In a recent study published in Frontiers in Endocrinology, researchers evaluated whether insulin-like peptide 3 (INSL3) could predict hypogonadism and age-associated morbidity in older males.

INSL3 is produced in males by the Leydig cells in the testes, and unlike testosterone, it is constitutively secreted into the bloodstream independent of pituitary gonadotropins. The production of INSL3 only reflects the absolute number and mature differentiation status of Leydig cells. Mature Leydig cells decline in later age, with blood INSL3 levels decreasing at about 15% per decade from 30-40 years onwards.

About the study

In the present study, researchers assessed whether INSL3 could predict testicular function and age-related morbidity using data from the European Male Aging Study (EMAS) cohort of community-dwelling males aged 40 to 79 from eight centers across Europe. Around 1252 serum samples from four centers of the first phase (2003-04) and 2283 serum samples from all centers of the second phase (2008-10) were available for the current analysis.

Postal questionnaires were used to determine the smoking status and alcohol consumption of participants. Total testosterone was measured by gas chromatography-mass spectrometry (GC-MS). Sex hormone-binding globulin (SHBG), follicular-stimulating hormone (FSH), and luteinizing hormone (LH) were quantified by an electro-chemiluminescent immunoassay.

INSL3 was evaluated by a specific time-resolved fluorescent immunoassay. Calculated free testosterone (cFT) was derived using the Vermeulen formula. Eugonadal males had high testosterone (> 10.5 nmol/l) and low/normal LH (< 9.4 U/l). Primary hypogonadal males had low testosterone (< 10.5 nmol/l) and high LH (> 9.4 U/l).

Secondary hypogonadal males had low testosterone and low/normal LH, and compensated hypogonadal males had high levels of testosterone and LH. Frailty was determined using the Physical Activity Scale for the Elderly (PASE) index. An ultrasound examination of the left calcaneal bone was performed to assess bone mineral density (BMD).

Subjects received a personal sexual function questionnaire (SFQ) with questions related to sexual function/dysfunction, sexual distress, and recent changes in sexual function. Self-reported morbidity, including cancer, cardiovascular disease (CVD), hypertension, and diabetes, was obtained through questionnaires. The Beck Depression Inventory was used to estimate depression.

Findings

Hypogonadal males had significantly lower INSL3 concentrations than eugonadal males. Multiple regression analysis revealed that age, body mass index (BMI), and smoking negatively contributed to the variance in physical activity; age, SHBG, and smoking negatively contributed to the variance in mean BMD, whereas BMI, testosterone, INSL3, and alcohol positively contributed.

FSH, SHBG, and smoking negatively contributed to variance in bone ultrasound attenuation (BUA), while INSL3 and BMI positively contributed. Unadjusted correlation analysis revealed that age, testosterone, INSL3, LH, FSH, testosterone/LH (T/LH) ratio, cFT, BMI, and alcohol intake were associated with overall sexual function (osf), change in sexual function (csf) [except testosterone], and masturbation frequency (m) [except BMI]. BMI, smoking, and T/LH ratio correlated with sexual function distress (sfd).

In multiple regression analysis, INSL3, FSH, and BMI did not affect osf; only BMI and smoking contributed to sfd; only age and alcohol consumption contributed to csf, and only INSL3, age, and alcohol intake contributed to m. Univariate binomial logistic regression was performed to compute unadjusted odds ratios (ORs) for the association of hormone parameters with self-reported morbidities.

LH, T/LH ratio, and cFT had no significant associations with morbidity, but INSL3 showed a significant association with depression. Contrastingly, testosterone showed a significant OR for osf, hypertension, diabetes, cancer, CVD, and ≥ 2 comorbidities. Furthermore, multivariate binomial logistic regression was used to determine hormonal and other factors most likely contributing to cohort variance.

BMI and age significantly contributed to seven of nine morbidity categories; smoking, SHGB, testosterone, and alcohol intake contributed to six, five, four, and four morbidity categories, respectively. Elevated SHBG was associated with a lower risk of diabetes, CVD, and hypertension and an increased risk of depression. Increased INSL3 was associated with CVD and hypertension and not with any other categories when age, BMI, and smoking were included in the equation.

Moreover, increased INSL3 negatively contributed to BMD loss. Finally, select hormone parameters from phase 1 samples were used in unadjusted binomial logistic regression for association with morbidity in phase 2. Reduced INSL3 was associated with seven morbidity categories. In contrast, lower testosterone and higher LH predicted three and five morbidity categories, respectively. 

Conclusions

The authors observed that reduced INSL3 significantly contributed to BMD and BUA, while testosterone alone contributed to BMD. In unadjusted analysis, INSL3 was a better predictor of osf than testosterone. However, this association was attenuated when adjusted for age. Similarly, INSL3 showed a significant correlation with csf when unadjusted, but adjusting for age eliminated the correlation.

INSL3 predicted the occurrence of morbidities similarly to LH, cFT, or T/LH ratio in univariate regression analysis. Nonetheless, when multivariate regression was applied, elevated INSL3 was only significantly associated with CVD and hypertension. Overall, the findings suggested INSL3 could represent an important parameter for primary Leydig cell insufficiency and, thus, primary hypogonadism.