Age-dependent elevations in plasma kisspeptin are observed in boys and girls when compared with adults

Abstract

Background

Kisspeptin is a hypothalamic neuropeptide playing a physiological role in human reproduction. Genetic over-activation of kisspeptin causes precocious puberty in children. Concentrations of circulating kisspeptin are low in adults. The concentrations of plasma kisspeptin in boys and girls have not been studied previously.

Methods

Blood was obtained from 51 children and 63 adults. Plasma samples were analysed using radioimmunoassay. Children were aged 2–18 years, and attending hospital for a medically requested blood test unrelated to reproductive development. Data on pubertal status were not collected due to ethical reasons.

Results

Mean plasma kisspeptin was significantly higher in children when compared with adults (mean plasma kisspeptin in pmol/L: 12.3 ± 0.9, adults; 40.9 ± 3.3, children, P < 0.001 vs. adults). Overall mean concentrations of plasma kisspeptin were not significantly different between sexes (mean plasma kisspeptin in pmol/L: 39.5 ± 3.2, boys; 44.3 ± 6.3, girls, P = 0.48). In both sexes, concentrations of plasma kisspeptin increased with age to peak concentrations between 9 and 12 years of age, before decreasing beyond 12 years of age to adulthood. Plasma kisspeptin concentrations were highly significantly elevated in both girls and boys aged 9–12 when compared with adults (mean plasma kisspeptin in pmol/L: 59.5 ± 18.3, girls, P < 0.01 vs. adult women; 43.8 ± 6.2, boys, P < 0.001 vs. adult men).

Conclusions

We report that circulating kisspeptin is elevated in both boys and girls when compared with adults. Furthermore both boys and girls may have distinct, age-dependent concentrations of circulating kisspeptin. Further studies may determine if plasma kisspeptin could be used as a clinically useful biochemical marker of reproductive development in children.

Introduction

Puberty is triggered by gonadotrophin releasing hormone (GnRH) secretion from the hypothalamus, which stimulates luteinising hormone (LH) and follicular stimulating hormone (FSH) secretion from the pituitary, which is needed for gonadal sex steroid synthesis and mature gamete release.

Kisspeptin is a neuropeptide encoded by the KISS1 gene, and released from specialized neurons which directly innervate hypothalamic GnRH neurons. ¹ Hypothalamic expression of KISS1 and the kisspeptin receptor (KISS1R) increase during puberty in rats and monkeys. ¹ Activating and inactivating mutations in KISS1 or KISS1R cause pubertal failure and precocious puberty in children, respectively.^2,3 Intravenous administration of kisspeptin potently stimulates pituitary secretion of LH and FSH in humans; ⁴ however, this action is blocked by pre-treatment with a GnRH antagonist in other mammalian species. ⁵ Concentrations of hypothalamic kisspeptin are therefore causally associated with GnRH activation and reproductive maturation in humans.

A number of animal studies have examined concentrations of expression of KISS1 during reproductive development. However, there is a comparative paucity of data examining circulating concentrations of kisspeptin during human reproductive development. This is mainly attributable to the technical challenge of measuring plasma kisspeptin in blood samples; rapid centrifugation and separation is required in order to avoid substantial degradation of kisspeptin in blood samples. ⁶ It is therefore not possible to analyse stored serum or plasma samples retrospectively for concentrations of kisspeptin.

Kisspeptin circulates at high concentrations during pregnancy due to placental secretion (mean concentrations, 10,000 pmol/L), but at relatively low concentrations in other adults (mean concentrations, 1–10 pmol/L). ⁷

Concentrations of circulating kisspeptin are raised in girls with premature breast development. ⁸ A novel biochemical marker of pubertal activation might therefore help to manage children with pubertal disorders. This study aimed to measure for the first time, concentrations of plasma kisspeptin in boys and girls, and make comparison with circulating kisspeptin concentrations in adults.

Methods

Following ethical approval (reference:10/Q0707/4), and informed written consent, blood was obtained from 51 children attending the outpatient department of Chelsea & Westminster Hospital, London, UK. Mean age was 11.5 ± 4.6 years and 27 subjects were male. Inclusion criteria were: age < 18years; attending hospital for an outpatient blood test. Exclusion criteria were: diagnosis of malignancy; severe illness requiring inpatient admission and needle phobia. In all volunteers, medical records were analysed, and height and weight were recorded. Clinical details and pre-existing medical conditions for all child participants are summarized in Table 1. Blood was also obtained from 63 adults attending the hospital outpatient department (ethics reference: 04/Q0406/80). Exclusion criteria were identical to those used in children.

Table 1

Characteristics of subjects included in study. SDS, standard deviation score; M, male; F, female.

Subject	Sex	Age (years)	Weight (kg)	Weight SDS	Height	Height SDS	BMI	BMI SDS	Diagnosis	Medications	Medical history
1	M	1.59	10.3	−1.157	80.8	−0.678	15.7	−1.005	Ulcerative colitis	Omeprazole, Ranitidine, Domperidone	Nil
2	M	2.20	8.8	−3.658	82.9	−1.78	12.8	−3.615	Congenital malnutrition	Aspirin, Lansoprazole, Thyroxine, Sodium chloride, Iron	Congenital heart defect, hemicolectomy
3	M	3.05	19.6	2.403	106.9	3.026	17.1	0.778	Congenital hypothyroidism	Thyroxine	Nil
4	F	4.16	21.7	2.018	122	4.72	14.5	−0.817	Ulcerative colitis	Sulphasalazine	Nil
5	M	4.30	18.5	0.640	107.2	0.621	16.0	0.344	Ulcerative colitis	Prednisolone, Azathioprine, Mesalazine	Nil
6	F	5.37	20.3	0.438	112.7	0.281	15.9	0.340	Thyroid nodule	Nil	Nil
7	F	5.82	23.0	0.921	118.6	0.910	16.3	0.539	Bilateral adrenal hyperplasia	Nil	Nil
8	M	6.04	19.5	−0.541	112.8	−0.693	15.3	−0.135	Investigation for colitis	Lactulose	Nil
9	F	6.12	18.6	−0.797	109	−1.442	15.6	0.098	Coeliac disease	Nil	Nil
10	F	6.22	15.7	−2.232	138	4.339	8.2	−9.532	Hypothyroidism	Levothyroxine	Nil
11	F	6.32	22.3	0.332	119.5	0.458	15.6	0.055	Ulcerative colitis	Prednisolone, Sulphasalazine, Calcium, Omeprazole, Azathioprine	Nil
12	M	6.37	20.9	−0.25	112.7	−1.098	16.4	0.658	Congenital hypothyroidism	Thyroxine	Dyspraxia
13	F	9.15	25.3	−0.91	118.5	−2.561	18.0	0.693	Ulcerative colitis and sclerosing cholangitis	Prednisolone, Mesalazine, Omeprazole, Vitamin K, Azathioprine	Nil
14	M	9.16	29.4	0.121	132.8	−0.223	16.6	0.326	Ulcerative colitis	Prednisolone, Ferrous sulphate, Omeprazole, Sulphasalazine Azathioprine	Asthma
15	M	9.76	33.8	0.582	137.4	0.041	17.9	0.788	Crohn's disease	Nil	Nil
16	M	9.82	28.2	−0.569	133.7	−0.616	15.7	−0.34	Crohn's disease, pancreatitis	Nil	Nil
17	M	9.88	27.2	−0.859	133.3	−0.729	15.3	−0.661	Crohn's disease	Azathioprine, Sodium chloride, Calcium supplements	Colostomy, poor nutrition
18	F	10.02	61	2.801	157	2.898	24.7	2.377	Investigation for back pain	Nil	Nil
19	M	10.58	36.5	0.534	138.6	−0.427	19.0	1.039	Crohn's disease	Ferrous sulphate, Azathioprine	Orofacial granulomatosis
20	M	11.25	33.4	−0.348	148.3	0.554	15.1	−1.119	Crohn's disease	Azathioprine	Nil
21	F	11.32	35	−0.342	133	−1.843	19.7	0.815	Ulcerative colitis	Prednisolone, Mesalazine, Azathioprine, Omeprazole	Nil
22	F	11.68	59	1.989	159	1.558	23.3	1.738	Hypothyroidism	Nil	Nil
23	F	11.69	61.7	2.177	157	1.268	25.0	2.104	Investigation for Crohn's disease	Mesalazine, Ergocalciferol	Nil
24	M	12.16	57	1.858	153	0.511	24.3	2.158	Ulcerative colitis	Mesalazine, Azathioprine, Calcium	Nil
25	M	12.18	31.4	−1.312	149.1	−0.039	14.1	−2.295	Ulcerative colitis	Azathioprine, Sulphasalazine, Supplements	Nil
26	F	12.41	40	−0.31	150	−0.295	17.7	−0.266	Congenital hypothyroidism	Levothyroxine	Nil
27	M	12.45	38.1	−0.292	160.8	1.305	14.7	−1.874	Crohn's disease	Iron supplements, Azathioprine	Nil
28	M	13.17	51.9	0.871	161.5	0.689	19.8	0.732	Ulcerative colitis	Omeprazole, Calcium, Ferrous sulphate, Mesalazine	Eczema, dust allergy
29	F	13.39	35.3	−1.797	144	−1.94	17.0	−0.923	Crohn's disease	Nil	Asthma
30	M	13.47	53.5	0.819	153.5	−0.594	22.7	1.532	Congenital adrenal hyperplasia	Nil	Nil
31	M	13.49	39	−0.922	145	−1.658	18.5	0.093	Ulcerative colitis	Prednisolone
32	M	13.61	49.9	0.36	149.9	−1.162	22.2	1.368	Ulcerative colitis	Nil	Nil
33	M	13.65	41	−0.753	153	−0.819	17.5	−0.465	Crohn's disease	Metronidazole	Nil
34	M	13.68	40.8	−0.804	154	−0.723	17.2	−0.646	Ulcerative colitis	Nil	Nil
35	F	14.41	44	−1.047	150	−1.701	19.5	−0.031	Crohn's disease, osteoporosis	Nil	Nil
36	F	14.67	62.9	1.15	153.6	−1.255	26.6	1.941	Investigation for colitis	Nil	Nil
37	F	14.79	54.7	0.23	169	1.147	19.1	−0.282	Ulcerative colitis	Mesalazine	Nil
38	F	14.84	48.4	−0.604	170.3	1.341	16.6	−1.518	Ulcerative colitis	Mesalazine	Nil
39	M	15.03	62.4	0.641	176.2	0.874	20.0	0.32	Ulcerative colitis	Sulphasalazine, Calcium, Azathioprine	Asthma, H. pylori gastritis
40	M	15.15	63	0.632	157	−1.582	25.5	1.889	Crohn's disease	Prednisolone, Azathioprine, Omeprazole	Nil
41	F	16.05	60.8	0.603	162	−0.205	23.1	0.904	Ulcerative colitis	Mesalazine, Azathioprine	Nil
42	F	16.12	45	−1.563	154.8	−1.394	18.7	−0.722	Crohn's disease	Nil	Nil
43	F	16.41	47.2	−1.266	154.6	−1.45	19.7	−0.346	Crohn's disease	Iron and food supplements, Modulen liquid diet	Pyostomatitis
44	M	16.46	95	2.479	185	1.385	27.7	2.137	Acne	Isotretinoin, Salbutamol	Asthma, chronic rhinosinusitis
45	F	16.65	86	2.644	165.8	0.386	31.2	2.552	PCOS	Ferrous sulphate, Oral contraceptive pill	Asthma
46	F	16.72	82	2.364	161.5	−0.326	31.4	2.57	Vitamin D deficiency, gastro-oesophageal reflux	Ranitidine, Vitamin D	Thinning hair, Weight gain
47	F	16.76	39	−2.878	146	−2.886	18.2	−1.072	Crohn's disease, right hemicolectomy	Azathioprine, Calcium	Hayfever
48	M	16.93	61.6	−0.273	171.0	−0.663	21.0	0.237	Crohn’s disease	Inhalers	Asthma
49	M	17.54	57.2	−1.031	158.8	−2.533	22.6	0.692	Ulcerative colitis	Prednisolone, Calcium Azathioprine,Omeprazole, Calcium	Eczema
50	F	20.97	37.9	−3.464	132.1	−5.227	21.7	−0.087	Ulcerative Colitis, Sclerosing cholangitis	Prednisolone, Mesalazine, Azathioprine, Ursodeoxycholic acid	Nil
51	M	11.99	50	1.374	152.7	0.611	21.4	1.507	Indeterminate colitis	Nil	Nil

SDS: standard deviation score; M: male; F: female.

Blood was collected into lithium heparin Vacutainer tubes containing 5000 KIU aprotinin (0.2 mL Trasylol; Bayer, Newbury, UK). Samples were centrifuged immediately (3000 rotation per minute), separated and stored at −20℃ until measurement of kisspeptin immunoreactivity using a previously described, in-house radioimmunoassay based on a sheep GQ2 antibody. ⁶ Assay sensitivity was 2pmol/L, and the intra- and inter-assay coefficients of variation were 8.3% and 10.2%, respectively.

Data are presented as mean ± standard error of mean unless stated otherwise. Pairs of means were compared using the unpaired two-tailed t-test. Multiple means were compared using the one-way analysis of variance test with Tukey’s post hoc test. Linear regression was used to analyse associations between parameters. Standard deviation scores (SDS) were used to analyse height weight and body mass index (BMI) when corrected for age.

Results

The observed ranges of plasma kisspeptin concentrations were <2–32 pmol/L in adults, and <2–128 pmol/L in children (Figure 1(a)). Mean concentrations of plasma kisspeptin were significantly higher in children when compared with adults (mean concentrations of plasma kisspeptin in pmol/L: 12.3 ± 0.9, adults; 40.9 ± 3.3, children, P < 0.001 vs. adults). No significant associations were observed between concentrations of plasma kisspeptin and height SDS (r² = 0.064; P = 0.08), weight SDS (r² = 0.0091; P = 0.52) or BMI SDS (r² = 0.0090; P = 0.52) of subjects (Figure 1(b)).

Figure 1.

Concentrations of plasma kisspeptin in children and adults. (a) Concentrations of plasma kisspeptin immunoreactivity presented in children (n = 51) and adults (n = 63) attending outpatient clinic visits. Mean ± SD is presented; ***P < 0.001. (b) Scatterplot of plasma kisspeptin vs.SDS of height, weight and BMI. Linear regression (solid lines) revealed no significant associations.

No clinical data on pubertal development were available for ethical reasons; therefore, we used subject age as a clinical surrogate of physical maturation in children. As expected, subject age was highly significantly and positively correlated with height (r² = 0.71; P < 0.0001), weight (r² = 0.60; P < 0.0001) and BMI (r² = 0.38; P < 0.0001) of subjects (Figure 2(a)).

Figure 2.

Age-dependent elevations in plasma kisspeptin in children when compared with adults. (a) Scatterplot of subject age vs. height, weight and BMI. Linear regression (solid lines) revealed highly significant associations between subject age and height (P < 0001), weight (P < 0.0001) and BMI (P < 0.0001). Mean plasma kisspeptin in males (b) and females (c) when classified by age. #P < 0.05 vs. adult concentrations. ##P < 0.01 vs. adult concentrations. ###P < 0.001 vs. adult concentrations. Data are mean ± SEM.

Overall mean concentrations of plasma kisspeptin were not significantly different between sexes (mean concentrations of plasma kisspeptin in pmol/L: 39.5 ± 3.2, boys; 44.3 ± 6.3, girls, P = 0.48) (data not shown); however, when subjects were classified into both sex and age, concentrations of plasma kisspeptin appeared slightly higher in girls when compared with boys (Figure 2(b) and (c)). In both sexes, concentrations of plasma kisspeptin appeared to increase with age to peak concentrations between 9 and 12 years of age, before decreasing beyond 12 years of age to adulthood. Plasma kisspeptin concentrations were highly significantly elevated in both girls and boys aged 9–12 when compared with adults (mean concentrations of plasma kisspeptin in pmol/L: 59.5 ± 18.3, girls, P < 0.01 vs. adult women; 43.8 ± 6.2, boys, P < 0.001 vs. adult men). Beyond 9–12 years of age, concentrations of plasma kisspeptin appeared progressively lower in children aged 12–15 and 15–18 years of age (Figure 2(b) and (c)).

Discussion

Animal and human genetic studies suggest that increased hypothalamic kisspeptin signalling is necessary for, and a trigger of pubertal maturation.^1–3 We report that plasma kisspeptin is elevated in children when compared with adults, and that concentrations are most significantly elevated in children during ages commonly associated with puberty. Girls with premature breast development have been recently reported to have elevated plasma kisspeptin when compared with age and sex-matched controls. ⁹ These data collectively suggest that measurement of plasma kisspeptin could be a novel biochemical marker of reproductive development in children.

It is interesting to consider the underlying explanation for elevated concentrations of plasma kisspeptin during reproductive development. Kisspeptin is a potent activator of endogenous GnRH secretion, which is an orchestrator of pubertal development. Furthermore, it is worth noting that the observed elevation in plasma kisspeptin occurred during childhood ages commonly associated with the early stage of puberty. By contrast, established gonadal markers of puberty such as inhibin B are maximal during later stages of puberty. ⁹ Our findings therefore support the hypothesis that plasma kisspeptin may reflect a central drive to initiate pubertal maturation. However KISS1 is also expressed in the human pituitary gland ¹⁰ and ovaries ¹¹ ; circulating kisspeptin may simply be a downstream marker representing concentrations of activity of these tissues during reproductive development.

We observed a non-significant elevation of plasma kisspeptin in girls when compared with boys. Furthermore, a recent report observed that plasma kisspeptin was marginally, but significantly, elevated in girls when compared with boys. Females have a higher number of hypothalamic kisspeptin neurons and higher concentrations of KISS1 expression when compared with males, in humans. ¹² It is therefore possible that girls may have elevated concentrations of plasma kisspeptin owing to higher concentrations of hypothalamic kisspeptin signalling. These observations are consistent with a model proposing that higher concentrations of hypothalamic kisspeptin expression in girls may partially explain why they undergo earlier puberty and are more prone to precocious pubertal development when compared with boys. ¹³ Further work is required to investigate to what extent central kisspeptin signalling may influence sexual dimorphism in pubertal development.

It is important to consider the results of our study in the context of two limitations. For ethical reasons, we were unable to obtain blood from children who did not require a blood test for clinical reasons. Many children included in the study had a chronic gastrointestinal disorder, which might have affected their physical development. The current study was also limited by an absence of pubertal clinical data, due to ethical constraints. Nevertheless, our data suggest that circulating concentrations of kisspeptin are elevated during human juvenile development in an age-dependent manner, thus highlighting its potential importance as a novel clinical marker.

In summary, we report that concentrations of circulating kisspeptin are elevated in both boys and girls, and that children have higher plasma kisspeptin concentrations when compared with adults.