Growth Hormone (GH) Treatment Decreases Plasma Kisspeptin Levels in GH-Deficient Adults with Prader–

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Clinical Medicine Article

Growth Hormone (GH) Treatment Decreases Plasma Kisspeptin Levels in GH-Deficient Adults with Prader–Willi Syndrome Olga Giménez-Palop 1,2,3,† , Laia Casamitjana 1,2,3,† , Raquel Corripio 2,3,4 , Susanna Esteba-Castillo 5,6 , Rocío Pareja 1,2 , Néstor Albiñana 2 , Mercedes Rigla 1,2,3 and Assumpta Caixàs 1,2,3, * 1

2

3 4 5

6

* †

Citation: Giménez-Palop, O.; Casamitjana, L.; Corripio, R.; Esteba-Castillo, S.; Pareja, R.; Albiñana, N.; Rigla, M.; Caixàs, A. Growth Hormone (GH) Treatment Decreases Plasma Kisspeptin Levels in GH-Deficient Adults with Prader–Willi Syndrome. J. Clin. Med. 2021, 10, 3054. https://doi.org/ 10.3390/jcm10143054 Academic Editor: Claudia Giavoli Received: 12 June 2021 Accepted: 8 July 2021 Published: 9 July 2021

Endocrinology and Nutrition Department, Hospital Universitari Parc Taulí, 08208 Sabadell, Spain; ogimenez@tauli.cat (O.G.-P.); lcasamitjana@tauli.cat (L.C.); rpareja@tauli.cat (R.P.); mrigla@tauli.cat (M.R.) Institut d’Investigació i Innovació Parc Taulí (I3PT), 08208 Sabadell, Spain; rcorripio@tauli.cat (R.C.); nalbinana@tauli.cat (N.A.) Department of Medicine, Universitat Autònoma de Barcelona, 08208 Sabadell, Spain Pediatrics Department, Hospital Universitari Parc Taulí, 08208 Sabadell, Spain Specialized Service in Mental Health and Intellectual Disability, Institut Assistència Sanitària (IAS), Parc Hospitalari Martí i Julià, 17190 Salt, Spain; susanna.esteba@ias.cat Neurodevelopment Group (Girona Biomedical Research Institute)—IDIBGI, Institute of Health Assistance (IAS), Parc Hospitalari Martí i Julià, 17190 Salt, Spain Correspondence: acaixas@tauli.cat Both authors contributed equally.

Abstract: Obesity and growth hormone (GH)-deficiency are consistent features of Prader–Willi syndrome (PWS). Centrally, kisspeptin is involved in regulating reproductive function and can stimulate hypothalamic hormones such as GH. Peripherally, kisspeptin signaling influences energy and metabolic status. We evaluated the effect of 12-month GH treatment on plasma kisspeptin levels in 27 GH-deficient adult PWS patients and analyzed its relationship with metabolic and anthropometric changes. Twenty-seven matched obese subjects and 22 healthy subjects were also studied.

Before treatment, plasma kisspeptin concentrations in PWS and obese subjects were similar (140.20 (23.5–156.8) pg/mL vs. 141.96 (113.9–165.6) pg/mL, respectively, p = 0.979)) and higher (p = 0.019) than in healthy subjects (124.58 (107.3–139.0) pg/mL); plasma leptin concentrations were similar in PWS and obese subjects (48.15 (28.80–67.10) ng/mL vs. 33.10 (20.50–67.30) ng/mL, respectively, p = 0.152) and higher (p < 0.001) than in healthy subjects (14.80 (11.37–67.30) ng/mL). After GH therapy, lean body mass increased 2.1% (p = 0.03), total fat mass decreased 1.6% (p = 0.005), and plasma kisspeptin decreased to levels observed in normal-weight subjects (125.1(106.2–153.4) pg/mL, p = 0.027). BMI and leptin levels remained unchanged. In conclusion, 12-month GH therapy improved body composition and decreased plasma kisspeptin in GH deficient adults with PWS. All data are expressed in median (interquartile range).

Keywords: Prader–Willi Syndrome; kisspeptin; leptin; growth hormone deficiency

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1. Introduction Kisspeptin is a hormone that promotes the onset of puberty by stimulating the secretion of gonadotropin-stimulating hormone (GnRH). Kisspeptin can also stimulate the release of other pituitary hormones such as prolactin, growth hormone (GH), oxytocin, and vasopressin [1].

Kisspeptin is mainly synthesized by neurons in the arcuate nucleus of the hypothalamus. Its synthesis is modulated by energy balance, decreasing in situations of insufficient weight (e.g., anorexia nervosa) or excess weight (e.g., obesity) [2]. Low kisspeptin levels are considered the cause of the hypogonadotropic hypogonadism seen in some patients with metabolic syndrome and obesity [2,3].

Kisspeptin-producing neurons express receptors for leptin, a hormone produced in adipose tissue that informs these neurons about the status of energy reserves [4,5].

J. Clin. Med. 2021, 10, 3054. https://doi.org/10.3390/jcm10143054

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J. Clin. Med. 2021, 10, 3054

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Kisspeptin is also expressed in peripheral tissues involved in metabolic functions such as the pancreas, liver, and adipose tissue, and emerging data suggests peripheral kisspeptin plays a role in the regulation of insulin secretion [6].

Prader–Willi Syndrome (PWS) is a rare genetic disease characterized by a series of disorders of hypothalamic functionalism: hyperphagia, obesity, hypogonadotropic hypogonadism, short stature due to GH deficiency [7–9], and cognitive, praxis, and behavioral disorders [10–12]. Up to 60% of adults with PWS have GH deficiency [13], which results in decreased muscle mass and strength and increased fat mass [14]. These changes can be mitigated with GH treatment, which induces metabolically beneficial changes in body composition [15,16].

Kisspeptin concentrations are decreased in patients with idiopathic hypogonadotropic hypogonadism, diabetes, or metabolic syndrome [2,4,5]. To our knowledge, kisspeptin has not been studied in patients with PWS; however, since these patients have impaired hypothalamic function, obesity (often associated with diabetes), central hypogonadism, and GH deficiency, they would be expected to have decreased kisspeptin levels. Moreover, it remains to be determined whether GH treatment could influence kisspeptin synthesis directly through a negative feedback mechanism or indirectly through changes in body composition.

We aimed to determine changes in plasma kisspeptin concentrations in 27 adult patients with PWS after 12 months of GH treatment and whether these changes are associated with changes in anthropometric or metabolic parameters.

2. Materials and Methods 2.1. Subjects We included 27 adults with PWS (15 women, median age: 26 years, range: 18–53) treated at our center between 1 January 2016 and 31 January 2019. Cytogenetic analysis revealed 7 had type 1 deletion, 10 had type 2 deletion, 6 had maternal uniparental disomy, 3 had imprinting defects and 1 had an atypical BP2-BP4 microdeletion. All had GH deficiency diagnosed with GHRH-arginine and/or glucagon stimulation tests [17–19]. The cut off for GH deficiency with GHRH-arginine test was: GH < 11 ng/mL if BMI < 25 kg/m2 , <8 ng/mL if BMI 25–30 kg/m2 , and <4 ng/mL if BMI ≥ 30 kg/m2 . The cut off for glucagon test was GH < 3 ng/mL at any time point.

Six patients had type 2 diabetes with good glycemic control (HbA1c < 7.5%) and 15 (7 women) were receiving sex steroids. None had precocious puberty. For comparison, we also evaluated 27 obese subjects matched for age, sex, and BMI and 22 healthy subjects.

The study complied with all provisions in the Declaration of Helsinki and was approved by the local ethics committee (Comitè d’Ètica d’Investigació amb medicaments del Parc Taulí). All patients with PWS agreed to participate after being informed together with their parents or caregivers; their legal guardians (usually their parents) signed the consent form before enrollment. All control participants provided written informed consent.

2.2. Methods Blood was extracted from all participants at 8 AM after overnight fasting. Plasma samples for kisspeptin and leptin measurements were kept at −80 ◦ C until analysis. We recorded subjects’ height, determined by a stadiometer (Harpenden, Holtain Ltd., Dyfed, UK); body weight, measured to the nearest 0.1 kg with standard equipment; body mass index (BMI); and body composition determined by dual-energy x-ray absorptiometry (Lunar Prodigy−963, Chicago, IL, USA).

To determine concentrations of kisspeptin-1 in plasma, we used the Kisspeptin-1 ELISA kit (Cloud-Clone Corp., Houston, TX, USA) (lower limit of detection, 9.27 pg/mL; intraassay coefficient of variation (CV) <10%; interassay CV < 12%). To determine concentrations of leptin in plasma, we used the Human Leptin ELISA Kit (Biorbyt, Cambridge, UK) (lower limit of detection, 10 pg/mL; intraassay CV < 7.6%; interassay CV < 8.4%). A routine automated analyzer was used for other laboratory tests. The homeostatic model as-

nar Prodigy−963, Chicago, IL, USA). recorded subjects’ height, determined a stadiometer (Harpenden, Holtain Dyfed, To determine concentrations of by kisspeptin-1 in plasma, we used the Ltd., Kisspeptin-1 2.2. Methods UK); body weight, measured to Houston, the nearest 0.1USA) kg with standard equipment; J. Clin. Med. 2021, 10, x FOR PEER REVIEW 3mass of 11 ELISA kit (Cloud-Clone Corp., TX, (lower limit of detection, body 9.27 pg/ml; Blood was extracted from all determined participantsby atdual-energy 8 AM after x-ray overnight fasting. Plasma index (BMI); and body composition absorptiometry (Luintraassay coefficient of variation (CV) <10%; interassay CV < 12%). To determine concensamples for kisspeptin andIL,leptin measurements were kept at −80 °C until analysis. We nar Prodigy−963, USA). trations of leptin Chicago, in plasma, we used the Human Leptin ELISA Kit (Biorbyt, Cambridge, J. Clin. Med. 2021, 10, 3054 3 of 11 recorded subjects’ height, determined by a stadiometer (Harpenden, Holtain Ltd., Dyfed, To determine concentrations of kisspeptin-1 plasma, used theCV Kisspeptin-1 UK) (lower limit of detection, 10 pg/ml; intraassayinCV < 7.6%;we interassay < 8.4%). A 2.2. Methods UK); body weight, measured toHouston, the nearest 0.1 kg with standard equipment; body mass ELISA kit (Cloud-Clone Corp., USA) (lower limit ofThe detection, 9.27 pg/ml; routine automated analyzer was forTX, other laboratory tests. homeostatic model Blood was extracted from allused participants at 8 AM after overnight fasting. Plasma index (BMI); and body composition determined by dual-energy x-ray absorptiometry (Luintraassay ofresistance variation (CV) <10%; interassay < 12%). To determine concenassessmentcoefficient for insulin (HOMA-IR) index wasCV calculated as fasting plasma glusamples for kisspeptin andIL, leptin measurements were kept at −80 °C until analysis. We nar Prodigy−963, Chicago, USA). trations of leptin in plasma, we used the Human Leptin ELISA Kit (Biorbyt, Cambridge, cose (mmol/L) x fasting insulin (µIU/mL))/22.5 [20]. sessment for insulin resistance (HOMA-IR) index was calculated as fasting plasma glucose recorded subjects’ height, determined by a stadiometer (Harpenden, Holtain Ltd., Dyfed, To determine concentrations of kisspeptin-1 in we used the UK) (lower limit of PWS detection, 10 pg/ml; intraassay CVplasma, < 7.6%; interassay CVKisspeptin-1 < 8.4%). A®, Patients with were treated with recombinant GH (Genotonorm Miniquick (mmol/L) × fasting insulin (µIU/mL)/22.5 [20].

UK); body weight, measured toHouston, the nearest 0.1 kg with standard equipment; bodypg/ml; mass® ELISA kit (Cloud-Clone Corp., TX, USA) (lower limit of detection, 9.27 routine automated analyzer was used with for other laboratory tests.

The homeostatic model Pfizer, New York, NY, USA), starting a dose of 0.2mg/day and adjusting the dose at , Patients with PWS were treated with recombinant GH (Genotonorm Miniquick index (BMI); and body composition determined by dual-energy x-ray absorptiometry (Luintraassay coefficient of variation(HOMA-IR) (CV) <10%; index interassay CV < 12%).as Tofasting determine concenassessment for insulin resistance was calculated plasma glu-at 1, 3, 6, and 12 months to achieve high-normal insulin-like growth factor-1 (IGF-1) levels Pfizer, New York, NY, USA), starting with a dose of 0.2 mg/day and adjusting the dose nar Prodigy−963, Chicago, IL, USA). trations of leptinfasting in plasma, we used the Human Leptin ELISA Kit (Biorbyt, Cambridge, cose (mmol/L) insulin (µIU/mL))/22.5 [20].

1, 3, 6,patient’s and 12xmonths achieve high-normal insulin-like growth factor-1 (IGF-1) levels for for the age. to To determine concentrations of kisspeptin-1 in plasma, we used theCV Kisspeptin-1 UK) (lower limit of detection, 10 pg/ml; intraassay CV < 7.6%; interassay < 8.4%). ®A Patients with PWS were treated with recombinant GH (Genotonorm Miniquick , the Only patient’s age.

PWS patients received GH treatment.

After 12 months’ GH treatment, patients’ ELISA kit (Cloud-Clone Corp., Houston, TX, USA) (lower limit of detection, 9.27 pg/ml; routine automated analyzer was used for other laboratory tests.

The homeostatic model Pfizer, New York, NY, USA), starting with a dose of 0.2mg/day and adjusting the dose at Only PWS patients received GH treatment.

After 12again months’ treatment, patients’ analytic and anthropometric parameters wereinterassay measured withGH thedetermine same protocol. intraassay coefficient variation (CV) <10%; < 12%).

To concenassessment formonths insulinof resistance (HOMA-IR) wasCV calculated as fasting plasma glu1, 3, 6, andand 12 to achieve high-normal insulin-like growth factor-1 (IGF-1) levels analytic anthropometric parameters wereindex measured again with the same protocol. trations of leptin in plasma, we(µIU/mL))/22.5 used the Human Leptin ELISA Kit (Biorbyt, Cambridge, cose (mmol/L) x fasting insulin [20]. for patient’s age.

2.3.the Statistical Analyses UK) (lower limit ofPWS detection, pg/ml; intraassay CV < 7.6%; interassay CVMiniquick < 8.4%). A ®, Patients with were 10 treated with recombinant GH (Genotonorm 2.3.Only Statistical Analyses PWS patients received GH treatment.

Afterand 12 months’ GH treatment, patients’ Continuous variables are reported as medians interquartile ranges (IQR).

CateroutineNew automated analyzer was usedwith for other laboratory tests.and Theadjusting homeostatic model Pfizer, York, NY, USA), starting a dose of 0.2mg/day the dose at analytic and anthropometric parameters were measured again with the same protocol.

Continuous variables are reported as medians and interquartile ranges (IQR).

Categorgorical variables are reported as frequencies and percentages.

To compare all the continassessment formonths insulin resistance (HOMA-IR) index was calculated as fasting plasma glu1, 3, 6, and 12 to achieve high-normal insulin-like growth factor-1 (IGF-1) levels ical variables are reported as frequencies and percentages.

To compare all the continuous uous variables xatfasting baseline, between the 3 groups, we used Kruskal–Wallis test followed cose (mmol/L) insulin (µIU/mL))/22.5 [20]. for the patient’s age. between the 3 groups, we used Kruskal–Wallis test followed by the 2.3.

Statistical variables at Analyses baseline, by the Mann–Whitney test with Bonferroni correction. To all variables before ®, Patients with PWSUreceived were treated with recombinant GHcompare (Genotonorm Miniquick Only PWS patients GH treatment.

After 12interquartile months’ GHranges treatment, patients’ Mann–Whitney U test with Bonferroni correction.

To compare allTo variables before and after Continuous variables are reported as medians and (IQR).

Cateand after GH treatment we used the Wilcoxon signed-rank test. study the relationship Pfizer, New NY, USA),parameters starting with a dose of 0.2mg/day and the adjusting the dose at analytic and York, anthropometric were measured again with same protocol.

GH treatment we used theSpearman's Wilcoxon signed-rank test.

To study the relationship between gorical variables arewe reported as frequencies and percentages.

To compare all significance the continbetween variables, used rank-order correlation test.

Statistical 1, 3, 6, and 12 months to achieve high-normal insulin-like growth factor-1 (IGF-1) levels variables, we used Spearman’s rank-order correlation test.

Statistical significance was fixed uous variables baseline, betweenwere the done 3 groups, used Kruskal–Wallis test followed was fixed at pAnalyses <at0.05.

All analyses withwe IBM SPSS Statistics for Windows, verfor the patient’s age.

2.3.

Statistical at p < 0.05.

All analyses were done with IBM SPSS Statistics for Windows, version 25.0 by the25.0 Mann–Whitney U test with Bonferroni correction. To compare all variables before sion (IBM Corp., Armonk, NY, Only PWS patients received GHUSA). treatment.

After 12 interquartile months’ GH treatment, patients’ (IBM Corp., Armonk, NY, USA).

Continuous variables are reported as medians and ranges (IQR).

Cateand after GH treatment we used the Wilcoxon signed-rank test. To study the relationship analyticvariables and anthropometric parameters wereand measured again To with the same protocol. gorical as frequencies percentages. compare the continbetween variables,are wereported used Spearman's rank-order correlation test.

Statisticalallsignificance 3.3.Results Results uous variables at baseline, between thedone 3 groups, we used Kruskal–Wallis test followed was fixed at p < 0.05.

All analyses were with IBM SPSS Statistics for Windows, ver2.3.

Statistical Analyses 3.1.

Baseline Findings 3.1.

Baseline Findings by the Mann–Whitney U test with Bonferroni correction.

To compare all variables before sion 25.0 (IBM Corp., Armonk, NY, USA).

Continuous variables reported as medians and interquartile ranges Cate3.1.1.

Patient 3.1.1.

Patient Characteristics and after GH Characteristics: treatment weare used the Wilcoxon signed-rank test. To study the (IQR). relationship gorical variables are reported as frequencies and percentages.

To compare all the continbetween variables, we used Spearman's rank-order correlation test.

Statistical significance Table 1 reports the baseline characteristics of subjects in the PWS, obese, and normal Table 1 reports the baseline characteristics of subjects in the PWS, obese, and normal 3. Results uous variables baseline, between the done 3 groups, used Kruskal–Wallis testpercentages followed was fixed atFindings p <at 0.05.

All analyses were with IBM SPSS Statistics for Windows, verweight groups.

The PWS and groups did not differ ininweight, BMI, waist, weight groups.

The PWS and obese groups did notwe differ weight, BMI, waist, percent3.1. Baseline by the Mann–Whitney U test with Bonferroni correction.

To compare all variables before sion 25.0 (IBM Corp., Armonk, NY, USA).HOMA-IR, of body fat and lean glucose, HOMA-IR, follicle-stimulating hormone, or testosterone ages of body fat and mass, lean mass, glucose, follicle-stimulating hormone, or tes3.1.1.

Patient Characteristics: and after GH treatment welevels. the Wilcoxon signed-rank test.

Tolower study the relationship (only measured in men) Females in the PWS group had levels oflevels estradiol tosterone (only measured inused men) levels.

Females in the PWS group had lower of between variables, we Spearman's correlation test.

Statistical significance Table 1 reports theused baseline characteristics ofinsubjects in the PWS, obese, and normal 3.

Results and luteinizing hormone than females in the obese group. estradiol and luteinizing hormone than rank-order females the obese group. wasBaseline fixed atFindings p <The 0.05.

All and analyses done with SPSS Statistics for waist, Windows, verweight groups.

PWS obesewere groups did notIBM differ in weight, BMI, percent3.1. sion of 25.0 (IBM Armonk, USA).

Table 1.and Baseline characteristics participants. follicle-stimulating hormone, or tesages body fat lean mass, NY, glucose, HOMA-IR, Table 1.Corp., Baseline characteristics ofofparticipants.

3.1.1. Patient Characteristics: tosterone (only measured in men) levels. Females in the PWS group had lower levels of PWS (n 27) Obese Obese Subjects (n 27) Healthy Subjects (n(n = 22) p-Value .

PWS Patients (n 27) Subjects (n ==females 27) Healthy Subjects = 22) Table 1= = reports the baseline characteristics of ingroup. the PWS, obese,p-Value and normal 3. Patients Results estradiol and luteinizing hormone than in subjects the obese SexSex (female) 15 (55.6%) 15 (55.6%) 11(50%) (50%) * percent0.907 weight groups.

The PWS and obese groups did not differ in weight, BMI, waist, (female) 15 (55.6%) 15 (55.6%) 11 * 0.907 3.1. Baseline Findings ages of body fat and lean mass, glucose, HOMA-IR, follicle-stimulating hormone, Age (years) 26 (24–37) 28 (24–37) 27.5 (21.5–37.5) * 0.909 Table 1.

Baseline characteristics of participants.

Age (years) 26 (24–37) 28 (24–37) 27.5 (21.5–37.5) * 0.909or tes3.1.1.

Patient Characteristics: tosterone (only measured in men) levels. Females in the PWS group had lower levels of * <0.001 *and <0.001 .

PWS Patients (n = 27) Obese Subjects (n = 27) Healthy Subjects (n = 22) p-Value Table 1 reports the baseline characteristics of subjects in the PWS, obese, normal estradiol and luteinizing hormone than females in the obese group. ⴕ 0.135 0.135 Sex Weight (female) 15 (55.6%) 15 (55.6%) 11(58.1–71.6) (50%) weight groups. The PWS95.5 and obese groups did not 65.8 differ in weight, BMI, waist,* 0.907 percent(kg) 89.6 (70.5–105.5) (81.8–121.1) Weight (kg) 89.6 (70.5–105.5) 95.5 (81.8–121.1) 65.8 (58.1–71.6) λλ<0.001 <0.001** ages of body and leancharacteristics mass, glucose, HOMA-IR, follicle-stimulating hormone, Age (years) 26 (24–37) 28 (24–37) 27.5 (21.5–37.5) * 0.909or tesTablefat 1. Baseline of participants. **<0.001 <0.001 tosterone (only measured in men) levels. Females in the PWS group had lower levels of * <0.001 * <0.001 .

PWS Patients = 27) Obesehormone Subjectsthan (n =females 27) Healthy Subjects (n = 22) p-Value estradiol(nand luteinizing in the obese group. ⴕ <0.001 0.135 * <0.001 SexHeight (female) 15 (55.6%) 15(162.0–173.0) (55.6%) 11 (50%) * 0.907 (cm) (148.0–165.0) 169 169 (164.5–181.5) Height (kg) (cm) 156156 (148.0–165.0) 169 (162.0–173.0) 169 Weight 89.6 (70.5–105.5) 95.5 (81.8–121.1) 65.8(164.5–181.5) (58.1–71.6) <0.001 λλ <0.001 ⴕ <0.001 Age (years) 26 (24–37) 28 (24–37) of participants. 27.5 (21.5–37.5) * 0.909 Table 1. Baseline characteristics 0.344 ****<0.001 * <0.001 * <0.001 .

PWS Patients (n = 27) Obese Subjects (n = 27) Healthy Subjects (n = 22) p-Value ⴕ 0.580 0.135 2) * <0.001 SexBMI (female) 15 (55.6%) 15 (55.6%) (50%) * 0.907 34.6 (30.9–41.3) 32.1 (29.3–41.9) 22.211 (20.9–22.9) (kg/m Height 156 169 169 (164.5–181.5) Weight(cm) (kg) 89.6(148.0–165.0) (70.5–105.5) 95.5(162.0–173.0) (81.8–121.1) 65.8 (58.1–71.6) λλ<0.001 <0.001** ⴕ <0.001 Age (years) 26 (24–37) 28 (24–37) 27.5 (21.5–37.5) * 0.909 **<0.001 <0.001 * <0.001 * <0.001 ⴕ 0.387 0.135 * <0.001 Waist (cm) 110.0 (101.0–124.0) 107.0 (99.0–121.0) 78.5 (71.5–83.7) Height 156 169 169 Weight (cm) (kg) 89.6(148.0–165.0) (70.5–105.5) 95.5(162.0–173.0) (81.8–121.1) 65.8(164.5–181.5) (58.1–71.6) λλ<0.001 ** <0.001 ⴕ <0.001 **<0.001 <0.001 Height (cm)

156 (148.0–165.0)

169 (162.0–173.0)

169 (164.5–181.5)

* <0.001 ⴕ <0.001

3. Results routine automated analyzer was for aother laboratory tests.

The homeostatic model Pfizer, New York, NY, USA), starting with dose ofdual-energy and adjusting the dose at Blood was extracted from allused participants at 8 0.2mg/day AM after overnight fasting.

Plasma analytic and anthropometric parameters measured again withTo protocol.

3the of same 11 uous variables at baseline, between the 3were groups, we used Kruskal–Wallis test followed index (BMI); and body composition determined by x-ray absorptiometry (Luwas fixed at p < 0.05.

All analyses were done with IBM SPSS Statistics for Windows, verintraassay coefficient of variation (CV) <10%; interassay CV < 12%). determine concenweight groups.

The PWS and obese groups did not differ in weight, BMI, waist, percentassessment for insulin resistance (HOMA-IR) index was calculated as fasting plasma glu3.1.

Baseline Findings 1, 3, 6, and 12 months to achieve high-normal insulin-like growth factor-1 (IGF-1) levels samples for kisspeptin and leptin measurements were kept at −80 °C until analysis.

We by the U test with Bonferroni correction.

To compare allhormone, variables before nar Prodigy−963, Chicago, IL, USA). sion 25.0 (IBM Corp., Armonk, NY, USA). trations of leptin in plasma, we used the HOMA-IR, Human Leptin ELISA Kit (Biorbyt, Cambridge, ages ofMann–Whitney body fat and lean mass, glucose, follicle-stimulating or tescose (mmol/L) xage. fasting insulin (µIU/mL))/22.5 [20]. for patient’s 2.3.the Statistical Analyses 3.1.1.

Patient Characteristics: recorded subjects’ height, determined by a stadiometer (Harpenden, Holtain Ltd., Dyfed, and after GH treatment we used the Wilcoxon signed-rank test.

To study the relationship To determine concentrations of kisspeptin-1 in plasma, we used the Kisspeptin-1 UK) Patients (lower limit ofPWS detection, 10 pg/ml; intraassay 7.6%; interassay CVMiniquick < 8.4%).

A ®, tosterone (only measured in men) levels.

Females inCV the< PWS group had lower levels of with were treated with recombinant GH (Genotonorm Only PWS patients received GH nearest treatment.

After 12 months’ GH treatment, patients’ UK); body measured toreported the 0.1 kgsubjects with standard equipment; body mass between variables, we Spearman's rank-order correlation test.

Statistical kit (Cloud-Clone Corp., Houston, USA) (lower limit of detection, 9.27 pg/ml; Continuous variables are asTX, medians and interquartile ranges (IQR).

CateTable 1weight, reports theused baseline characteristics of in the PWS, obese, significance and normal Results routine automated analyzer was used for other laboratory tests.

The homeostatic model 2.2. Methods 3.ELISA estradiol and luteinizing hormone than females in the obese group.

Pfizer, New York, NY, composition USA),parameters starting with a dose of 0.2mg/day andthe adjusting the dose at analytic and anthropometric were measured again with same index (BMI); body determined by dual-energy x-ray absorptiometry (LuJ. Clin. Med. 2021, 10, 3054 4verof 11 was fixed at pand <insulin 0.05.

All analyses were done with IBM Statistics for Windows, gorical variables arePWS reported as (HOMA-IR) frequencies and percentages.

To compare allprotocol. thepercentcontinintraassay coefficient ofresistance variation (CV) <10%; interassay CV 12%).

To determine concenweight groups.

The and obese groups did not differ in<weight, BMI, waist, assessment for index wasSPSS calculated as fasting plasma glu3.1.

Baseline Findings 1, 3, 6, and 12 months to achieve high-normal insulin-like growth factor-1 (IGF-1) levels Blood was extracted from all participants at 8 AM after overnight fasting.

Plasma nar Prodigy−963, Chicago, IL, USA). sion 25.0 (IBM Armonk, NY, USA). uous baseline, between the 3HOMA-IR, groups, we used Kruskal–Wallis test followed trations of leptin in plasma, we used theparticipants.

Human Leptin ELISA Kit (Biorbyt, Cambridge, ages ofvariables body fat and lean mass, glucose, follicle-stimulating hormone, or tesTable 1.Corp., Baseline characteristics of cose (mmol/L) xat fasting insulin (µIU/mL))/22.5 [20]. for the patient’s age. samples for kisspeptin andCharacteristics: leptin measurements were kept at −80 °C until analysis.

We 2.3.

Statistical Analyses 3.1.1.

Patient To determine concentrations of kisspeptin-1 in plasma, we used the Kisspeptin-1 by the Mann–Whitney U test with Bonferroni correction.

To compare all variables before UK) (lower limit of detection, 10 pg/ml; intraassay CV < 7.6%; interassay CV < 8.4%).

A ®, tosterone (only measured in men) levels.

Females in the PWS group had lower levels of Patients with PWSby were treated with recombinant GH Ltd., (Genotonorm Miniquick Only PWS patients received GH(ntreatment.

After 12 months’ GH treatment, patients’ recordedPWS subjects’ height, a stadiometer (Harpenden, Holtain Dyfed, .

Patients (n =determined 27) Obese Subjects =for 27) Healthy Subjects (n =detection, 22) p-Value ELISA kit Corp., Houston, TX, USA) (lower of 9.27 pg/ml; Continuous variables are reported as medians and interquartile ranges (IQR).

CateTable 1(Cloud-Clone reports the baseline characteristics of subjects inlimit the PWS, obese, and normal 3.

Results and after GH treatment we used the Wilcoxon signed-rank test.

To study the relationship routine automated analyzer was used other laboratory tests.

The homeostatic model estradiol and luteinizing hormone than females in the obese group.

Pfizer, New York, NY,nearest USA), starting with a dose ofequipment; 0.2mg/day and the adjusting the dose at analytic and anthropometric parameters were measured again with same protocol.

UK); body weight, measured toare the 0.1 kg with standard body mass Table 1. Cont.

Sex (female) 15 (55.6%) 15Spearman's (55.6%) (50%) *percent0.907 gorical variables reported as frequencies and percentages.

To compare all plasma the continintraassay coefficient ofresistance variation (CV) <10%; interassay CV <weight, 12%).

To determine concenweight groups.

The PWS and obese groups did not differ in BMI, waist, between variables, we used rank-order correlation test.

Statistical significance assessment for insulin (HOMA-IR) index was11 calculated as fasting glu3.1.

Baseline Findings 1, 3, 6, and 12 months to achieve high-normal insulin-like growth factor-1 (IGF-1) levels index (BMI); and body composition determined by the dual-energy x-ray absorptiometry (Luuous variables at baseline, between 3 groups, we used Kruskal–Wallis test followed trations of leptin in plasma, we used the Human Leptin ELISA Kit (Biorbyt, Cambridge, ages of body fat and lean mass, glucose, HOMA-IR, follicle-stimulating hormone, or tesAge (years) 26 (24–37) 28 (24–37) 27.5 (21.5–37.5) * 0.909 Table 1.

Baseline characteristics of participants. was fixed at=pCharacteristics: 0.05.Obese Allinsulin analyses were done with SPSS Statistics cose (mmol/L) x< age. fasting (µIU/mL))/22.5 [20].IBMSubjects PWS Patients (n 27) Subjects (n = 27) Healthy (n = 22) for Windows, p-Value verfor the patient’s 2.3.

Statistical Analyses 3.1.1.

Patient nar Prodigy−963, Chicago, IL, USA). by the Mann–Whitney U test with Bonferroni correction.

To compare all variables before UK) (lower limit ofPWS detection, 10 pg/ml; intraassay 7.6%; interassay CVMiniquick < levels 8.4%). ®, tosterone (only measured in men) levels.

Females inCV the<PWS group had lower ofA * <0.001 sion 25.0 (IBM Corp., Armonk, NY, USA).

Patients with were treated with recombinant GH (Genotonorm Only PWS patients received GH treatment.

After 12 months’ GH treatment, patients’ * 0.006 .

Patients (n 27) Obese Subjects (n 27)other Healthy Subjects (n = 22) p-Value To PWS determine concentrations of we kisspeptin-1 in=for plasma, used the Kisspeptin-1 Continuous variables are reported as medians and interquartile ranges (IQR).

CateTable 1= reports the baseline characteristics ofwe subjects in the PWS, obese, and normal and after GH treatment used the Wilcoxon signed-rank test.

To study the relationship routine automated analyzer was used laboratory tests.

The homeostatic model estradiol and luteinizing hormone than females in the obese group. ⴕprotocol.

0.135 Pfizer, New York, NY, USA),parameters starting with a dose of 0.2mg/day and adjusting the dose at 0.827 analytic and anthropometric were measured again with the same ELISA kit (Cloud-Clone Corp., Houston, TX, (lower limit of detection, 9.27 pg/ml; Sex (female) 15 (55.6%) 15 (55.6%) 11 (50%) * percent0.907 gorical variables are reported asUSA) frequencies and percentages.

To compare allsignificance the continweight groups.

The PWS and obese groups did not differ in weight, BMI, waist, Glucose (mmol/L) 5.00 (4.55–6.99) 4.83 (4.77–5.49) 4.36 (4.16–5.02) between variables, we used Spearman's rank-order correlation test.

Statistical assessment formonths insulin resistance (HOMA-IR) index was calculated as fasting plasma glu3.(70.5–105.5) Results 1, 3, 6, and 12 to achieve high-normal insulin-like growth factor-1 (IGF-1) levels Weight (kg) 89.6 95.5 (81.8–121.1) 65.8 (58.1–71.6) λλ 0.004 ** <0.001 intraassay coefficient of variation (CV) <10%; interassay CV < 12%).

To determine concenuous variables at baseline, between the 3 groups, we used Kruskal–Wallis test followed ages of body fat and lean mass, glucose, HOMA-IR, follicle-stimulating hormone, orvertesAge (years) 26 (24–37) 28 (24–37) 27.5 (21.5–37.5) * 0.909 Table 1.

Baseline characteristics of participants. was fixed at p < 0.05.

All analyses were done with IBM SPSS Statistics for Windows, cose (mmol/L) x fasting insulin (µIU/mL))/22.5 [20].

0.006 for the patient’s age. ** <0.001 2.3.

Analyses 3.1.inStatistical Baseline Findings trations of leptin plasma, we used the Human Leptin ELISA Kit (Biorbyt, Cambridge, by the Mann–Whitney U test with Bonferroni correction.

To compare all variables before ®, tosterone (only measured in men) levels.

Females in the PWS group had lower levels of * <0.001 sion 25.0 (IBM Corp., Armonk, USA).

Patients with PWS were NY, treated with recombinant GH (Genotonorm Miniquick Only PWS patients received GH treatment.

After 12 months’ GH treatment, patients’ * <0.001 .

PWSlimit Patients (n = 27) Obese Subjects (n = 27) Healthy Subjects (n = 22) p-Value Continuous variables are reported as medians and interquartile ranges (IQR).

Cate3.1.1.

Patient Characteristics: UK) (lower of detection, 10 pg/ml; intraassay CV < 7.6%; interassay CV < 8.4%).

A and after GH treatment we used the Wilcoxon signed-rank test.

To study the relationship estradiol and luteinizing hormone than females inof the obese group. ⴕprotocol.

0.135 Pfizer, New NY, USA), starting with a dose 0.2mg/day and the adjusting the dose at analytic and York, anthropometric parameters were measured again with same <0.001 Sex (female) 15 (55.6%) 15Spearman's (55.6%) (50%) *0.710 0.907 gorical variables are reported as frequencies and To compare all*significance the continHOMA-IR 2.26 (1.52–4.80) 2.58 (1.79–3.96) 1.3711(0.84–1.84) routine automated analyzer was used for other laboratory tests.

The homeostatic model between variables, we used rank-order correlation test.

Statistical Table 112reports the baseline characteristics ofpercentages. subjects in the PWS, obese, normal 3.

Results Height 156 (148.0–165.0) 169 (162.0–173.0) 169 1, 3, 6, and months to achieve high-normal insulin-like growth factor-1 (IGF-1) levels Weight (cm) (kg) 89.6 (70.5–105.5) 95.5 (81.8–121.1) 65.8(164.5–181.5) (58.1–71.6) λλand 0.001 ** <0.001 ⴕ <0.001 uous variables at baseline, between the 3 groups, we used Kruskal–Wallis test followed Age (years)assessment for26 (24–37) 28 (24–37) 27.5 (21.5–37.5) * 0.909 insulin resistance (HOMA-IR) index was calculated as fasting plasma gluTable 1.

Baseline characteristics of participants. was fixed at p < 0.05.

All analyses were done with IBM SPSS Statistics for Windows, weight groups.

The PWS and obese groups did not differ in weight, BMI, waist, percent<0.001 for the patient’s age. ** <0.001ver2.3.Baseline Statistical Analyses 3.1.

Findings by the Mann–Whitney U test with Bonferroni correction.

To compare all hormone, variables before cose (mmol/L)sion x fasting insulin (µIU/mL))/22.5 [20]. * <0.001 25.0 (IBM Corp., Armonk, NY, USA). ages of body fat and lean mass, glucose, HOMA-IR, follicle-stimulating or tesOnly PWS patients received GH(ntreatment.

After 12Subjects months’(n GH treatment, patients’ * <0.001 .

PWS 3.1.1.

Patients (n = 27) Obese Subjects = 27) Healthy = 22) p-Value Continuous variables are reported as medians and interquartile ranges (IQR).

CatePatient Characteristics: ® and after GH treatment we used thelevels.

Wilcoxon signed-rank test.Miniquick To study the relationship Patients with PWS were treated with recombinant GH (Genotonorm , ⴕ 0.135 tosterone (only measured in men) Females in the PWS group had lower levels of =0.007 analytic and anthropometric parameters wereand measured again with the same protocol. * <0.001 Sex (female) 15 (55.6%) 15 (55.6%) 11 (50%) * 0.907 gorical variables are reported as frequencies percentages.

To compare all the continIGF-I (ng/mL) 143.0 (95.0–188.0) 171 (139.5–257.5) 226.0 (192.7–312.0) between variables, we used Spearman's rank-order correlation test.

Statistical significance Table 1 reports the baseline characteristics of subjects in the PWS, obese, and normal Pfizer, New York, NY, USA), starting with a dose of 0.2mg/day and adjusting the dose at 3.

Results Height (cm) 156 (148.0–165.0) 169 (162.0–173.0) 169 (164.5–181.5) Weight (kg) 89.6 estradiol (70.5–105.5) 95.5 (81.8–121.1) (58.1–71.6) λλ<0.001 <0.001** and luteinizing hormone than females in 65.8 the obese group. ⴕ* <0.001 uous variables at0.05. baseline, between the done 3 groups, used test followed Age (years) 26 (24–37) 28 (24–37) 27.5 (21.5–37.5) 0.909 was fixed pAnalyses <The All and analyses were with IBM SPSS Statistics for waist, Windows, vergroups.

PWS obese groups did notwe differ in Kruskal–Wallis weight, BMI, percent1, 3, 6, and 12weight months toatachieve high-normal insulin-like growth factor-1 (IGF-1) levels ** 0.084 <0.001 2.3.

Statistical 3.1.

Baseline Findings by the Mann–Whitney U test with Bonferroni correction.

To compare all variables before * <0.001 sion 25.0 (IBM Corp., Armonk, NY, USA). ages of body fat and lean mass, glucose, HOMA-IR, follicle-stimulating hormone, or tesfor the patient’s age.

Table 1. Baseline characteristics of participants. * 0.001CateContinuous variables reported as medians and interquartile ranges (IQR).

3.1.1.

Patient Characteristics: and after GH treatment weinare used Wilcoxon signed-rank test.group Topatients’ study relationship ⴕ 0.001 0.135 tosterone (only measured men)the levels. in thetreatment, PWS had the lower levels of Only PWS patients received GH treatment.

After 12Females months’ GH <0.001 gorical variables are reported as characteristics frequencies and percentages.

ToPWS, compare all*significance the normal continLH. (IU/L) 1.21 (0,36–6.29) 6.01 (4.1–7.75) 5.48 (3.5–12.75) PWS Patients (n = 27) Obese Subjects (n = 27) Healthy Subjects (n = 22) p-Value between variables, we used Spearman's rank-order correlation test.

Statistical Table 1 reports the baseline of subjects in the obese, and 3.

Results Height 156 (148.0–165.0) 169 (162.0–173.0) 169 (164.5–181.5) Weight(cm) (kg) 89.6 (70.5–105.5) 95.5 (81.8–121.1) 65.8 (58.1–71.6) λλ0.001 ** <0.001 estradiol and luteinizing hormone than females the obese group. analytic and anthropometric parameters were measured againinwith the same protocol. ⴕ <0.001 uous variables baseline, between the done 3 groups, used Kruskal–Wallis test0.947 followed Sex (female) 15 (55.6%) 15 (55.6%) 11 (50%) *percent0.907 was fixed atFindings p <at 0.05.

All analyses were with IBM SPSS Statistics for Windows, verweight groups.

The PWS and obese groups did notwe differ in weight, BMI, waist, ** <0.001 3.1. Baseline by the Mann–Whitney U test with Bonferroni correction.

To compare all variables before sion 25.0 (IBM Corp., Armonk, NY, USA). ages of body and lean mass, glucose, HOMA-IR,3.31 follicle-stimulating hormone, or tesAge (years) 26 (24–37) 28 (2.9–6.76) (24–37) 27.5 (21.5–37.5) 0.909 Table fat 1. Baseline characteristics of participants.

2.3. Statistical Analyses FSH (IU/L) 3.63 (0.3–7.28) 5.19 (2.3–7.22) **0.301 3.1.1.

Patient Characteristics: and after GH treatment weinused thelevels.

Wilcoxon signed-rank test.group To study relationship tosterone (only measured men) Females in the PWS hadthe lower levels of * <0.001 **significance <0.001 variables are reported as medians ranges (IQR).

Cate<0.001 . (cm) Continuous PWS (nvariables, =127) Obese Subjects (nand = rank-order 27)interquartile Healthy Subjects (n =Statistical 22) between we Spearman's correlation test.

Table reports theused baseline characteristics of ingroup. the PWS, obese,p-Value and normal 3.

Results Height 156Patients (148.0–165.0) 169 (162.0–173.0) 169 (164.5–181.5) estradiol and luteinizing hormone than females insubjects the obese 0.135 Testosterone, gorical in malesvariables are reported as frequencies and percentages. To compare all the contin- ⴕⴕ<0.001 0.75 Sex (female) 15 (55.6%) 15 (55.6%) 11 (50%) * 0.907 was fixed at p < 0.05.

All analyses were done with IBM SPSS Statistics for Windows, verweight groups.

The PWS 3.82 and(2.47–4.44) obese groups did not 5.63(4.82–8.11) differ in weight, BMI, waist, percent2.08 Baseline (0.51–3.61) 3.1.

Findings Weight (kg) 89.6 (70.5–105.5) 95.5 (81.8–121.1) 65.8 (58.1–71.6) (ng/mL) λλ <0.001 ** <0.001 uous variables at baseline, between the 3 groups, we used Kruskal–Wallis test followed sion 25.0 (IBMfat Armonk, NY, USA). ages of body and lean mass, glucose, HOMA-IR, follicle-stimulating hormone, Age (years) 26 (24–37) 28 (24–37) 27.5 (21.5–37.5) * 0.909or tesTable 1.Corp., Baseline characteristics of participants. <0.001 3.1.1. Patient Characteristics: by the Mann–Whitney U test with Bonferroni correction. To compare all variables before **0.002 tosterone (only measured in men) levels. Females in the PWS group had lower levels of * <0.001 * 0.001 and afterPWS GH treatment we used the Wilcoxon signed-rank To study thethe relationship .

Patients (nand 27) Obese Subjects (n =females 27) test.

Healthy Subjects (n = 22) Table 1= reports the baseline characteristics of subjects in PWS, obese,p-Value and normal 3.

Results estradiol luteinizing hormone than in the obese group.

0.135 Estradiol, in between females variables, we used Spearman's rank-order correlation test. Statistical significance ⴕ* <0.001 <0.001 Sex (female) 15 (55.6%) 15 (55.6%) 11 (50%) * 0.907 groups.

The PWS and obese groups did not169 differ in weight, BMI, waist, percent23Baseline (16–44) 94 (60–164) 63 (5–168) 3.1.

Findings Height (cm) 156weight (148.0–165.0) 169 (162.0–173.0) Weight (kg) 89.6 95.5 (81.8–121.1) 65.8 (164.5–181.5) (58.1–71.6) (pg/mL) 0.253 ** λλ <0.001 was fixed at p 26 <(70.5–105.5) 0.05. analyses done with IBM SPSS Statistics for Windows, verⴕ* 0.909 <0.001 ages ofAll body fat andwere leancharacteristics mass, glucose, HOMA-IR, follicle-stimulating hormone, or tesAge (years) (24–37) 28 (24–37) 27.5 (21.5–37.5) Table 1. Baseline of participants.

0.287 ** <0.001 Characteristics: sion 25.0 (IBM3.1.1.

Corp.,Patient Armonk, NY, USA).in men) levels. Females in the PWS group had lower tosterone (only measured levels of * <0.001 *and <0.001 .

PWS Patients (n 27) Obese Subjects (n =females 27) Healthy Subjects = 22) Table 1= reports the baseline characteristics of ingroup. the (n PWS, obese,p-Value normal estradiol and luteinizing hormone than insubjects the obese ⴕ 0.640 0.135 * <0.001 SexFat (female) 15 (55.6%) 15 (41.9–61.9) (55.6%) 11 (50%) *percent0.907 Results weight groups.

The PWS and obese groups did not differ in weight, BMI, waist, mass 3. (%) 56.3 (49.3–61.1) 50.8 38.7(28.1–39.5) Height 156 169 169 (164.5–181.5) Weight(cm) (kg) 89.6(148.0–165.0) (70.5–105.5) 95.5(162.0–173.0) (81.8–121.1) 65.8 (58.1–71.6) λλ<0.001 <0.001** ⴕ *<0.001 ages of body and leancharacteristics mass, glucose, HOMA-IR,27.5 follicle-stimulating hormone, or tesAge (years) 26 (24–37) 28 (24–37) (21.5–37.5) 0.909 Tablefat 1. Baseline of participants.

3.1. Baseline Findings <0.001 ** <0.001 tosterone (only measured in men) levels. Females in the PWS group had lower levels * <0.001 of 3.1.1. Patient * < 0.001 .

PWSCharacteristics: Patients = 27) Obesehormone Subjectsthan (n =females 27) Healthy Subjects (n = 22) p-Value estradiol(n and luteinizing in the obese group. ⴕ 0.891 0.135 the baseline characteristics of subjects in the PWS, obese, Sex (female) 15 (55.6%) 15 (55.6%) (50%)and normal * <0.001 * 0.907 Lean mass (%) Table 1 reports 43.6(38.9–50.2) 49.2 (36.4–51.3) 69.211 (56.4–71.8) Height 156 169 169 Weight (cm) (kg) 89.6(148.0–165.0) (70.5–105.5) 95.5(162.0–173.0) (81.8–121.1) 65.8(164.5–181.5) (58.1–71.6) λλ<0.001 ** <0.001 weight groups.26The PWS and1.obese groups did not differ in weight, BMI, waist, percent- ⴕ* <0.001 Age (years) (24–37) 28 (24–37) 27.5 (21.5–37.5) 0.909 Table Baseline characteristics of participants. <0.001 <0.001 ages of body fat and lean mass, glucose, HOMA-IR, follicle-stimulating hormone, or tes- ** * <0.001 * 0.094 .

PWS Patients (n = 27) Obese Subjects (n = 27) Healthy Subjects (n = 22) p-Value tosterone (only measured in men) levels. Females in the PWS group had lower levels of ⴕ 0.135 0.979 * <0.001 Sex (female) 15 (55.6%) 15 (55.6%) (50%) * 0.907 Kisspeptin (pg/mL) 140.20 (123.5–156.8) 141.96 (113.9–165.6) 124.5811 (107.3–139.0) estradiol and luteinizing hormone than females in the obese group.

Height 156 (148.0–165.0) 169 (162.0–173.0) 169 Weight (cm) (kg) 89.6 (70.5–105.5) 95.5 (81.8–121.1) 65.8(164.5–181.5) (58.1–71.6) λλ0.019 ** <0.001 ⴕ* <0.001 Age (years) 26 (24–37) 28 (24–37) 27.5 (21.5–37.5) 0.909 **0.154 <0.001 Table 1. Baseline characteristics of participants. * <0.001 * <0.001 ⴕ 0.152 0.135 .

PWS Patients (n = 27) Obese Subjects (n = 27) Healthy Subjects (n = 22) p-Value * <0.001 Leptin (ng/mL) 48.15 (28.80–67.10) 33.10 (20.50–67.30) 14.8 (11.4–67.3) Height (cm) 156 (148.0–165.0) 169 (162.0–173.0) 169 (164.5–181.5) λλ<0.001 <0.001** x (female) Weight (kg) 15 (55.6%) 89.6 (70.5–105.5)15 (55.6%) 95.5 (81.8–121.1) 11 (50%) 65.8 (58.1–71.6) * 0.907 ⴕ <0.001 **<0.001 <0.001 ge (years) 26 (24–37) 28 (24–37) 27.5 (21.5–37.5) * 0.909 PWS = Prader–Willi syndrome; BMI = body mass index; HOMAR-IR = homeostatic model assessment for insulin resistance; * <0.001 LH = luteinizing hormone; FSH = follicle-stimulating hormone. p-values are marked with * Kruskall–Wallis test for comparisons of * <0.001 ⴕ PWS 0.135vs. Obese, λ PWS vs.

Height 156Mann–Whitney (148.0–165.0) 169 (162.0–173.0) 169 (164.5–181.5) PWS vs.(cm) Obese vs. Healthy.

U test with Bonferroni correction for the following comparisons: ⴕ <0.001 Healthy,89.6 and (70.5–105.5) ** Obese vs. Healthy. All variables except for sex are reported as median (interquartile range). λ <0.001 eight (kg) 95.5 (81.8–121.1) 65.8 (58.1–71.6) ** <0.001

Med. 2021, 10, x FOR PEER REVIEW

eight (cm)

156 (148.0–165.0)

169 (162.0–173.0)

169 (164.5–181.5)

* <0.001 ⴕ <0.001

PWS = Prader–Willi syndrome; BMI = body mass index; HOMAR-IR = homeostatic model assessment for insul resistance; LH = luteinizing hormone; FSH = follicle-stimulating hormone. p-values are marked with * Kruskall–Wallis te for comparisons of PWS vs. Obese vs. Healthy. Mann–Whitney U test with Bonferroni correction for the followin comparisons: ⴕ PWS vs. Obese, λ PWS vs. Healthy, and ** Obese vs. Healthy. All variables except for sex are reported J. Clin. Med. 2021, 10, 3054median (interquartile range).

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3.1.2. Kisspeptin Levels:

Overall, kisspeptin levels did not differ between sexes (132.9 pg/mL (107.8–16 3.1.2. Kisspeptin Levels females vs. 135.3 pg/mL (119.9–162.2) in males, p = 0.488).

Overall, kisspeptin levels did notbaseline differ between sexes (132.9 pg/mL (107.8–161.8) In PWS subjects, kisspeptin levels were similar to those ininthe obese females vs. 135.3 pg/mL (119.9–162.2) in males, p = 0.488). and higher than those in the healthy group (Table 1, Figure 1). There were no diffe In PWS subjects, baseline kisspeptin levels were similar to those in the obese group between diabetic and non-diabetic subjects (141.1 pg/mL (121.1–152.6) vs. 140.2 and higher than(128.2–163.6), those in the healthy group (Table 1, Figure 1).

There were no respectively, p = 0.798). Patients treated with sex differences steroids had higher between diabetic and non-diabetic subjects (141.1 pg/mL (121.1–152.6) vs. 140.2 pg/mL of kisspeptin (149.4 pg/mL (138.3–165.3) vs. 133.6 pg/mL (117.8–142.2) than tho (128.2–163.6), respectively, p = 0.798). Patients treated with sex steroids had higher levels of treated with sex steroids, p = 0.01), but these two groups did not differ in terms of kisspeptin (149.4 pg/mL (138.3–165.3) vs. 133.6 pg/mL (117.8–142.2) than those not treated = 0.981), testosterone levels in men, or estradiol levels in women (data not shown). with sex steroids, p = 0.01), but these two groups did not differ in terms of BMI (p = 0.981), testosterone levels in men, or estradiol levels in women (data not shown).

Figure 1. Kisspeptin levels at baseline in the three groups (blue) and after GH treatment in PWS (red).

GH = growth hormone.

Willcoxon signed-rank test, after p = 0.027 between baseline postFigure 1. Kisspeptin levels at baseline in *the three groups (blue) and GH treatment in PWSand (red).

GH = grow GH treatment. ** Mann–Whitney U test with Bonferroni correction, p = 0.019 between PWS and U test wi hormone. * Willcoxon signed-rank test, p = 0.027 between baseline and post-GH treatment. ** Mann–Whitney groups.

Outliers are represented as circle symbols.

Bonferronihealthy correction, p = 0.019 between PWS and healthy groups.

3.1.3. Leptin Levels 3.1.3. Leptin Levels: Overall, leptin levels were higher in females than in males (43.95 ng/mL (22.75–73.05) Overall, leptin levels were higher in females than in males (43.95 ng/mL (22.75 vs. 22.80 ng/mL (11.80–29.50) respectively, p < 0.001). vs. 22.80 ng/mL (11.80–29.50) respectively, p < 0.001).

In PWS subjects, baseline leptin levels were similar to those in the obese group and In PWS subjects, baseline leptin levels were similar to those in the obese grou higher than those in the healthy group (Table 1, Figure 2). Leptin levels did not differ higher than those in the healthy group (Table 1, Figure 2). Leptin levels did not between diabetic and non-diabetic subjects (45.30 ng/mL (19.60–61.80) vs. 51.0 ng/mL between diabetic and non-diabetic subjects (45.30 ng/mL (19.60–61.80) vs. 51.0 (29.50–82.40), respectively, p = 0.866) or between those treated with sex steroids and those (29.50–82.40), 0.866) or between those treated(26.70–82.40), with sex steroids and not treated with sex steroidsrespectively, (51.0 ng/mLp =(31.20–62.0) vs. 45.2.0 ng/mL respectively, p = 0.919).

3.2. Comparison between Baseline and Post-GH-Treatment Findings in the PWS Group After GH treatment, no significant changes were observed in anthropometric measures, glucose metabolism, gonadotropins, or sex steroids. As expected, IGF-1 levels increased (143(95–188) ng/mL vs. 217(160–254) ng/mL, p < 0.001) (Table 2). Kisspeptin levels were significantly lower than at baseline (p = 0.027) and were similar to those in the healthy group (125.1 pg/mL (106.2–153.4) vs. 124.58 pg/mL (107.3–139.0), respectively, p = 0.936) (Table 2, Figure 1). Body composition improved after treatment: total body fat was 1.6% lower (p = 0.005) and lean body mass was 2.1% higher (p = 0.005) (Table 2).

J. Clin. Med. 2021, 10, 3054

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J. Clin. Med. 2021, 10, 3054

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not treated with sex steroids (51.0 ng/mL (31.20–62.0) vs. 45.2.0 ng/mL (26.70–82.40), respectively, p = 0.919).

Figure 2. Leptin levels at baseline in the three groups (blue) and after GH treatment in PWS (red).* p < at 0.001 Differences in baseline leptinand levels the three groups (Kruskall–Wallis test).

Figure 2. Leptin levels baseline in the three groups (blue) afterbetween GH treatment in PWS (red).* p < 0.001 Differences in baseline leptin between three in groups (Kruskall–Wallis levels did(Willcoxon not changesigned-rank in Prader–Willi Leptinlevels levels did notthe change Prader–Willi subjectstest). afterLeptin GH treatment test, subjects afterp GH treatment (Willcoxon signed-rankas test, p = 0.144). = 0.144).

Outliers are represented circle symbols.

Table 2. Anthropometrics, body composition, and plasma between hormone levels and in patients with PWS before andin after months’ 3.2. Comparison Baseline Post-GH-Treatment Findings the 12 PWS Group: growth hormone treatment.

After GH treatment, no significant changes were observed in anthropometric

Weight (kg) BMI (kg/m2 ) Waist (cm) Glucose (mmol/L) HOMA-IR

measures, glucose metabolism, gonadotropins, or sex steroids. As expected, IGF-1 levels Before GH Treatmentng/mL vs.

After 12 Months’ng/mL, GH Treatment increased (143(95–188) 217(160–254) p < 0.001) (Table 2). Kisspeptin p-Value * Median (IQR) Median (IQR) levels were significantly lower than at baseline (p = 0.027) and were similar to those in the 89.6 (70.5–105.5) 0.990 healthy group (125.1 pg/mL (106.2–153.4)87.7 vs. (74.3–100.2) 124.58 pg/mL (107.3–139.0), respectively, p= 0.936) 34.6 (Table 2, Figure 1).

Body composition improved after treatment: total body fat was (30.9–41.3) 34.0 (31.8–41.6) 0.692 1.6% lower (p = 0.005) and lean body mass was 2.1% higher (p = 0.005) (Table 2).

110.0 (101.0–124.0) 112.0 (104.0–123.5) 0.602 (Table 2, By contrast, leptin levels were not significantly different after treatment Figure5.00 2) and no significant changes in bone mineral density were observed; (4.55–6.99) 4.83 (4.33–5.49) 0.107 Z-scores remained lower than expected for age (Table 2).

2.26 (1.52–4.80) 2.95 (1.99–6.14) 0.209

HbA1c (%) 2. Anthropometrics, body composition, 5.6 (5.3–6.9)and plasma hormone levels 5.6 in (5.3–6.2) 0.294after 12 Table patients with PWS before and months’ growth hormone treatment.

LH (IU/L)

1.21 (0.36–6.29) Before GH Treatment 3.63 (0.3–7.28) Median (IQR) Testosterone, in males (ng/mL) 2.08 (0.51–3.61) Weight (kg) 89.6 (70.5–105.5) Estradiol, in females (pg/mL) 23 (16–44) BMI (kg/m2) 34.6 (30.9–41.3) 110.0 (101.0–124.0) IGF-1 (ng/mL) Waist (cm) 143(95–188) Glucose (mmol/L) 5.00 (4.55–6.99) Fat mass (%) 56.3 (49.3–61.1) HOMA-IR 2.26 (1.52–4.80) Lean mass (%) 43.6(38.9–50.2) HbA1c (%) 5.6 (5.3–6.9) Total body water (kg) 37.6(32.2–43.0) LH (IU/L) 1.21 (0.36–6.29) FSH (IU/L) 3.63 (0.3–7.28) Extremities/trunk body fat index 0.92(0.70–1.09) Testosterone, in males (ng/mL) 2.08 (0.51–3.61) Appendicular skeletal muscle mass index 6.6(5.6–7.7) 2 Estradiol, in females (pg/mL) 23 (16–44) (kg/m ) IGF-1 (ng/mL) 143(95–188) Total femur bone mineral density −1.31(−1.8–(−0.65)) (Z-score) FSH (IU/L)

2.71(0.3–5.81) 0.841 After 12 Months’ GH Treatment 0.015 5.82 (0.92–9.03) p-Value * Median (IQR) 1.43 (0.43–3.22) 0.814 87.7 (74.3–100.2) 0.990 16 (8–31) 0.124 0.692 34.0 (31.8–41.6) 112.0 (104.0–123.5) 217 (160–254) <0.001 0.602 4.83 (4.33–5.49) 52.1 (49.9–59.2) 0.028 0.107 2.95 (1.99–6.14) 0.209 47.9 (40.8–50.1) 0.002 5.6 (5.3–6.2) 0.294 37.1 (32.9–43.6) 0.342 0.841 2.71(0.3–5.81) 5.82 (0.92–9.03) 0.86 (0.75–1.11) 0.637 0.015 1.43 (0.43–3.22) 0.814 7.2 (6.1–8.4) 0.059 0.124 16 (8–31) 217 (160–254) <0.001 −1.22 (−1.77–(−0.43)) 0.485

Total spine bone mineral density Z-score

−1.61(−2.20–(−0.56))

−1.90 (−2.49–(−0.51))

0.927

Kisspeptin (pg/mL)

140.20 (123.5–156.8)

125.1 (106.2–153.4)

0.027

Leptin (ng/mL)

48.15 (28.80–67.10)

43.00 (21.75–68.32)

0.144

IQR = interquartile range; BMI = body mass index; HOMAR-IR = homeostatic model assessment for insulin resistance; LH = luteinizing hormone; FSH = follicle-stimulating hormone; IGF−1 = insulin-like growth factor−1. * Willcoxon signed-rank test.

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By contrast, leptin levels were not significantly different after treatment (Table 2, Figure 2) and no significant changes in bone mineral density were observed; Z-scores remained lower than expected for age (Table 2).

4. Correlations Overall, baseline kisspeptin did not correlate significantly with weight, BMI, percentage of body fat or total body fat, percentage of lean mass or total lean mass, waist circumference, glucose, insulin, HOMA-IR, glycated hemoglobin, gonadotropins, or sex steroids.

Baseline kisspeptin and leptin levels correlated only in the healthy group (r = 0.414, p = 0.05).

In the PWS group, baseline kisspeptin did not correlate significantly with any of the above variables or with IGF-1, bone mineral density (total femur Z-score, total spine Z-score), extremities/trunk body fat index, or appendicular skeletal muscle mass index.

Moreover, the percentage of change in kisspeptin did not correlate with the percentage of change in body fat (whether measured in % or kg), lean body mass (whether measured in % or kg), HOMA-IR, insulin, glucose, or IGF-1.

Overall, baseline leptin levels correlated with percentage of body fat (r = 0.721, p < 0.001) and total body fat (r = 0.762, p < 0.001).

In the PWS group, baseline leptin levels correlated with HOMA-IR (r = 0.317, p = 0.006), but after GH treatment, leptin levels did not correlate with HOMA-IR (r = 0.110, p = 0.602).

Baseline leptin levels also correlated with percentage of body fat (r = 0.705, p < 0.001) and after treatment, with percentage of body fat (r = 0.692, p < 0.001), as well as with total body fat (r = 0.498, p = 0.01).

The percentage of change in IGF-1 did not correlate with the percentage of change in total body fat or in lean body mass.

5. Discussion To our knowledge, this is the first study to evaluate plasma kisspeptin levels in PWS patients. We found that plasma kisspeptin levels in PWS patients with GH deficiency were similar to those in obese subjects matched by age, sex, and BMI and were higher than those in healthy controls. After 12 months of treatment with GH, plasma kisspeptin levels in PWS patients decreased to levels similar to those observed in healthy subjects.

The relationship between obesity and kisspeptin levels is unclear. Hestiantoro et al. [21] found kisspeptin levels were lower in obese than in normal-weight menopausal women.

Pita et al. [22] found kisspeptin levels were higher in obese than in normal-weight prepubertal girls, but similar in obese and normal-weight prepubertal boys. Sitticharoon et al. [23] observed higher kisspeptin levels in obese than in non-obese men. In other studies, this group found no differences in kisspeptin levels between obese and normalweight women [24] or between obese and normal-weight girls with central precocious puberty [25]. Taken together, these findings suggest that kisspeptin levels may be influenced more by hormonal status than by obesity per se. Moreover, kisspeptin levels in women differ across the menstrual cycle, being highest in the luteal phase, followed by the preovulation phase and the follicular phase, suggesting that kisspeptin might play a role in peripheral reproductive regulation [26]. In the present study, kisspeptin levels were higher in obese patients (with or without PWS), but did not differ between sexes.

However, all our PWS patients had some degree of hypogonadism, and not all of them were being treated with sex hormones. Furthermore, we did not take women’s menstrual phase into account because most of these patients do not ovulate. Nevertheless, kisspeptin levels were higher in those with hormone replacement therapy than in those without, suggesting sex hormones might affect peripheral kisspeptin. These findings are in line with those reported in vitro experiments where estrogens stimulated Kiss-1 gene expression in cultured kisspeptin neurons, suggesting a central interaction between sex hormones and kisspeptin-producing cells [27].

The relationship between kisspeptin and BMI is also unclear. Some studies have found positive correlations. One study in men found that kisspeptin levels correlated positively

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with BMI and weight [23], and another in children found they correlated positively with BMI, weight, and waist circumference [28]. However, other studies have found negative correlations between kisspeptin levels and BMI and waist circumference in non-diabetic men and women [29] and between kisspeptin and BMI in anorectic women [30]. In the present study, we found no correlations between kisspeptin and BMI or waist circumference in the whole group or in the PWS group. Discrepancies between studies might be due to the inaccuracy of BMI and waist circumference as measures of body fat and/or to the heterogeneity and the small size of the cohorts studied.

In the PWS group, GH treatment resulted in a decrease in total body fat and an increase in lean body mass, as reported by other authors [31,32]. Because adipocytes appear to be a source of circulating kisspeptin, we expected decreases in body fat mass to result in decreases in kisspeptin levels. Moreover, adipocytes express the kisspeptin-1 receptor (KISS1R), indicating that kisspeptin secreted by adipose tissue could act as an adipokine or as autocrine/paracrine regulator of adipocyte function [33]. However, we found no correlations between the change in kisspeptin and the change in fat mass, lean mass, extremities/trunk body fat index, or appendicular lean mass index after treatment, suggesting there is no clear relationship between kisspeptin and body composition parameters. Furthermore, kisspeptin appears to regulate GH, but the role of kisspeptin in GH release remains unclear, especially due to discrepancies between in vivo and in vitro findings [1], although the data suggest that kiss1/KISS1R could play a role in a short or ultra-short feedback loop that regulates the function of somatotrophs [1]. Such a role would help explain why kisspeptin levels decreased in GH-deficient patients with PWS after one year of GH therapy. Nevertheless, we found no correlation between the changes in kisspeptin and IGF-1 levels. Further studies are necessary to test this hypothesis.

Leptin is mainly produced by adipose tissue [34], and its concentration in serum correlates with body energy reserves [35]. Leptin may signal peripheral energy status to the hypothalamus, thus influencing food intake and reproductive function [36]. Kisspeptin neurons in the hypothalamus have leptin receptors, and leptin increases Kiss1 mRNA expression in those neurons, thus supporting the existence of a leptin-kisspeptin-GnRH pathway in which kisspeptin would mediate between energy reserves and the maturation of the hypothalamic-pituitary-gonadal axis [2].

In our study, plasma leptin levels correlated with body fat mass in all groups at baseline, and no significant changes in leptin levels occurred in the PWS group after GH treatment. These findings are in line with those reported by Höybe et al. [37] in 2003 in a study with 17 adult patients with PWS, but discrepant with those reported by Myers et al. [38], who reported decreased leptin levels after GH treatment in children with PWS. Moreover, we found no correlation between plasma leptin and kisspeptin levels, indicating that these two peptides could be less interrelated peripherally than in the central nervous system.

Inactivation of kiss1r in mice results in obesity and diabetic phenotype [39], and kisspeptin and its receptor are expressed in metabolic tissues (e.g., fat, liver and pancreatic tissues) and likely plays a role in regulating insulin secretion. [6]. However, kisspeptin stimulates insulin secretion only when glucose levels are elevated, suggesting kisspeptin’s role involves correcting hyperglycemia [40]. Glucagon secreted from pancreatic α-cells provokes kiss1 expression in the liver, increasing secretion of kisspeptin from the liver and thereby suppressing glucose-stimulated insulin secretion from pancreatic β-cells [33].

For all these reasons, we also analyzed the relationship between kisspeptin and glucose metabolism in our cohort of patients with PWS, 6 (22.2%) of whom had diabetes with good glycemic control (HbA1c < 7.5%). We found no difference in kisspeptin levels between diabetic and non-diabetic PWS patients, and kisspeptin levels did not correlate with plasma glucose, insulin, glycated hemoglobin, or HOMA-IR index. After GH treatment, no changes in these parameters or correlations between these parameters and kisspeptin were observed.

These results corroborate those of a previous study that showed no effect of GH treatment on glycemic control in adults with PWS [32].

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The limitations of our study are mainly due to the characteristics of the sample. The small size of the groups precluded subanalyses by sex or by genetic subtype. Moreover, in both the PWS group and the matched obese controls, obesity was heterogeneous, ranging from low risk (class 1) to high risk (class 3). Only a small proportion of patients were receiving sex hormones, and we did not take the menstrual cycle phase into consideration.

All these limitations may have contributed to the lack of significant correlations between variables, and larger studies would be necessary to draw conclusions.

The authors want to emphasize that the recent development of more sensitive methods to detect plasma kisspeptin concentrations leading to better quantitative results instead of undetectable values may have influenced the opposite findings to the initial hypothesis of the present study.

In summary, we found that plasma kisspeptin levels in our cohort of GH-deficient adults with PWS did not differ from those in matched obese patients and were higher than those in a group of normal weight subjects. After one year of GH treatment, kisspeptin levels decreased to levels similar to those in healthy controls. Although the mechanism through which this decrease occurs and its clinical significance remain to be determined, we speculate that GH treatment might decrease kisspeptin levels directly through a negative feedback mechanism, since we found no relationship between changes in kisspeptin levels and changes in anthropometric or metabolic parameters or in leptin levels. Further studies are required to corroborate this hypothesis.

Author Contributions: O.G.-P., L.C., and A.C. designed the study, recruited the patients, and wrote the manuscript. R.C. and S.E.-C. also recruited some patients and contributed to additional versions of the manuscript. R.P. obtained and stored all the blood samples and contributed to the methodology section of the manuscript. N.A. contributed to laboratory tasks and to methodology section. M.R. contributed to the final version of the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding: This research was funded by Instituto de Salud Carlos III and Fondo Europeo de Desarrollo Regional “Feder: Otra manera de hacer Europa” grant number PI18/00734. Also by Fundació Parc Taulí, grant number (CIR2018/001).

Institutional Review Board Statement: The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the Ethics Committee “Comitè d’Ètica d’Investigació amb medicaments” (CEIm) del Parc Taulí (Ethical Committee approval on 4 July 2019—Code CIR2018/001).

Informed Consent Statement: Informed consent was obtained from all subjects involved in the study.

Data Availability Statement: Data supporting reported results can be found in Parc Taulí server (contact jcoliva@tauli.cat or the corresponding author acaixas@tauli.cat).

Acknowledgments: We thank the Associació Catalana de Síndrome de Prader-Willi for crowdfunding and all the patients and their families for kindly agreeing to participate. We also thank Neus Gómez-Gerique for kisspeptin determinations and Joan Carles Oliva for data analysis.

Conflicts of Interest: The authors have no conflict of interest.

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