Insulin-like peptide 3 (INSL3) as an indicator of leydig cell insufficiency (LCI) in Middle-aged and older men with hypogonadism: reference range and threshold

Figures & data

Table 1. INSL3 Concentrations in diverse control populations of community-dwelling adult men measured using different assays.

code	matrix	number of subjects	age (mean ± SD/range)	ethnicity	country	assay type/source	INSL3 (mean ± SD/range)	reference
1	serum	40	29.4 ± 3.4	Caucasian	Italy	RIA (Phoenix)	0.56 ± 0.16	[17]
2	serum	135	19 (18-25)^a	Caucasian	Denmark	TRFIA version 1	0.99 (0.55-1.73)^b	[14]
3	serum	1183	55.0 ± 11.6	Caucasian	Australia	TRFIA version 1	35-44 yrs: 1.29 ± 0.47 75-80 yrs: 0.79 ± 0.39	[25]
4	serum	30	54.4 ± 7.0	Caucasian	Italy	EIA (Phoenix)	1.5 ± 0.7	[30]
5	serum	32	24.4 ± 3.0	Caucasian	France	EIA (Phoenix)	0.75 (0.39-1.13)^c	[31]
6	serum	71	30.0 ± 9.0	Caucasian	Italy	RIA (Phoenix)	0.66 ± 0.28	[32]
7	serum	500	29.1 (23.0-38.0)^b	Chinese	China	RIA (Phoenix)	0.94 + 0.30	[33]
8	plasma	35	32.3 ± 0.6^d	Chinese	Taiwan	RIA (Phoenix)	0.64 ± 0.06^d	[34]
9	serum	111 98	19-50 70-90	Caucasian Caucasian	New Zealand	EIA (Phoenix)	young: 1.8 ± 1.1 old: 1.0 ± 1.0	[35]
10	plasma	37	32.7 ± 0.6^d	Chinese	Taiwan	RIA (Phoenix)	0.64 ± 0.43^d	[36]
11	serum	60	33.7 ± 9.7	Caucasian	Italy	RIA (Phoenix)	0.46 ± 0.18	[37]
12	serum	675	17.8 − 60.9^a	Caucasian	Denmark	LC-MS/MS	19-40 yrs: 1.3 (0.9-2.7)^b 51-60 yrs: 1.2 (0.9-2.5)^b	[19]
13	serum	302 43	18.2 ± 0.4 39.0 ± 6.1	Caucasian Caucasian	Sweden Australia	TRFIA version 2	2.15 ± 0.86 1.78 ± 0.82	[26]
14	serum	886	31.3 ± 3.8	Chinese	China	EIA (Phoenix)	1.67 (0.11-4.77)^a	[38]
15	serum	2283	63.0 ± 10.5	Caucasian	mixed European	TRFIA version 2	0.99 ± 0.50	[24]

^a range.

^b 95% CI.

^c 5/95^th percentiles.

^d geometric mean.

Figure 1. Means and 95%CI for different age groups derived from the Swedish [26], FAMAS [25] and EMAS [24] cohorts, as indicated. Each age-range is non-overlapping, i.e. 35-45 y represents subjects aged from 35.0 to 44.9 years, etc. The horizontal black dotted line represents the 0.4 ng/ml threshold below which indicates hypogonadism. The horizontal red dashed lines indicate the means assessed by the LC-MS/MS method for a Danish cohort [19]. INSL3 concentration is represented as a Log₂ scale.

Figure 2. A. Interpolated 65-year-old means and 95% CI calculated from age vs INSL3 regressions for subjects assessed as being eugonadal (T > 10.5 nmol/l; LH ≤ 9.4 nmol/l), primary hypogonadal (T ≤ 10.5 nmol/l; LH > 9.4 nmol/l), secondary hypogonadal (T ≤ 10.5 nmol/l; LH ≤ 9.4 nmol/l), or compensated hypogonadal (T > 10.5 nmol/l; LH > 9.4 nmol/l) according to the criteria of Tajar et al. [4]. Different lower-case lettering indicates significant difference at p < 0.05 (from [10]). B. Individual INSL3 values from the second phase of the EMAS cohort from all centres plotted against age. INSL3 concentration is represented as a Log₂ scale. The magenta slope indicates the significant regression approximating a decline in INSL3 of approximately 14% per decade. The black horizontal dashed line indicates the proposed 0.4 ng/ml threshold below which hypogonadism is prevalent (from [24]).

Figure 3. Unadjusted Odds Ratios for the association of low phase 1 INSL3, T, or cFT concentrations with the incidence of various morbidities, as indicated, in the EMAS cohort in phase 2, mean 4.3 years later (adapted from [10]). Abbreviations: BDI, Beck Depression Index; PASE, Physical Activity Scale for the Elderly; CVD, cardiovascular disease; BMD, bone mineral density; SFQ-osf, sexual function questionnaire – overall sexual function.

Figure 4. Violin plots of the Overall Sexual Function (OSF) index [29], with means and quartiles (red dashed and dotted horizontal lines within the violins), for phase 2 of the EMAS cohort comparing different thresholds of INSL3, testosterone (T), or calculated free T (cFT) (as indicated on the x-axis). No shading indicates subjects below the threshold (hypogonadal), grey shading above the threshold (eugonadal). The black horizontal dashed line shows the 25th OSF percentile for all subjects. See text for further details.

Table 2. Comparisons of INSL3, testosterone (T), and calculated free T (cFT) as leydig cell biomarkers to discriminate men with functional hypogonadism (hypogonadism with sexual symptoms) from the eugonadal population (using data from phase 2 of the EMAS cohort). The OSF threshold was set at the 25^th percentile (OSF index ≤16).

discriminator	% hypogonadal	OSF threshold	false positive	false negative
INSL3 (≤0.4 ng/ml)	5.0%	25% (16)	41.0%	24.1%
INSL3 (≤0.5 ng/ml)	10.8%	25% (16)	49.3%	22.8%
T (≤10.5 nmol/l)	14.3%	25% (16)	62.4%	23.3%
cFT (<220 pmol/l)	14.7%	25% (16)	59.0%	21.5%

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