for hyperthyroidism in men was a decrease in SHBG, without changes in free T, gonadotropins, or the T:E2 ratio. In hypothyroid men under LT4 treatment, apart from increased SHBG, the gonadotropins also increased, probably secondary to normalization of prolactin. These findings support the assessment of SHBG when evaluating the male gonadal axis in the setting of thyroid dysfunction.
Supplementary materials
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the work reported.
Funding
This work was funded by research grants from the Research Foundation Flanders (FWO) (G085413N, 1800923N, and 11P9024N).
Author contribution statement
VORP conceptualized the study, led the formal analysis, administered the project, wrote the original draft of the manuscript, and reviewed and edited the same. SS conceptualized the study. NN dealt with resources and reviewed and edited the manuscript. NJ critically reviewed the manuscript. NS collected the data and critically reviewed the manuscript. LA conceptualized this study, collected and curated the data, and reviewed and edited the manuscript. DV conceptualized this study, collected the data, reviewed and edited the manuscript, and acquired funding. DK conceptualized this study, led the formal analysis, and reviewed and edited the manuscript. BD conceptualized this study, supervised and administered the project, wrote the original draft of the manuscript, and reviewed and edited the same.
References
- 1.Krassas GE, Poppe K & Glinoer D. Thyroid function and human reproductive health. Endocr Rev 2010. 31 702–755. ( 10.1210/er.2009-0041) [DOI] [PubMed] [Google Scholar]
- 2.Chaker L, Razvi S, Bensenor IM, et al. Hypothyroidism. Nat Rev Dis Primers 2022. 8 30. ( 10.1038/s41572-022-00357-7) [DOI] [PubMed] [Google Scholar]
- 3.Gabrielson AT, Sartor RA & Hellstrom WJG. The impact of thyroid disease on sexual dysfunction in men and women. Sex Med Rev 2019. 7 57–70. ( 10.1016/j.sxmr.2018.05.002) [DOI] [PubMed] [Google Scholar]
- 4.De Silva NL, Papanikolaou N, Grossmann M, et al. Male hypogonadism: pathogenesis, diagnosis, and management. Lancet Diabetes Endocrinol 2024. 12 761–774. ( 10.1016/s2213-8587(24)00199-2) [DOI] [PubMed] [Google Scholar]
- 5.Narinx N, David K, Walravens J, et al. Role of sex hormone-binding globulin in the free hormone hypothesis and the relevance of free testosterone in androgen physiology. Cell Mol Life Sci 2022. 79 543. ( 10.1007/s00018-022-04562-1) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Nyamaah JA, Narinx N, Antonio L, et al. Use of calculated free testosterone in men: advantages and limitations. Curr Opin Endocrinol Diabetes Obes 2024. 31 230–235. ( 10.1097/med.0000000000000891) [DOI] [PubMed] [Google Scholar]
- 7.Selva DM & Hammond GL. Thyroid hormones act indirectly to increase sex hormone-binding globulin production by liver via hepatocyte nuclear factor-4α. J Mol Endocrinol 2009. 43 19–27. ( 10.1677/jme-09-0025) [DOI] [PubMed] [Google Scholar]
- 8.Forest MG, Lecoq A & Saez JM. Kinetics of human chorionic gonadotropin-induced steroidogenic response of the human testis. II. Plasma 17α-hydroxyprogesterone, Δ4-androstenedione, estrone, and 17β-estradiol: evidence for the action of human chorionic gonadotropin on intermediate enzymes implicated in steroid biosynthesis. J Clin Endocrinol Metab 1979. 49 284–291. ( 10.1210/jcem-49-2-284) [DOI] [PubMed] [Google Scholar]
- 9.Chopra IJ & Tulchinsky DAN. Status of estrogen-androgen balance in hyperthyroid men with Graves’ disease. J Clin Endocrinol Metab 1974. 38 269–277. ( 10.1210/jcem-38-2-269) [DOI] [PubMed] [Google Scholar]
- 10.Ridgway EC, Maloof F & Longcope C. Androgen and oestrogen dynamics in hyperthyroidism. J Endocrinol 1982. 95 105–115. ( 10.1677/joe.0.0950105) [DOI] [PubMed] [Google Scholar]
- 11.Abalovich M, Levalle O, Hermes R, et al. Hypothalamic-pituitary-testicular axis and seminal parameters in hyperthyroid males. Thyroid 1999. 9 857–863. ( 10.1089/thy.1999.9.857) [DOI] [PubMed] [Google Scholar]
- 12.Velázquez EM & Arata GB. Effects of thyroid status on pituitary gonadotropin and testicular reserve in men. Arch Androl 1997. 38 85–92. ( 10.3109/01485019708988535) [DOI] [PubMed] [Google Scholar]
- 13.Kanakis GA, Nordkap L, Bang AK, et al. EAA clinical practice guidelines—gynecomastia evaluation and management. Andrology 2019. 7 778–793. ( 10.1111/andr.12636) [DOI] [PubMed] [Google Scholar]
- 14.Rosner W, Hankinson SE, Sluss PM, et al. Challenges to the measurement of estradiol: an endocrine society position statement. J Clin Endocrinol Metab 2013. 98 1376–1387. ( 10.1210/jc.2012-3780) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Pauwels S, Antonio L, Jans I, et al. Sensitive routine liquid chromatography-tandem mass spectrometry method for serum estradiol and estrone without derivatization. Anal Bioanal Chem 2013. 405 8569–8577. ( 10.1007/s00216-013-7259-5) [DOI] [PubMed] [Google Scholar]
- 16.Narinx N, Nyamaah JA, David K, et al. A survey on measurement and reporting of total testosterone, sex hormone-binding globulin and free testosterone in clinical laboratories in Europe. Clin Chem Lab Med 2025. 63 1561–1572. ( 10.1515/cclm-2024-1237) [DOI] [PubMed] [Google Scholar]
- 17.French D. Clinical utility of laboratory developed mass spectrometry assays for steroid hormone testing. J Mass Spectrom Adv Clin Lab 2023. 28 13–19. ( 10.1016/j.jmsacl.2023.01.006) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Hampl R, Kancheva R, Hill M, et al. Interpretation of sex hormone-binding globulin levels in thyroid disorders. Thyroid 2003. 13 755–760. ( 10.1089/105072503768499644) [DOI] [PubMed] [Google Scholar]
- 19.Zähringer S, Tomova A, Von Werder K, et al. The influence of hyperthyroidism on the hypothalamic-pituitary-gonadal axis. Exp Clin Endocrinol Diabetes 2000. 108 282–289. ( 10.1055/s-2000-7756) [DOI] [PubMed] [Google Scholar]
- 20.Kjaergaard AD, Marouli E, Papadopoulou A, et al. Thyroid function, sex hormones and sexual function: a Mendelian randomization study. Eur J Epidemiol 2021. 36 335–344. ( 10.1007/s10654-021-00721-z) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Rastrelli G, Antonio L, Carrier S, et al. The hormonal regulation of men’s sexual desire, arousal, and penile erection: recommendations from the fifth international consultation on sexual medicine (ICSM 2024). Sex Med Rev 2025. 13 433–455. ( 10.1093/sxmrev/qeaf025) [DOI] [PubMed] [Google Scholar]
- 22.Ringel MD, Sosa JA, Baloch Z, et al. 2025 American Thyroid Association Management Guidelines for adult patients with differentiated thyroid cancer. Thyroid 2025. 35 841–985. ( 10.1177/10507256251363120) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Stein RB & Price L. Evaluation of adjusted total thyroxine (free thyroxine index) as a measure of thyroid function. J Clin Endocrinol Metab 1972. 34 225–228. ( 10.1210/jcem-34-1-225) [DOI] [PubMed] [Google Scholar]
- 24.Vermeulen A, Verdonck L & Kaufman JM. A critical evaluation of simple methods for the estimation of free testosterone in serum. J Clin Endocrinol Metab 1999. 84 3666–3672. ( 10.1210/jcem.84.10.6079) [DOI] [PubMed] [Google Scholar]
- 25.Moskovic DJ, Eisenberg ML & Lipshultz LI. Seasonal fluctuations in testosterone-estrogen ratio in men from the Southwest United States. J Androl 2012. 33 1298–1304. ( 10.2164/jandrol.112.016386) [DOI] [PubMed] [Google Scholar]
- 26.Koal T, Schmiederer D, Pham-Tuan H, et al. Standardized LC-MS/MS based steroid hormone profile-analysis. J Steroid Biochem Mol Biol 2012. 129 129–138. ( 10.1016/j.jsbmb.2011.12.001) [DOI] [PubMed] [Google Scholar]
- 27.Lovejoy JC, Smith SR, Bray GA, et al. Effects of experimentally induced mild hyperthyroidism on growth hormone and insulin secretion and sex steroid levels in healthy young men. Metabolism 1997. 46 1424–1428. ( 10.1016/s0026-0495(97)90142-6) [DOI] [PubMed] [Google Scholar]
- 28.Van Der Spoel E, Cornet S, Zutinic A, et al. Effect of thyroid status modulation on pituitary and peripheral hormone concentrations in healthy older subjects. Neuroendocrinology 2024. 115 1–12. ( 10.1159/000542832) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 29.Liu S & Sun Q. Sex differences, endogenous sex hormones, sex hormone–binding globulin, and exogenous disruptors in diabetes and related metabolic outcomes. J Diabetes 2018. 10 428–441. ( 10.1111/1753-0407.12517) [DOI] [PubMed] [Google Scholar]
- 30.Huhtaniemi IT, Tajar A, Lee DM, et al. Comparison of serum testosterone and estradiol measurements in 3174 European men using platform immunoassay and mass spectrometry; relevance for diagnostics in aging men. Eur J Endocrinol 2012. 166 983–991. ( 10.1530/eje-11-1051) [DOI] [PubMed] [Google Scholar]
- 31.Longcope C, Kato T & Horton R. Conversion of blood androgens to estrogens in normal adult men and women. J Clin Invest 1969. 48 2191–2201. ( 10.1172/JCI106185) [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.van den Beld AW, de Jong FH, Grobbee DE, et al. Measures of bioavailable serum testosterone and estradiol and their relationships with muscle strength, bone density, and body composition in elderly men. J Clin Endocrinol Metab 2000. 85 3276–3282. ( 10.1210/jcem.85.9.6825) [DOI] [PubMed] [Google Scholar]
- 33.Grasa MM, Gulfo J, Camps N, et al. Modulation of SHBG binding to testosterone and estradiol by sex and morbid obesity. Eur J Endocrinol 2017. 176 393–404. ( 10.1530/eje-16-0834) [DOI] [PubMed] [Google Scholar]
- 34.Carani C, Isidori AM, Granata A, et al. Multicenter study on the prevalence of sexual symptoms in male hypo- and hyperthyroid patients. J Clin Endocrinol Metab 2005. 90 6472–6479. ( 10.1210/jc.2005-1135) [DOI] [PubMed] [Google Scholar]
- 35.Donnelly P & White C. Testicular dysfunction in men with primary hypothyroidism; reversal of hypogonadotrophic hypogonadism with replacement thyroxine. Clin Endocrinol 2000. 52 197–201. ( 10.1046/j.1365-2265.2000.00918.x) [DOI] [PubMed] [Google Scholar]
- 36.Hekimsoy Z, Kafesçiler S, Güçlü F, et al. The prevalence of hyperprolactinaemia in overt and subclinical hypothyroidism. Endocr J 2010. 57 1011–1015. ( 10.1507/endocrj.k10e-215) [DOI] [PubMed] [Google Scholar]
- 37.Bičíková M, Hampl R, Hill M, et al. Steroids, sex hormone-binding globulin, homocysteine, selected hormones and markers of lipid and carbohydrate metabolism in patients with severe hypothyroidism and their changes following thyroid hormone supplementation. Clin Chem Lab Med 2003. 41 284–292. ( 10.1515/cclm.2003.045) [DOI] [PubMed] [Google Scholar]
- 38.Angervo M, Toivonen J, Leinonen P, et al. Thyroxine withdrawal is accompanied by decreased circulating levels of insulin-like growth factor-binding protein-1 in thyroidectomized patients. J Clin Endocrinol Metab 1993. 76 1199–1201. ( 10.1210/jcem.76.5.7684392) [DOI] [PubMed] [Google Scholar]
Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.