Ibuprofen alters human testicular physiology to produce a state of compensated hypogonadism

Abstract

Concern has been raised over increased male reproductive disorders in the Western world, and the disruption of male endocrinology has been suggested to play a central role. Several studies have shown that mild analgesics exposure during fetal life is associated with antiandrogenic effects and congenital malformations, but the effects on the adult man remain largely unknown. Through a clinical trial with young men exposed to ibuprofen, we show that the analgesic resulted in the clinical condition named "compensated hypogonadism," a condition prevalent among elderly men and associated with reproductive and physical disorders. In the men, luteinizing hormone (LH) and ibuprofen plasma levels were positively correlated, and the testosterone/LH ratio decreased. Using adult testis explants exposed or not exposed to ibuprofen, we demonstrate that the endocrine capabilities from testicular Leydig and Sertoli cells, including testosterone production, were suppressed through transcriptional repression. This effect was also observed in a human steroidogenic cell line. Our data demonstrate that ibuprofen alters the endocrine system via selective transcriptional repression in the human testes, thereby inducing compensated hypogonadism.

Keywords: endocrine disruption; endocrinology; hypogonadism; ibuprofen; reproduction.

Fig. 1.

Ibuprofen increases gonadotropins and decreases AMH in adult men. (A–D) Fold change in the Leydig cell–pituitary axis (A and C) and the Sertoli cell–pituitary axis (B and D) compared with baseline after 14 d (A and B) and at the end of the intervention after 44 d (C and D). Values are means ± SEM, and differences were analyzed with an unpaired Student’s t test. (E) Correlation between absolute ibuprofen levels in plasma (x axis) and fold change in LH levels compared with baseline levels (y axis) 14 d after intervention. The slope and P value were calculated with a Pearson product-moment coefficient correlation. AMH, anti-Müllerian hormone; Free T, free testosterone; Inh B, inhibin B; SHBG, sex hormone-binding protein; Total T, total testosterone. *P ≤ 0.05, **P ≤ 0.01.

Fig. 2.

Ibuprofen inhibits Leydig cell hormone production in human testicular explants. (A) Dose effect of ibuprofen for 24 and 48 h on testosterone production by the adult human testis. Values are means ± SEM of five independent experiments from different donors. Dose responses were analyzed for significance with the Mann–Whitney U test. The slopes and P values of these results were calculated with Spearman correlation. (B) Effect of 10⁻⁵ and 10⁻⁴ M ibuprofen on the Δ4 and Δ5 steroidogenic pathways. Steroid precursors were measured by GC-MS/MS. Values are means ± SEM of three independent experiments from different donors. Dose effects were analyzed for significance with the Mann–Whitney U test. DHEA, dehydroepiandrosterone. (C) Effect of 10⁻⁵ and 10⁻⁴ M ibuprofen on the gene expression of steroidogenic enzymes after 48-h exposure. Values are means ± SEM of five independent experiments from different donors. Each bar represents the mean ± SEM of the fold change in target gene expression relative to the reference genes BZW1 and GUSB. Dose responses were analyzed for significance with the Mann–Whitney U test. (D) Dose effect of ibuprofen for 24 and 48 h on INSL3 production by the adult human testis. Values are means ± SEM of three independent experiments from different donors. Dose responses were analyzed for significance with the Mann–Whitney U test. (E) Specific Leydig cell hormone gene expression. Values are means ± SEM of five independent experiments from different donors. Each bar represents the mean ± SEM of the fold change in target gene expression relative to the reference genes BZW1 and GUSB. Dose responses were analyzed for significance with the Mann–Whitney U test. BZW1, basic leucine zipper and W2 domains 1; CYP11A1, cytochrome P450 family 11 subfamily A member 1; CYP17A1, cytochrome P450 family 17 subfamily A member 1; CYP19A1, cytochrome P450 family 19 subfamily A member 1; GUSB, β-glucuronidase; INSL3, insulin-like factor 3; HSD17B3, hydroxysteroid 17-β dehydrogenase 3; HSD3B2, hydroxy-δ-5-steroid dehydrogenase 3 β- and steroid δ-isomerase 2; LHCGR, luteinizing hormone/choriogonadotropin receptor; StAR, steroidogenic acute regulatory protein; TSPO: translocation protein. *P ≤ 0.05, **P ≤ 0.01.

Fig. 3.

The steroid screen identifies ibuprofen as an inhibitor of steroidogenesis in the human NCI-H295R cell line. (A) Effects of ibuprofen (red trace; n = 9–18) and abiraterone (blue trace; n = 6–15) exposure on the relative steroidogenic hormone production in the dose ranges of 10⁻⁶–10⁻³ M and 3.15 × 10⁻¹¹–3.15 × 10⁻⁶ M (x axis), respectively, in the human NCI-H295R cell line, according to OECD standards. The position of each graph corresponds to the position of that particular steroid hormone in steroidogenesis. Steroid concentrations (y axis; % of control) are depicted as mean ± SEM with key enzymes shown in blue boxes. For statistics see Table S1. (B–D) Quantitative RT-PCR screen of steroidogenic gene expression in NCI-H295R cells after 48 h of culture with 10⁻⁷–10⁻³ M ibuprofen. Values are means ± SEM of three independent experiments analyzed with one-way ANOVA followed by a post hoc Dunnett’s test. CYP11A1, cytochrome P450 family 11 subfamily A member 1; CYP17A1, cytochrome P450 family 17 subfamily A member 1; CYP19A1, cytochrome P450 family 19 subfamily A member 1; HSD17B3, hydroxysteroid 17-β dehydrogenase 3; HSD3B2, hydroxy-δ-5-steroid dehydrogenase 3 β- and steroid δ-isomerase 2; StAR, steroidogenic acute regulatory protein; TSPO, translocation protein. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.

Fig. 4.

Ibuprofen affects Sertoli cell activity in human testicular explants. (A and B) Dose effect of ibuprofen on the production of inhibin B after 24 and 48 h (A) and anti-Müllerian hormone (AMH) after 48 h (B) by the adult human testis. Values are means ± SEM of three independent experiments from different donors. Slopes and P values of Spearman correlation are indicated. (C) Quantitative RT-PCR performed after 48 h of culture treated with 10⁻⁵ and 10⁻⁴ M ibuprofen for specific Sertoli cell gene expression. Values are means ± SEM of five independent experiments from different donors. Each bar represents the mean ± SEM of the fold change in target gene expression relative to the reference genes BZW1 and GUSB. Dose responses were analyzed for significance with the Mann–Whitney U test. AMH, anti-Müllerian hormone; BZW1, basic leucine zipper and W2 domains 1; FSHR, follicle-stimulating hormone receptor; GUSB, β-glucuronidase; INHBB, inhibin B subunit B; LAMA5, laminin subunit α5. *P ≤ 0.05, **P ≤ 0.01.

Fig. 5.

Ibuprofen decreases gene expression in peritubular cells but does not affect germ cells or morphology in human testicular explants. (A and B) Quantitative RT-PCR performed after 48 h of culture treated with 10⁻⁵ and 10⁻⁴ M ibuprofen for gene expression in peritubular cells (A) and germ cells (B). Each bar represents the mean ± SEM of the fold change in target gene expression relative to the reference genes BZW1 and GUSB. Values are means ± SEM of five independent experiments from different donors. A Mann–Whitney U test was performed. (C) Number of apoptotic germ cells. Values are means ± SEM of caspase⁺ cells in three independent experiments from different donors. (D) Immunostaining of apoptotic germ cells in testis explants cultured for 48 h in the presence of DMSO (control) or 10⁻⁵ or 10⁻⁴ M ibuprofen. Each micrograph shows representative areas of ibuprofen-induced morphology compared with corresponding area. (Scale bars: 50 µm.) ACTA2, actin α2 smooth muscle aorta; ALPP, alkaline phosphatase, placental; BZW1, basic leucine zipper and W2 domains 1; GUSB, β-glucuronidase; KCNIP4, potassium voltage-gated channel interacting protein 4; MYH11, myosin heavy polypeptide 11, smooth muscle; PGK2, phosphoglycerate kinase 2; PRM2, protamine 2; THY1, Thy-1 cell-surface antigen. **P ≤ 0.01.

Fig. 6.

Ibuprofen decreases PGE2 and PGD2 production and PTGS gene expression in human testicular explants. (A and B) Dose effect of ibuprofen exposure after 24 and 48 h on PGD2 (A) and PGE2 (B) production by adult human testicular explants. Values are means ± SEM of five independent experiments from different donors. Dose responses were analyzed for significance with the Mann–Whitney U test. Slopes and P values of Spearman correlation are indicated. (C) Quantitative RT-PCR was performed after 48 h of culture treated with 10⁻⁵ and 10⁻⁴ M ibuprofen. Each bar represents the mean ± SEM of the fold change in target gene expression relative to the reference genes BZW1 and GUSB. Values are means ± SEM of five independent experiments from different donors. Differences in gene expression were analyzed with a Mann–Whitney U test. BZW1, basic leucine zipper and W2 domains 1; GUSB, β-glucuronidase; PTGS, prostaglandin-endoperoxide synthase. *P ≤ 0.05, **P ≤ 0.01.

Fig. 7.

Ibuprofen dose-dependently reduces prostaglandin levels and mRNA expression in human endocrine NCI-H295R cells. (A) Effects of ibuprofen on general prostaglandin production from NCI-H295R cells after 24 h. Values are means ± SEM of three independent experiments analyzed with one-way ANOVA followed by a post hoc Dunnett’s test. (B) Quantitative RT-PCR screen of steroidogenic and PTGS gene expression in NCI-H295R cells after 48 h of culture with 10⁻⁷–10⁻³ M ibuprofen. Values are means ± SEM of three independent experiments analyzed with one-way ANOVA followed by a post hoc Dunnett’s test. PTGS, prostaglandin-endoperoxide synthase. *P ≤ 0.05, **P ≤ 0.01.