Investigation of the Novel Hormone Kisspeptin in Disorders Of

Investigation of the novel hormone kisspeptin in

disorders of reproduction

Gurjinder Monica Kaur Nijher

Department of Investigative Medicine

Thesis submitted for the degree of Doctor of Philosophy

Imperial College London

2012

Abstract

The novel hormone kisspeptin has been identified to play a pivotal role in the regulation of the hypothalamo-pituitary-gonadal axis. Previous studies from our laboratory have demonstrated that a bolus administration of kisspeptin-54 can acutely stimulate gonadotrophin release in healthy men and women. However, no previous studies have examined the effects of kisspeptin-54 to women with infertility. In this study I have examined the effects of acute and chronic administration of kisspeptin-54 on women with infertility due to hypothalamic amenorrhoea. This study has identified that acute administration of kisspeptin to women with hypothalamic amenorrhoea results in stimulation of reproductive hormones, but chronic administration for two weeks of twice daily injections of kisspeptin-54 at a dose of 6.4 nmol/kg, results in tachyphylaxis. I have conducted further studies to examine the time course of desensitisation of the kisspeptin receptor. I have also determined that a dosing regime of twice weekly administration of kisspeptin-54 results in sustained stimulation of gonadotrophin release. I have performed the first study of kisspeptin-10 administration to men and women. These results demonstrate that kisspeptin-10 stimulates gonadotrophin release in men as well as women during the preovulatory phase of the menstrual cycle; but fails to stimulate gonadotrophin release in women during the follicular phase. This reveals a sexual dimorphism in response to the kisspeptin-10. These findings have important ramifications for the potential use of kisspeptin-54 and kisspeptin-10 as a therapeutic agent in disorders of reproduction.

Contents

Abstract ...... 2 List of Figures ...... 5 List of Tables ...... 8 Abbreviations ...... 9 Declaration of contributors ...... 10 Acknowledgements ...... 12 Chapter 1 ...... 13 General Introduction ...... 13 1.1 The hypothalamo-pituitary-gonadal axis ...... 14 1.2 Kisspeptin ...... 21 1.3 The kisspeptin receptor and kisspeptin signaling ...... 24 1.4 Kisspeptin administration results in stimulation of reproductive hormone release ...... 26 1.5 Kisspeptin and ovulation ...... 27 1.6 Metabolic regulation of the reproductive axis ...... 29 1.7 Potential therapeutic applications of kisspeptin ...... 31 1.8 Hypothesis and Aims ...... 33 Chapter 2 ...... 36 The effects of kisspeptin on the hypothalamo pituitary gonadal axis in women with hypothalamic amenorrhoea ...... 36 2.1 Introduction ...... 37 2.2 Hypothesis and Aims ...... 38 2.3 Methods ...... 39 2.3.1 Subjects ...... 39 2.3 Kisspeptin 54 ...... 40 3.Results ...... 49 Study 3.2: Effect of chronic kisspeptin administration on GnRH sensitivity in women with hypothalamic amenorrhoea ...... 68 4. Discussion ...... 70 Chapter 3 ...... 74 The effects of chronic twice weekly kisspeptin administration in women with hypothalamic amenorrhoea ...... 74 3.1 Introduction ...... 75 3.2 Hypothesis and Aims ...... 76 3.3 Methods ...... 77 3.3.1 Subjects ...... 77 3.3.2 Study 1: Determination of the time-course of desensitisation to the effects of kisspeptin on gonadotophin release in women with HA ...... 78 3.3.3 Study 2: Effects of reduced dose and increased dose-interval on serum reproductive hormone release in women with HA administered kisspeptin injections for 2 weeks ...... 80 3.3.4 Study 3: Effects of twice weekly kisspeptin-54 (6.4nmol/kg) administration for 8 weeks on reproductive hormone release in women with HA ...... 80 3.3.5 Kisspeptin-54 ...... 83 3.3.6 Injections of Kisspeptin-54...... 84 3.3.7 Collection and processing of blood samples ...... 84 3.3.8 Analytical methods ...... 85 3.3.9 Data analysis ...... 85 3.4 Results ...... 86 Study 1: Time course of desensitisation to the effects of kisspeptin on reproductive hormone release in women with HA ...... 87 Study 2: Effects of reduced dose and increased dose-interval on serum reproductive hormones in women with HA, during a 2-week protocol of kisspeptin injections ...... 89

Study 3: Effects of twice-weekly kisspeptin-54 administration for 8 weeks on reproductive hormone levels in women with HA...... 100 5. Discussion ...... 107 Chapter 4 ...... 111 The Effects of Kisspeptin-10 on Reproductive Hormone Release Show Sexual Dimorphism in Humans ...... 111 4.1 Introduction ...... 112 4.2 Hypothesis and Aim ...... 113 4.3 Methods ...... 113 4.3.1 Subjects ...... 113 4.3.2 Kisspeptin-10 and -54 peptide synthesis ...... 114 4.3.3 Study Days ...... 115 4.3.4 Serum reproductive hormone measurement ...... 115 4.3.5 Kisspeptin measurement ...... 116 4.3.6 Study 1: The effects of intravenous bolus injection of saline or kisspeptin-10 in healthy male volunteers on plasma kisspeptin IR and reproductive hormones ...... 117 4.3.7 Study 2: Effects of intravenous bolus injection of saline, kisspeptin-10 or kisspeptin-54 in healthy female volunteers ...... 118 4.3.8 Study 3: Effects of subcutaneous bolus injection of saline or kisspeptin-10 on plasma kisspeptin-IR and serum reproductive hormones in healthy female volunteers in the follicular phase of menstrual cycle ...... 118 4.3.9 Study 4: Effects of intravenous infusion of saline or kisspeptin-10 on plasma kisspeptin-IR and serum reproductive hormones in healthy female volunteers ...... 118 4.3.9.1 Study 5: Pharmacokinetic profile of kisspeptin-IR during intravenous infusion of kisspeptin-10 ...... 119 4.3.9.2 Data Analysis ...... 119 4.4 Results ...... 120 4.4.1 Study 1: Effects of IV bolus injection of saline or kisspeptin-10 in healthy male volunteers ...... 122 4.4.2 Study 2: Effects of intravenous bolus injection of saline, kisspeptin-10 or kisspeptin-54 in healthy female volunteers ...... 130 4.4.3 Study 3: Effects of subcutaneous bolus injection of saline or kisspeptin-10 on plasma kisspeptin-IR and serum reproductive hormones in healthy female volunteers ...... 136 4.4.4 Study 4: Effects of intravenous infusion of kisspeptin-10 on plasma kisspeptin-IR and serum reproductive hormones in healthy female volunteers ...... 140 4.4.5 Study 5: Pharmacokinetic profile of kisspeptin-IR during intravenous infusion of kisspeptin- 10 ...... 143 5. Discussion ...... 147 Chapter 5 ...... 154 General Discussion ...... 154 References ...... 163 Appendix 1 Principle of radioimmunoassay ...... 175 Kisspeptin radioimmunoassay: ...... 177 Appendix 2 Original Publications ...... 179

List of Figures

Figure 1. The amino acid sequence of GnRH decapeptide...... 14

Figure 2. The hypothalamic pituitary gonadal (HPG) axis (Conn & Ulloa-Aguirre 2010)...... 15

Figure 3. The human female menstrual cycle (Aitken et al. 2008)...... 17

Figure 4. The amino acid structure of the kisspeptins...... 22

Figure 5. The 8 week study protocol...... 42

Figure 6. The four hour study protocol...... 44

Figure 7. The GnRH study protocol...... 46

Figure 8. Plasma kisspeptin immunoreactivity (IR) levels over 4 hours post injection of kisspeptin-54 (KP54) 6.4 nmol/kg or saline control on the first day of the study...... 51

Figure 9. Plasma kisspeptin immunoreactivity (IR) levels over 4 hours post injection of kisspeptin-54 6.4 nmol/kg or saline control on the last day of two weeks treatment with either twice daily kisspeptin-54 (KP54) 6.4 nmol/kg or saline injections...... 53

Figure 10. Weekly plasma kisspeptin IR levels in women with HA randomised to receive subcutaneous injections of (A) kisspeptin-54 6.4 nmol/kg or (B) saline...... 55

Figure 11. Changes in serum levels of LH (A), FSH (B) and oestradiol (E2) (C) from baseline after administration of subcutaneous injection of either saline or kisspeptin-54 6.4 nmol/kg

(KP54) on the first day of the treatment period...... 57

Figure 12. Changes in serum levels of LH (A), FSH (B) and oestradiol (E2) (C) from baseline after administration of subcutaneous injection of either saline or kisspeptin-54 6.4 nmol/kg on the last day of the treatment period...... 59

Figure 13. Comparison of the mean area under curve (AUC) increases in LH (A), FSH (B) oestradiol E2 (C) during the four hours post sc injection of kisspeptin-54 6.4 nmol/kg on the first and last injection days on the treatment period...... 61

Figure 14. Mean gonadotrophin response to GnRH 100 mcg bolus pre (A) and post (B) treatment with twice daily injections of kisspeptin-54 (KP54) 6.4 nmol/kg...... 69

Figure 15. The four hour study protocol...... 79

Figure 16. Protocol to investigate the effects of twice-weekly kisspeptin-54 administration for

8 weeks in women with HA...... 82

Figure 17. Time course of desensitisation to the effects of kisspeptin-54 (6.4 nmol/kg) on reproductive hormone release administered twice-daily for 2 weeks in women with HA...... 88

Figure 18. Serum reproductive hormone levels following twice-daily regimens of kisspeptin-

54 6.4 nmol/kg injection in 5 women with HA...... 90

Figure 19. Serum reproductive hormone levels following once daily dosing regimens of kisspeptin-54 1 nmol/kg injection in 5 women with HA...... 92

Figure 20. Serum reproductive hormone levels following once daily dosing regimens of kisspeptin-54 0.3 nmol/kg injection in 5 women with HA...... 94

Figure 21. Serum reproductive hormone levels following twice weekly daily dosing regimens of kisspeptin-54 6.4 nmol/kg injection in 5 women with HA...... 96

Figure 22. Serum reproductive hormone levels following different 2 week dosing regimens of kisspeptin-54 injection in women with HA...... 98

Figure 23. Serum reproductive hormone levels following twice-weekly saline injection for 8 weeks in women with HA...... 101

Figure 24. Serum reproductive hormone levels following twice weekly kisspeptin-54 injection for 8 weeks in women with HA...... 103

Figure 25. Plasma kisspeptin levels post intravenous bolus of saline or kisspeptin-10 in healthy men...... 123

Figure 26. Serum LH levels following intravenous bolus administration of saline or kisspeptin-10 in healthy men...... 125

Figure 27. Serum FSH levels following intravenous bolus (IVB) administration of saline or kisspeptin-10 in healthy men...... 127

Figure 28 Serum testosterone levels following intravenous bolus (IVB) administration of saline or kisspeptin-10 in healthy men...... 129

Figure 29 Plasma kisspeptin levels following intravenous bolus (IVB) administration of saline or kisspeptin-10 in healthy women...... 131

Figure 30. Serum LH levels following intravenous bolus (IVB) administration of saline or kisspeptin-10 in healthy women...... 133

Figure 31. Serum FSH levels following intravenous bolus (IVB) administration of saline or kisspeptin-10 in healthy women...... 134

Figure 32. Serum oestradiol levels following intravenous bolus (IVB) administration of saline or kisspeptin-10 in healthy women...... 135

Figure 33. Plasma kisspeptin immunoreactivity (IR) following subcutaneous bolus (sc) injection of kisspeptin-10 to healthy women...... 137

Figure 34. Serum reproductive hormone levels following subcutaneous bolus injection of kisspeptin-10 to healthy women...... 138

Figure 35. Plasma kisspeptin immunoreactivity (IR) during a 90 minute intravenous infusion of kisspeptin-10 to healthy women...... 141

Figure 36. Serum reproductive hormone levels during intravenous infusion of kisspeptin-10 to healthy women...... 142

Figure 37. Plasma kisspeptin IR before, during and after a 90 minute infusion of 360 pmol/kg/min kisspeptin-10...... 144

Figure 38. Natural Logarithm kisspeptin IR against time immediately after cessation of a 90 minute infusion of 360 pmol/kg/min kisspeptin-10...... 146

List of Tables

Table 1. Neurotransmitters acting on Gonadotrophin releasing hormone (GnRH) neurons. 20

Table 2. Characteristics of the women in the kisspeptin and saline control groups...... 50

Table 3. Comparison of basal serum reproductive hormone levels at baseline, during and after 2 weeks treatment with either saline or kisspeptin injections...... 63

Table 4. Comparison of LH pulsatility before and after 2 weeks treatment with saline or kisspeptin-54 6.4 nmol/kg injections in women with HA...... 65

Table 5. Summary of ultrasound parameters at baseline, during and after 2 weeks treatment with saline or kisspeptin injections in women with HA...... 67

Table 6. Comparison of baseline characteristics of women with hypothalamic amenorrhea 86

Table 7. Comparison of basal serum reproductive hormone levels at baseline, during and 8 weeks of treatment with either saline or kisspeptin injections...... 105

Table 8. Summary of ultrasound parameters at baseline and throughout the 8 week treatment with saline or kisspeptin injections, in women with HA...... 106

Table 9. Baseline characteristics of healthy male and female volunteers recruited to the study...... 121

Table 10. Volumes of reactants for addition to kisspeptin radioimmunoassay...... 178

Abbreviations

AN anorexia nervosa ARC arcuate nucleus AUC area under the curve AVPV anteroventral periventricular nucleus of hypothalamus BP blood pressure CRH corticotrophin releasing hormone DBP diastolic blood pressure ER oestrogen receptor FSH follicular stimulating hormone GABA gamma-aminobutyric acid GAD glutamic acid decarboxylase GnIH gonadotrophin inhibitory hormone GnRH gonadotrophin releasing hormone GPR54 G-protein coupled receptor 54 HA hypothalamic amenorrhoea HPG hypothalamo-pituitary-gonadal HPLC high performance liquid chromatography E2 oestradiol FEI free oestradiol index GH growth hormone HR heart rate IHH idiopathic hypogonadotrophic hypogonadism icv intracerebroventricular iv intravenous ivb intravenous bolus IVF in vitro fertilisation IR immunoreactivity KP54 ` kisspeptin-54 KISS1R kisspeptin receptor LH luteinising hormone MBH mediobasal hypothalamus MCH melanin-concentrating hormone MMP2 matrix metalloprotease 2 mTOR mammalian target of rapamycin protein NMDA N-methyl D,L-aspartate NPY neuropeptide tyrosine / neuropeptide Y OCP oral contraceptive pill OHSS ovarian hyperstimulation syndrome POA preoptic area POMC pro-opiomelanocortin PYY peptide tyrosine-tyrosine / peptide YY RFRP RF-related peptide RIA radioimmunoassay SBP systolic blood pressure sc subcutaneous SHBG sex hormone binding globulin

Declaration of contributors

Declaration of originality: The work described in this thesis is my own. Any collaboration and assistance is described below. Contributors are within Section of Investigative Medicine,

Imperial College London unless stated otherwise

Chapter 2: The study protocol was designed by Professor. W.S. Dhillo. I recruited and obtained consent from all subjects with the assistance of Drs. C. Jayasena and A. Ranger. I performed these studies with the assistance of Drs. C. Jayasena and A. Ranger, V. Salem and R. Ramachandran. Drs. K.G. Murphy and O.B. Chaudhri aliquoted and tested the kisspeptin-54 peptide for bioactivity and toxicity. Histological analysis during toxicity analysis was performed by Prof. G. Stamp (Department of Histopathology, Imperial College London).

Ultrasound examinations were performed by Dr. A. Lim and Mrs. D. Patel (Imaging

Department, Charing Cross Hospital, Imperial College Healthcare NHS Trust). Screening

MRI scans were performed by Ms. C. Todd and Dr. A. Mehta Patel (Imaging Department,

Charing Cross Hospital, Imperial College Healthcare NHS Trust). All serum samples were analysed by myself (Department of Biochemistry, Charing Cross Hospital, Imperial College

Healthcare NHS Trust). I performed all radioimmunoassays under the guidance of Prof. M.A.

Ghatei.

Chapter 3: The study protocol was designed by Professor. W.S. Dhillo, Dr C. Jayasena and I.

I recruited and obtained consent from all subjects with the assistance of Drs. C. Jayasena, S.

Hameed and S. Zac-Varghese. I performed these studies with the assistance of Drs. C.

Jayasena, and A Abbara. Dr. K.G. Murphy assisted me with aliquoting of kisspeptin-54 peptide. Bioactivity and toxicity testing of peptide, ultrasound and MRI scans, and serum analyses were performed as in Chapter 2.

Chapter 4: The study protocol was designed by Professor. W.S Dhillo, Dr C. Jayasena and I.

I recruited, obtained consent from subjects with the assistance of Drs. C. Jayasena, A.

Abbara, and A. Comninos. I performed these studies with the assistance of Drs. C.

Jayasena, A. Comninos, A. Abbara, A. Januszewki, L. Sriskandarajah L and Ms M Vaal and

Ms Z Farzad. I performed all radio-immunoassays with Dr C. Jayasena under the guidance of Prof. M.A. Ghatei. All serum samples were analysed by myself (Department of

Biochemistry, Charing Cross Hospital, Imperial College Healthcare NHS Trust).

Acknowledgements

I would like to thank for my supervisor Professor Waljit Dhillo for his endless enthusiasm, guidance and support throughout this project. I would also like to thank Sir Bloom, Dr

Murphy and Professor Ghatei for their expert advice. I was privileged to be working with Dr

Channa Jayasena who has been a thoughtful and intelligent partner in this study. I have also had the opportunity to work with a number of excellent colleagues and would like to particularly thank Dr. Alex Comninos, Dr Akila De Silva and Dr Ali Abbara. This thesis was conducted with support from a Wellcome Trust Clinical Training Fellowship.

I would like to thank my family for their support and love throughout, especially my husband

Rumant and children Manraj and Baby Grewal.

This thesis is dedicated to the memory of my mother Mrs. Jasbir Kaur Nijher.

Chapter 1

General Introduction

1.1 The hypothalamo-pituitary-gonadal axis

The regulation of reproductive function is controlled by the hypothalamo pituitary gonadal

(HPG) axis. A pulsatile secretion of gonadotrophin releasing hormone (GnRH), a ten amino acid peptide (Figure 1), synthesized in hypothalamic neurons situated in the preoptic area

(POA) and anterior hypothalamus, stimulates gonadotrophs via GnRH receptors in the anterior pituitary gland to release luteinizing hormone (LH) and follicle stimulating hormone

(FSH) into the circulation (Figure 2) (Gore 2002). The secretion of LH also demonstrates a pulsatile pattern and reflects the pulsatile secretion of GnRH (Dierschke et al. 1970). LH and

FSH in turn act on the gonads to activate gametogenesis and sex steroid production. Sex steroids and gonadotrophins inhibit further GnRH secretion, in a negative feedback system.

A failure at any point in the HPG axis or metabolic stress and extremes in energy reserves may result in disordered reproductive function and infertility (Neill J.D 2006).

One in seven couples in the United Kingdom suffers with infertility (HEFA Fertility Facts and

Figures 2008). The resulting impact on physical, psychological and social functioning can be dramatic. Current therapies for infertility are associated with limitations both in terms of side effects and drug delivery. Improving our understanding of the physiology of the HPG axis may lead to the development of novel therapies for infertility.

Figure 1. The amino acid sequence of GnRH decapeptide. pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2

Figure 2. The hypothalamic pituitary gonadal (HPG) axis (Conn & Ulloa-Aguirre 2010).

The HPG axis is controlled in a classical endocrine feedback loop. Gonadotrophin releasing hormone (GnRH) is synthesised and secreted by specialised neurons located in the preoptic and arcuate nucleus of the hypothalamus. GnRH neurons have projections to the median eminence (ME) where GnRH is released into the hypophyseal-portal capillary circulation to act on gonadotrope cells of the adenohypophysis (AH). This stimulates the release of luteinizing hormone (LH) and follicle stimulating hormone (FSH), which in turn act on the gonads to activate gametogenesis and sex steroid production. The pulsatile release of GnRH, LH, and FSH are positively or negatively regulated by several hypothalamic neurotransmitters and sex steroids.

In the male, LH binds to LH receptors on the Leydig cells of the testis resulting in the stimulation of the synthesis and secretion of androgens, primarily testosterone. Testosterone stimulates spermatogenesis and the development of secondary sexual characteristics. In the female, LH stimulates secretion of testosterone from the theca cells of the ovary; this is converted into oestrogens by neighbouring granulosa cells. Follicle stimulating hormone

(FSH) stimulates folliculogenesis. A mid-cycle surge of LH in the pre-ovulatory phase of the menstrual cycle induces ovulation (Levi-Setti et al. 2004). Although generally LH release is suppressed by negative feedback from ovarian steroids, during the pre-ovulatory phase the high levels of oestradiol secreted from the developing ovarian follicle and progesterone result in positive feedback and stimulate LH release. Following ovulation the corpus luteum forms in the follicle, which secretes progesterone and oestradiol. Throughout the menstrual cycle the changing levels of LH and FSH (figure 3) act in a synergistic manner to promote follicular development, oestradiol synthesis, follicular rupture and ovulation (Levi-Setti et al.

2004)

Figure 3. The human female menstrual cycle (Aitken et al. 2008).

The follicular phase of the menstrual cycle begins on the first day of menstrual bleeding. A decrease in levels of oestrogen and progesterone results in the breakdown and shedding of the endometrium. Follicle stimulating hormone (FSH) levels increase, stimulating the development of several oocyte follicles. FSH levels subsequently decrease and only one or two follicles continue to develop. The developing follicles release oestrogen, which initiates thickening of the endometrium. The second phase of the menstrual cycle is the ovulatory phase which begins at approximately day 13, levels of LH and FSH increase dramatically; levels of oestrogen also peak at this time and levels of progesterone begin to increase. The high levels of LH stimulate ovulation. During the luteal phase, levels of LH and FSH decrease and the ruptured follicle forms the corpus luteum, which produces progesterone. The corpus luteum degenerates without fertilisation and the decreased levels of progesterone and oestrogen, initiates a new menstrual cycle.

GnRH displays structural conservation across species and all mammals, except the guinea pig, have an identical decapeptide sequence (Gore 2002). In the guinea pig GnRH sequence, tyrosine replaces histidine at position 2 and valine replaces leucine present at position 7

(Gore 2002). The secretion of GnRH from hypothalamic neurons varies throughout human development. Pulsatile secretion of GnRH is noted in early gestation and continues until six months of age in boys and two years in girls. Re-emergence of GnRH secretion does not occur until puberty is approached, when pulsatile secretion of GnRH occurs first during sleep followed by the addition of daytime pulses. Thus the pulse interval and amplitude of GnRH pulses varies throughout development and also during the female menstrual cycle (Neill J.D

2006). LH release stimulated by GnRH is lower in the luteal phase of the menstrual cycle compared with the follicular phase. Pulse interval is 110 minutes in the early follicular, 70 minutes in the mid-follicular and 65 minutes in periovulatory phase. This is more frequent than the luteal phase pulses, which are 100, 200 and 300 minutes in the early, middle and late luteal phase (Gore 2002). Due to other factors such as the action of inhibin, the secretion of FSH, unlike that of LH, does not strictly reflect that of pulsatile GnRH secretion

(Clarke, Moore, & Veldhuis 2002;Clarke et al. 1986). The pulsatile secretion of GnRH is necessary for normal reproductive function. Continuous chronic administration of GnRH causes downregulation of the GnRH receptor, resulting in a failure to stimulate pituitary LH and FSH release (Belchetz et al. 1978). Thus GnRH has a pivotal role in regulating reproduction and disordered secretion of GnRH can result in a failure of normal reproductive function.

GnRH neurons have projections to the median eminence where GnRH is released into the hypophyseal-portal capillary circulation (Gore 2002). The onset of puberty and normal reproductive function throughout adulthood is dependent on the pulstatile release of GnRH from the anterior hypothalamic region acting on the anterior pituitary gland to stimulate gonadotrophin release (Belchetz et al. 1978). This “pulse generator” appears to be contained either in the GnRH neuronal network or in the GnRH neurone. In vitro studies have

18 demonstrated that the hypothalamic neuronal cell line GT1-7 secretes GnRH in a spontaneous pulsatile fashion (Wetsel et al. 1992). In addition GnRH neurones release

GnRH in a pulsatile manner without hypothalamic inputs (Funabashi et al. 2000;Terasawa et al. 1999). In vivo a number of excitatory and inhibitory factors influence GnRH secretion; these include noradrenaline, neuropeptide Y (NPY), glutamate, gamma-aminobutyric acid

(GABA) and endogenous opioids, nitric oxide, cyclic adenosine monophosphate (cAMP), and adenosine triphosphate (ATP) may modify GnRH release without synaptic input (Evans

1999;Terasawa 2001) (Table 1). The precise mechanisms controlling the pulsatile release of

GnRH are not fully established. Sex steroid feedback, stress and nutrition also have a role in the regulation of the hypothalamo pituitary gonadal axis. Recently kisspeptin has been identified as a key player in the regulation of GnRH secretion and reproduction.

Table 1. Neurotransmitters acting on Gonadotrophin releasing hormone (GnRH) neurons.

A number of neurotransmitters have either inhibitory, stimulatory or both inhibitory and stimulatory actions on GnRH neurones.

Inhibitory Stimulatory Acetycholine Acetycholine

Cholecystokinin Angiotensin II

CRF Cholecystokinin

Dopamine Delta sleep inducing peptide

GABA Dopamine

Opioids Endothelin

Oxytocin Galanin

Serotonin Glutamate

Vasopressin Histamine

Leptin

Neuropeptide Y

Neurotensin

Norepinephrine

Oxytocin

Serotonin

Substance P

VIP

Vasopressin

1.2 Kisspeptin

In 2003 the kisspeptins and their receptor GPR54 (KISS1R) were identified to play a pivotal role in the regulation of reproduction and puberty (de Roux N. et al. 2003;Seminara et al.

2003). In humans the kisspeptins are the neuropeptide products of the KISS1 gene, and are the ligands for the G-protein-coupled receptor KISS1R (GPR54) (Kotani et al. 2001; Lee et al.

1999;Muir et al. 2001; Ohtaki et al. 2001). The KISS1 gene encodes a 145 precursor amino acid peptide which undergoes proteolytic processing to produce shorter peptides. The kisspeptins are named according to amino acid number, kisspeptin-10, 13, 14 and 54; and all share a common C-terminal decapeptide necessary for receptor binding (Kotani et al.

2001) (Figure 4). The kisspeptin peptide hormones all have an arginine-phenylalanine residue present at the carboxy terminal (Clements et al. 2001; Kotani et al. 2001), and thus belong to the RF family of peptides.

Figure 4. The amino acid structure of the kisspeptins.

The kisspeptin hormones all have a common carboxy-terminal decapeptide sequence highlighted in grey. This sequence is necessary for biological activity.

Kisspeptin-54

GTSLSPPPESSGSRQQPGLSAPHSRQIPAPQGAVLVQREKDLPNYNWNSFGLRF-NH2

Kisspeptin-14

DLPNYNWNSFGLRF-NH2

Kisspeptin-13

LPNYNWNSFGLRF-NH2

Kisspeptin-10

YNWNSFGLRF-NH2

Kisspeptin 10 is highly conserved between humans and mice, with only one amino acid replacement; tyrosine to phenylalanine (Stafford et al. 2002). The KISS1 gene was initially defined as a metastasis suppressor gene (Lee & Welch 1997) and thus kisspeptin was originally termed metastin. In vivo studies have demonstrated that KiSS-1 suppresses metastasis in human melanoma cells and breast carcinomas (Lee & Welch 1997).

Kisspeptin-54 has also been shown to inhibit pulmonary metastasis in a murine melanoma model (Ohtaki et al. 2001). Kisspeptin inhibits the chemotaxis, motility and growth of Chinese

Hamster Ovary (CHO) cells with KISS1R, but not that of mock transfectants, thus its actions appear to be mediated via the KISS1R (Ohtaki et al. 2001). The kisspeptin/GPR54 system may have a role in cancer progression (Dhar et al. 2004; Masui et al. 2004;Shirasaki et al.

2001), GPR54 is over-expressed in various forms of malignant tissue compared with normal tissue (Muir et al. 2001; Ohtaki et al. 2001). Plasma kisspeptin levels have been shown to be

22 elevated in women with placental malignancies such as gestational trophoblastic neoplasia

(Dhillo et al. 2006). The precise mechanisms by which KiSS-1 expression or GPR54 activation influence metastasis are not yet fully understood.

Data from rodents, sheep, primates and humans has demonstrated KISS1 mRNA to be expressed both centrally in the anteroventral periventricular nucleus (AVPV) or preoptic area and the arcuate or infundibular nucleus; as well as peripheral expression in a number of organs including the placenta, gonads, pancreas and liver (Adachi et al. 2007; Estrada et al.

2006; Gottsch et al. 2004; Kauffman et al. 2007; Kotani et al. 2001; Lee et al. 1999; Muir et al. 2001; Navarro et al. 2004; Ohtaki et al. 2001;Rometo et al. 2007; Shahab et al. 2005;

Smith et al. 2007).

The kisspeptin/GPR54 system has an essential role in the reproductive development of humans (de Roux N. et al. 2003; Seminara et al. 2003). Inactivating mutations in the GPR54 gene result in hypogonadotrophic hypogonadism and a failure to undergo puberty in humans and mice (de Roux N. et al. 2003; Funes et al. 2003; Seminara et al. 2003). GPR54 null mice have anatomically normal GnRH neurons with normal levels of hypothalamic GnRH.

However, they display phenotypical features of hypogonadotrophic hypogonadism and do not go through puberty, they are phenotypically normal in all other respects (de Roux N. et al.

2003; Funes et al. 2003; Seminara et al. 2003). Administration of GnRH to GPR54 null mice stimulates pituitary gonadotrophin release (Funes et al. 2003; Seminara et al. 2003). In contrast administration of kisspeptin to GPR54 null mice at a dose equivalent to stimulating gonadotrophin release in wild type mice, does not result in gonadotrophin release (Messager et al. 2005). Humans with inactivating mutations in the GPR54 gene have clinical features of hypogonadotrophic hypogonadism (Cerrato et al. 2006; de Roux N. et al. 2003; Lanfranco et al. 2005; Pallais et al. 2006; Seminara et al. 2003; Semple et al. 2005; Tenenbaum-Rakover et al. 2007). They do not display any features associated with abnormal GnRH neuron migration (such as anosmia), and apart from reduced gonadotrophin release, have normal anterior pituitary function (Seminara et al. 2003). Treatment of patients with GPR54

23 inactivating mutations with GnRH or gonadotrophins has allowed some of these patients to undergo gametogenesis, successful placental function during gestation and lactation

(Lanfranco et al. 2005; Pallais et al. 2006; Tenenbaum-Rakover et al. 2007). Thus these findings suggest that inactivating mutations of the GPR54 gene result in disordered GnRH secretion and subsequently hypogonadotrophic hypogonadism, but that the GPR54 may not be vital to maintain pregnancy.

Precocious puberty can be stimulated with administration of kisspeptin to immature prepubertal female rats, as demonstrated by signs of premature vaginal opening, increased uterine weight and stimulation of ovulation (Matsui et al. 2004; Navarro et al. 2004).

Kisspeptin can also induce pulsatile LH secretion in juvenile rhesus monkeys, these results appear to be secondary to GnRH stimulation, as the utilisation of GnRH antagonists abolished the effects of kisspeptin (Plant, Ramaswamy, & Dipietro 2006). In humans an activating mutation of GPR54 has been reported to result in central precocious puberty

(Teles et al. 2008). The substitution of proline for arginine at amino acid 386 resulted in extended activation of GPR54 receptor with kisspeptin administration (Teles et al. 2008).

Three further KISS1R mutations, P74S and H90D (Silveira et al. 2010), and Pro196His

(Luan et al. 2007); and a kisspeptin variant Pro110Thr (Luan et al. 2007; Luan et al. 2007); have been identified in idiopathic central precocious puberty. Thus the kisspeptin / GPR54 system has an essential role in pubertal regulation.

1.3 The kisspeptin receptor and kisspeptin signaling

The kisspeptin receptor also known as the GPR54 (abbreviated to KISS1R in humans) is a

G protein coupled receptor (GPCR). The KISS1R shares 45% amino acid similarity to the galanin receptor (Lee et al. 1999), despite this homology galanin is not a ligand for the

KISS1R.

Binding kisspeptin to its receptor stimulates phospholipase C which in turn activates second messenger systems inositol 1,4,5-trisphosphate and diaclyglycerol resulting in increased intracellular calcium (Kotani et al. 2001; Muir et al. 2001; Stafford et al. 2002). The minimum sequence length of kisspeptin required for activation is the ten amino acid sequence at the C terminal (Kotani et al. 2001). Although in vitro studies have demonstrated similar biological activity of the kisspeptins (Kotani et al. 2001); in vivo studies suggest that kisspeptin 54 (KP-

54) has a higher potency (Thompson et al. 2006).

In humans there is widespread distribution of the kisspeptin receptor. Highest levels are expressed in the placenta, but the receptor is distributed throughout the body in a number or organs, the central nervous system and peripheral vasculature; these include the cerebral cortex, thalamus, medulla, cerebellum pituitary, pancreas, spinal cord, heart, muscle, kidney, liver, aorta, coronary arteries and umbilical vein (Clements et al. 2001; Kotani et al. 2001;

Mead et al. 2007; Muir et al. 2001; Ohtaki et al. 2001).

G protein coupled receptors exhibit receptor desensitisation (Lefkowitz 1998; Ritter & Hall

2009). Acutely desensitisation occurs via loss of signaling to secondary messenger systems due to persistent ligand binding and subsequently receptor internalisation. Degradation rather than recycling of these receptors results in chronic desensitisation (Ferguson 2001).

Recently it has been shown that the sequence variant R368P which clinically results in precocious puberty (Teles et al. 2008), reduces the rate of desensitisation of the kisspeptin receptor by decreasing the degradation of the receptor resulting in prolonged responsiveness to kisspeptin (Bianco et al. 2011). Degradation of the kisspeptin receptor appears to be via proteasomes rather than the more common pathway of GPCR degradation which is via lysosomes (Bianco et al. 2011).

1.4 Kisspeptin administration results in stimulation of reproductive hormone release

Kisspeptin administration either via central or peripheral routes stimulates the HPG axis in a number of animal models including rodents, sheep, pigs, primates and humans (Arreguin-

Arevalo et al. 2007; Caraty et al. 2007; Dhillo et al. 2005; Dhillo et al. 2007; Gottsch et al.

2004; Irwig et al. 2004; Lents et al. 2008; Matsui et al. 2004; Messager et al. 2005; Navarro et al. 2004; Plant, Ramaswamy, & Dipietro 2006; Ramaswamy et al. 2007; Seminara et al.

2006; Thompson et al. 2004). Evidence suggests this effect appears to be predominantly mediated via the release of GnRH (Gottsch et al. 2004; Irwig et al. 2004; Matsui et al. 2004;

Messager et al. 2005). The use of GnRH antagonists inhibits kisspeptin stimulated increases in gonadotrophins (Gottsch et al. 2004; Irwig et al. 2004; Matsui et al. 2004; Shahab et al.

2005). Intracerebroventricular administration of kisspeptin to sheep results in an increase in cerebrospinal fluid GnRH (Messager et al. 2005). Administration of GnRH to humans and mice with GPR54 mutations results in pituitary release of gonadotrophins suggesting that the kisspeptin/GPR54 system acts upstream of GnRH neurons (Seminara et al. 2003). GPR54 expression has been co-localized to GnRH neurons in rodents (Irwig et al. 2004; Messager et al. 2005) and kisspeptin axons project to GnRH neurons (Clarkson & Herbison 2006).

Intracerebroventricular administration of kisspeptin to mice results in increased expression of neuronal markers of activation in GnRH neurons (Irwig et al. 2004). In addition the use of a kisspeptin antagonist at the GPR54 receptor inhibits activation of GnRH neurons and inhibits the release of GnRH and subsequently gonadotrophin release (Roseweir et al. 2009).

Kisspeptin is released into the stalk median eminence in a pulsatile fashion with a pattern that demonstrates high concordance with the release of GnRH (Keen et al. 2008).

Sex steroids exert both a negative and positive feedback on GnRH secretion. However the precise mechanisms that regulate these effects are unknown. GnRH neurons do not express oestrogen alpha or androgen receptors (Clarkson & Herbison 2006). In contrast the majority of Kiss1 neurons in the hypothalamus of rodents do express oestrogen alpha receptors

(Smith et al. 2005; Smith et al. 2005) and also express androgen receptors (Smith et al.

2005). Thus kisspeptin neurons may relay sex steroid signals to GnRH neurons. In rodents low circulating sex steroids result in an increase in Kiss1 mRNA expression in the arcuate nucleus but a decrease in Kiss1 mRNA expression in the anteroventral periventricular nucleus (Smith et al. 2005a; Smith et al. 2005b). This configuration of kisspeptin expression is reversed with replacement of sex steroids. Thus this rodent model suggests that kisspeptin neurons in the arcuate nucleus may be involved in negative feedback regulation of HPG axis and those in the AVPV with positive feedback regulation of GnRH secretion

(Smith et al. 2005a; Smith et al. 2005b). Studies from other species show that gonadectomised animals with subsequently low sex steroid levels exhibit increased Kiss1 expression in the arcuate or infundibular nucleus and increased gonadotrophin levels due to the removal of negative feedback of sex steroids on the HPG axis (Kauffman et al. 2007;

Shibata et al. 2007; Smith et al. 2005a; Smith et al. 2005b). Elevated levels of gonadotrophins are not found in gonadectomised GPR54 null mice, even though Kiss1 gene expression is increased in their arcuate nuclei, suggesting that the kisspeptin GPR54 system is pivotal in the negative feedback stimulation of sex steroids on the HPG axis (Dungan et al.

2007).

The use of a kisspeptin antagonist has been demonstrated to inhibit the post castration rise in LH in male mice, which occurs due to removal of negative feedback of sex steroids on the

HPG axis. Thus kisspeptin appears to be pivotal in relaying signals between sex steroids and GnRH neuron modulation (Roseweir et al. 2009b).

1.5 Kisspeptin and ovulation

In females positive feedback of oestrogen and progesterone on GnRH secretion triggers the

LH surge required for ovulation. Data from rodents suggests that the AVPV is integral in this preovulatory GnRH/LH rise (Herbison 2008; Smith et al. 2006). Rodent models have

27 demonstrated sexual differentiation in the expression of Kiss1, with an increased expression of Kiss1 mRNA and kisspeptin in the AVPV of females (Adachi et al. 2007; Clarkson &

Herbison 2006; Kauffman et al. 2007). In female mice there are an increased number of kisspeptin fibre projections to GnRH neurons compared to male mice (Clarkson & Herbison

2006). Evidence suggests that kisspeptin neurons in the AVPV may be involved in the regulation of ovulation. In female rats the injection of a specific kisspeptin monoclonal antibody into the preoptic area close to GnRH neuron cell bodies, results in elimination of the preovulatory LH surge and inhibits oestrous cyclicity (Kinoshita et al. 2005). Differential hypothalamic expression of KISS-1 is noted during proestrous or experimentally evoked LH surge in female rats; with increased expression of kiss1 and elevated c-fos expression noted in the anteroventral periventricular nucleus in direct contrast to expression in the arcuate nucleus (Smith et al. 2006). In females oestrogen administration results in an increased expression of kiss1 mRNA in the AVPV (Adachi et al. 2007; Smith et al. 2005a; Smith et al.

2006). In addition the majority of kisspeptin neurons in the AVPV express oestrogen receptor alpha, which is proposed to mediate the positive feedback of oestrogen (Glidewell-Kenney et al. 2007; Smith et al. 2005a; Smith et al. 2005b; Wintermantel et al. 2006). Using dual label immunofluorescence techniques Kiss1 neurons appear to have axonal projections to GnRH neurons, with an increase in the number of kisspeptin neuron fibres juxtaposing GnRH neuron cell bodies occurring prior to puberty in the rostral preoptic area of the mouse hypothalamus. However, the absolute numbers of GnRH neurons receiving kisspeptin neuron synaptic connections have not been clarified and inputs to GnRH neuron dendrites has not been established (Clarkson & Herbison 2006). Confirmation of synapse apposition is required with other methods such as electron microscopy. Similar findings have been reported in experiments on female mice, with the identification that kisspeptin neurons in the rostal periventricular area of the third ventricle express oestrogen receptor alpha and progesterone receptors and that these kisspeptin neurons are activated during the GnRH/LH surge (Clarkson et al. 2008).

Importantly, wild type mice which are ovariectomised and supplemented with oestrogen and progesterone are able to generate LH surges, however GPR54 null and Kiss-1 null mice which underwent the same procedure were unable to mount a LH surge and did not display

GnRH neuron activation (Clarkson et al. 2008). This highlights the essential role of the kisspeptin/GPR54 system in linking sex steroid signals and GnRH neuron activation required for ovulation. However, a previous study has reported that GPR54 null mice are able to mount an oestrogen induced LH surge, with simultaneous c-Fos induction in GnRH neurons

(Dungan et al. 2007).These two studies differed both in the methods used to generate

GPR54 knockout mice and in the experimental protocols used to induce and LH surge which may account for the conflicting results (Clarkson et al. 2008; Dungan et al. 2007).

Kisspeptin administration can stimulate ovulation in gonadotrophin primed prepubertal rats with comparable rates to human chorionic gonadotropin (hCG) treatment (Matsui et al. 2004).

In addition infusions of kisspeptin can induce timed LH surges and ovulation in progesterone primed cyclical ewes (Caraty et al. 2007); even during anestrous kisspeptin infusions were able to elicit ovulation in acyclic ewes (Caraty et al. 2007). In keeping with these findings in animals, peripheral administration of kisspeptin stimulates gonadotrophin release during each phase of the menstrual cycle in healthy women but its effects were most potent during the preovulatory phase (Dhillo et al. 2007).

1.6 Metabolic regulation of the reproductive axis

Idiopathic hypogonadotrophic hypogonadism, resulting from either a hypothalamic defect in secretion of GnRH or the defective action of GnRH on the pituitary, results in a failure to undergo puberty and infertility. Women with functional hypothalamic amenorrhoea (HA) are hypogonadal or eugonadal, have low oestrogen levels and a lack of menses without any associated organic pathology. As a result these women are infertile and studies indicate that hypothalamic amenorrhoea is a major cause of infertility, accounting for 30% of cases of

29 amenorrhoea (Reindollar 1986). Hypothalamic amenorrhoea results from a failure of pulsatile GnRH secretion from the hypothalamus and may be triggered by energy deficits associated with weight loss, exercise and eating disorders as well as psychological stress.

Recent studies have identified that certain women with rare variants in genes associated with idiopathic hypogonadotrophic hypogonadism may have increased genetic susceptibility for hypothalamic amenorrhoea (Caronia 2011).

Fertility and body nutritional status are closely interwoven, during periods of under nutrition or a lean body weight GnRH production is decreased and thus reproductive ability is reduced or absent (Schneider 2004). By contrast states of obesity can also negatively impact reproductive ability. The hormone leptin is synthesised and secreted by adipocytes in accordance to body energy stores, circulating leptin is proportional to fat stores and falls after weight loss. Leptin has been postulated to relay signals between nutritional status and reproductive functioning. Leptin deficient ob/ob rodent models have hypogonadotrophic hypogonadism with a resultant failure to undergo puberty and infertility (Zhang et al. 1994).

Humans with mutations in either the leptin receptor or leptin also display the phenotype of hypogonadotrophic hypogonadism (Farooqi & O'rahilly 2004). LH secretion is suppressed with fasting but these effects are overcome with the administration of leptin in both rodent and primate leptin deficient models (Finn et al. 1998; Nagatani et al. 1998). Leptin administration to juvenile mice stimulates the onset of puberty (Ahima et al. 1996 Chehab,

Lim, & Lu 1996). Healthy men and women exposed to calorie restriction have hypoleptinaemia (Chan et al. 2003; Schurgin et al. 2004). Women with hypothalamic amenorrhoea; including those of normal weight, (Miller et al. 1998) have reduced plasma levels of leptin, and treatment with twice daily injections of leptin in these women results in stimulation of gonadotrophin release and ovulation (Chou et al. 2011; Welt et al. 2004).

Leptin does not appear to have a direct action on GnRH neurons (Quennell et al. 2009) and

GnRH neurons in the hypothalamus do not express the leptin receptor. Kisspeptin has been proposed as a potential mediator between leptin and GnRH signaling. Forty percent of

30 kisspeptin neurons in the hypothalamus express the leptin receptor (Smith 2006). In rodent models of leptin deficiency, hypothalamic kiss1 expression is reduced and leptin replacement results in an increase in expression (Smith 2006). Short term fasting leads to decrease in hypothalamic expression of KiSS-1 mRNA and an increase in receptor GPR54 mRNA in prepubertal rats (Castellano et al. 2005). Administration of kisspeptin to rats with delayed puberty due to under nutrition results in restoration of pubertal signs as measured by vaginal opening and gonadotrophin secretion (Castellano et al. 2005). Conversely a recent study has reported that mice lacking the leptin receptor on kisspeptin neurons had normal pubertal development (Donato, Jr. et al. 2011). However, the effects of leptin on kisspeptin neurons may differ in mechanism in specific regions of the hypothalamus, with those kisspeptin neurons in the rodent arcuate nucleus having a direct leptin action on kisspeptin neurons and those in the rostral periventricular area of the third ventricle (RP3V) having an indirect action mediated by currently unknown upstream leptin sensitive neurons

(Quennell et al. 2011).

1.7 Potential therapeutic applications of kisspeptin

Kisspeptin may have therapeutic potential in manipulating the human HPG axis via release of GnRH. Various animal models have demonstrated that kisspeptin can stimulate the HPG axis. Two studies in healthy male and female volunteers have also demonstrated its potential in human use with no reported side effects (Dhillo et al. 2005; Dhillo et al. 2007).

The first study investigated the effects of intravenous kisspeptin-54 administration to six healthy males in a double blind placebo controlled trial (Dhillo et al. 2005). Compared to placebo, infusion of kisspeptin-54 at a dose of 4 pmol/kg/min resulted in increases of mean plasma LH by 100%, and FSH 18%. The second study of subcutaneous administration of kisspeptin-54 to healthy females demonstrated that kisspeptin can stimulate the HPG axis in females in a dose dependent manner with significant increases in mean plasma LH and FSH; and its effects were most pronounced in the preovulatory phase of the menstrual cycle

(Dhillo et al. 2007). Both these studies confirmed that kisspeptin, similar to animal models, can stimulate reproductive hormone release in humans. They also reported no adverse effects associated with kisspeptin administration. Kisspeptin has been shown to have vasoconstrictor actions in vitro (Mead et al. 2007) however in vivo studies in humans have not observed any changes in blood pressure or heart rate following kisspeptin administration

(Nijher et al. 2010).

Thus kisspeptin has a number of possible therapeutic uses. The stimulation of the HPG axis is required for ovulation and kisspeptin has a potential role in the treatment of infertility and in vitro fertilisation treatment. Kisspeptin stimulates the release of endogenous gonadotrophins, so potentially may offer a more controlled HPG axis stimulation without the risk of excessive stimulation of gonadotrophin release and possible subsequent ovarian hyperstimulation syndrome.

In addition the mode of administration of kisspeptin may determine its therapeutic use; acute administration of kisspeptin stimulates the HPG axis whilst chronic administration of kisspeptin may downregulate the HPG axis (Ramaswamy et al. 2007; Seminara et al. 2006;

Thompson et al. 2006). In certain clinical cases such as sex steroid dependent tumours chronic administration of kisspeptin or long acting KISS1R agonists may have a role in achieving medical castration. Exciting studies on the development of kisspeptin antagonists and agonists, will further our understanding of the role of kisspeptin in the regulation of gonadotrophin release and potential therapeutic uses. Specific kisspeptin receptor antagonists have been shown to inhibit the simulation of GnRH neuron firing by kisspeptin

(Roseweir et al. 2009). These studies demonstrated that kisspeptin antagonists can inhibit

GnRH pulses but do not affect basal GnRH levels in pubertal female rhesus monkeys; additionally there were no effects on basal LH levels but the stimulation of gonadotrophin release by kisspeptin was reduced in uncastrated male mice pretreated with a kisspeptin antagonist. Castration and the subsequent reduction in sex steroids result in removal of negative feedback on the HPG axis and an increase in gonadotrophins; kisspeptin

32 antagonists were able to inhibit this increase in LH in castrated male mice in a dose dependent manner. Central administration of a kisspeptin antagonist resulted in a reduced

LH pulse amplitude in ovariectomised ewes. These findings illustrate that kisspeptin is required for pulsatile GnRH secretion in terms of both frequency and amplitude. The effects of kisspeptin antagonist on basal LH are exciting as they offer a therapeutic method of inhibiting pulsatile gonadotrophin release without reducing basal levels. This would offer a potential new method of contraception (Roseweir et al. 2009).

The kisspeptins share a 10 amino acid c-terminal region required for receptor activation

(Kotani et al. 2001). The development of smaller peptides with GPR54 agonist properties have demonstrated that the c-terminal five amino acid residues are necessary for GPR54 receptor activation, with the generation of pentapeptide analogues as GPR54 agonists (Niida et al. 2006; Tomita et al. 2006; Tomita et al. 2007; Tomita et al. 2007). Novel studies of structure and biological activity of kisspeptin-10 analogues have identified the importance of combined in vitro and in vivo experiments. Amino acids 6 and 10 appear to be essential for in vitro kisspeptin-10 GPR54 receptor binding (Gutierrez-Pascual et al. 2009). However in vivo studies demonstrate that kisspeptin-10 analogues with Ala point substitutions at amino acid 6 still function as partial agonists with an ability to stimulate gonadotrophin release in adult male Wistar rats (Gutierrez-Pascual et al. 2009). The development of low molecular weight oral KiSS-1R agonists and antagonists may offer practical advantages to current treatments for disorders of reproductive function in humans (Gutierrez-Pascual et al. 2009;

Niida et al. 2006; Orsini et al. 2007; Tomita et al. 2006; Tomita et al. 2008).

1.8 Hypothesis and Aims

Kisspeptin has been identified to be a key regulator in the control of the hypothalamo- pituitary-gonadal axis. The 145-amino acid precursor of kissepeptin-54 encoded by KISS1 is also processed to 14, 13 and 10 amino acid sequences (Kotani et al. 2001). Previous work from our laboratory has shown that kisspeptin-54 stimulates gonadotrophin release in healthy men and women without any adverse effects (Dhillo et al. 2005, Dhillo et al 2007).

However, no previous studies have examined the effects of kisspeptin in women with infertility due to hypothalamic amenorrhoea. Rodent models of hypothalamic amenorrhoea suggest that there is a decrease in hypothalamic expression of kisspeptin in this condition

(Castellano et al. 2005). I hypothesise that women with hypothalamic amenorrhoea, may also have low hypothalamic kisspeptin expression and kisspeptin may restore the functioning of their HPG axis. It is therefore essential that we examine the effects of both acute and chronic kisspeptin administration, as well as different dosing regimes, on women with hypothalamic amenorrhoea.

Animal models suggest that kisspeptin-10 and kisspeptin-54 act similarly to stimulate reproductive hormone release however, kisspeptin-10 is characterised by a shorter half-life.

Recently it has been reported that an intravenous bolus of kisspeptin-10 potently stimulates

LH secretion while continuous infusion also increases testosterone, LH pulse frequency and amplitude in healthy men (George et al. 2011). In addition kisspeptin-10 appears to reset the

GnRH pulsatile clock in men (Chan et al. 2011). The shorter amino acid sequence of kisspeptin-10 makes it simpler and cheaper to synthesize than kisspeptin-54, therefore it may be a more attractive pharmaceutical agent than kisspeptin-54. However, there have been no previous studies on the effects of kisspeptin-10 on women. I hypothesise that kisspeptin-10 will stimulate reproductive hormone release in healthy men and women in a similar fashion to kisspeptin-54.

The aims of this thesis are to investigate the role of kisspeptin in disorders of reproduction specifically investigating:

 The acute and chronic effects of kisspeptin-54 administration on the HPG axis

in women with hypothalamic amenorrhoea.

 To compare the effects of different dosing regimens of kisspeptin-54

administration on the HPG axis in women with hypothalamic amenorrhoea.

 To investigate the effects of kisspeptin-10 on the HPG axis in healthy men

and women.

Chapter 2

The effects of kisspeptin on the hypothalamo pituitary gonadal axis in women with hypothalamic amenorrhoea

2.1 Introduction

Hypothalamic amenorrhoea is a common cause of infertility in women, accounting for 30% of cases of secondary amenorrhoea (Reindollar et al. 1986). In this condition, abnormal GnRH secretion due to deficient GnRH secretion from the hypothalamus or altered pulsatility patterns results in a failure of the synthesis and secretion of gonadotrophins from the pituitary and subsequently a failure in stimulation of the gonads to release sex steroids. This is similar to the phenotype described in hypogonadotrophic hypogonadism. Functional hypothalamic amenorrhoea describes cases where although there is no structural or organic defect identified in the hypothalamic pituitary gonadal axis, patients have amenorrhoea, low or normal serum gonadotrophins and low oestrogen (Reindollar et al. 1986). Functional hypothalamic amenorrhoea may be caused by energy deficient states caused by excessive exercise (Warren 1980), eating disorders (Boyar et al. 1974), weight loss (Frisch 1996;

Frisch & McArthur 1974), chronic illness and psychological stress (Giles & Berga 1993)

(Meczekalski et al. 2008). There also appears to be a genetic predisposition to hypothalamic amenorrhoea, recently genetic variants in the genes associated with hypogonadotrophic hypogonadism have been identified which may result in increased susceptibility to hypothalamic amenorrhoea in women carrying them (Caronia et al. 2011).

Women with hypothalamic amenorrhoea appear to have leptin deficiency and when given leptin demonstrate restoration of GnRH pulsatility and menstruation (Welt et al. 2004). In a randomised double blinded, placebo controlled 36 week trial of twenty women with hypothalamic amenorrhoea; the administration of subcutaneous injections of human recombinant leptin (metreleptin) to the treatment group resulted in restoration of menstrual cyclicity, with over 50% of cycles being ovulatory (Chou et al. 2011). These authors also note that in addition to biochemical evidence for normalisation of the HPG axis, thyroid and adrenal axis, there were also positive changes in markers of bone formation and resorption.

With regards to treatment, oral contraceptive pills are given to those women who require oestrogen replacement to avoid long term side effects of oestrogen deficiency; these of

37 course do not restore gonadotrophin release and ovulation and thus do not confer fertility.

There is also no evidence as yet that oestrogen and progesterone replacement result in increased bone density (Gordon 2010).

For those women being treating for infertility due to hypothalamic amenorrhoea the options are, clomiphene, GnRH pump therapy or in vitro fertilisation (IVF) with gonadotrophin injections. Women with hypothalamic amenorrhoea usually have low serum oestrogen levels

(Gordon 2010), thus clomiphene has a poor rate of ovulation induction in these women, as its pharmacologic actions rely on removing oestradiol-mediated negative feedback on pituitary gonadotrophin release and thus increasing circulating gonadotrophin level. Both

GnRH pump therapy and exogenous gonadotrophins result in similar rates of ovulation induction in women with hypogonadotrophic amenorrhea (Martin et al. 1993). Although efficacious, both treatment options have individual flaws associated. GnRH pump therapy has practical delivery problems associated with it. Gonadotrophin therapies are associated with risk of ovarian hyperstimulation syndrome (OHSS). Mild forms of ovarian hyperstimulation are common occurring in a third of women having IVF. However, 3-8% of women will have moderate or severe OHSS (HFEA Fertility Facts & Figures 2008). Ovarian hyperstimulation syndrome is a potentially life threatening condition, which can result in massive ovarian enlargement, ovarian torsion, ascites, hydrothorax, liver dysfunction, thromboembolism, electrolyte imbalance, renal failure and acute respiratory distress syndrome and death.

2.2 Hypothesis and Aims

Acute central and peripheral administration of kisspeptin results in release of gonadotrophin hormone release in animals. To date human studies on kisspeptin have demonstrated that kisspeptin administration acutely stimulates gonadotrophin release in healthy men and women. Rodent models of hypothalamic amenorrhoea have shown that administration of

38 kisspeptin-10 restores gonadotrophin secretion and associated pubertal signs (Castellano

2005). Based on these data, I hypothesised that chronic administration of kisspeptin would stimulate reproductive hormone release and restore menstrual cyclicity in human female subjects with HA.

The aim of this study was to determine the acute and chronic effects of subcutaneous administration of kisspeptin-54 compared with placebo saline injections on reproductive hormone release and ovulation in women with hypothalamic amenorrhoea.

2.3 Methods

2.3.1 Subjects

Ethical approval was granted by the Hammersmith and Queen Charlotte‟s and Chelsea

Hospitals Research Ethics Committee. The study was carried out in accordance with the declaration of Helsinki. All subjects gave full informed written consent prior to study commencement. Recruitment was via advertisements in the local press. After an initial telephone interview, participants were invited for a medical screening. This consisted of a clinical history including a detailed menstrual history, medical examination, electrocardiogram and blood tests. Blood samples were sent to the Imperial College London

NHS Trust haematology and biochemistry laboratories and the following assays were conducted: full blood count, renal profile, liver profile, bone profile, thyroid profile, glucose, gonadotrophins, oestradiol, progesterone, androstenedione, dehydroepiandrosterone, testosterone, sex hormone binding globulin, prolactin, 17- hydroxyprogesterone and cortisol.

Ten women with functional hypothalamic amenorrhorea were recruited. Hypothalamic amenorrhorea was diagnosed if the following criteria were met: secondary amenorrhorea of at least 6 months duration, body mass index (BMI) of below 25 kg/m2 with a stable body weight over the previous 6 months, age between 18–40 years, absence of hormonal contraceptive therapy for one year, absence of systemic disease co-morbidity or active

39 psychiatric illness; absence of therapeutic or recreational drug use; absence of clinical or biochemical hyperandrogenemia; structurally normal hypothalamus and pituitary region assessed by magnetic resonance imaging; structurally normal female reproductive tract visualised on ultrasound; absence of polycystic ovarian appearances on ultrasound; thyroid function biochemistry and serum prolactin levels within reference range and serum LH:FSH ratio <1.5.

2.3 Kisspeptin 54

Kisspeptin-54 was synthesised by the Advanced Biotechnology Centre, Imperial College

London and purified by reverse-phase high performance liquid chromatography (HPLC).

Electrospray mass spectroscopy and amino acid analysis confirmed the identity of the peptide. Toxicology testing in animals was conducted prior to administration to volunteers.

The Limulus Amoebocyte Lysate assay test for pyrogen (LAL; Associates of Cape Cod,

Liverpool, UK) was negative, and the peptide was sterile on culture (Microbiology

Department, Hammersmith Hospital, London, UK).

Study 1: Effect of chronic kisspeptin administration on reproductive hormone secretion in women with hypothalamic amenorrhoea

This was a double-blind, placebo-controlled study, which was conducted over eight weeks.

Each subject (n = 10) received a subcutaneous (sc) injection of kisspeptin-54 or a control injection of 0.9% saline solution. The dose of kisspeptin-54 used was 6.4 nmol/kg. This dose of kisspeptin-54 was chosen as human studies from our laboratories have identified previously that this dose stimulated the highest gonadotrophin release in healthy women

(Dhillo et al. 2007).

Pre study training: each volunteer was trained how to self administer subcutaneous injections into the lower abdominal region. Saline injections were used for training. Subjects were provided with all the necessary equipment to safely self administer their injections and dispose of needles. Subjects were also trained to reconstitute the freeze dried vials containing either control saline or kisspeptin-54 with 0.5ml of 0.9% saline. Each subject was given a specific volume which was calculated according to their weight, to self administer.

Subjects were instructed to keep the vials refrigerated.

The study lasted eight weeks in total and this was divided into a 4 week baseline period, followed by a 2 week treatment phase and then a 2 week post treatment observation period.

During the 8 week study subjects attended the clinical investigation unit for a review and blood tests twice per week. Measurements of serum gonadotrophin levels and sex hormones and plasma kisspeptin were made at each visit. In addition, pelvic ultrasound scans were performed once a week. These twice weekly measurements allowed calculation of mean levels of LH, FSH and oestradiol during the baseline, treatment and post treatment periods. Compliance was assessed with measurement of plasma kisspeptin immunoreactivity (IR). At each visit pregnancy was excluded using a urine hCG test

(Clearview easy HCG, Inverness Medical Innovations Inc. Waltham, MA) (Figure 5).

Figure 5. The 8 week study protocol.

The baseline period was from weeks 1- 4, weeks 5 and 6 were the treatment period (shaded black) and the post treatment period was week 7 and 8. During weeks 1 and 2 all subjects were given saline injections to acclimatise them to the study protocol. Each subject was randomised to receive twice daily double blinded saline or kisspeptin-54 (KP54) 6.4 nmol/kg injections during weeks 5 and 6. Following the first kisspeptin or saline injection, on day 1 of week 5, a four hour blood sampling study was performed. A second 4 hour study was performed on the last injection day which was on day 7, week 6 of the treatment period. LH pulsatility was assessed at the start of the study and after the treatment period. Ultrasound scans were performed once a week during the 8 week study. Twice weekly blood samples were taken for measurement of LH, FSH, E2 and kisspeptin-IR.

4 HOUR BLOOD 4 HOUR BLOOD SAMPLING POST- SAMPLING POST- INJECTON INJECTON ASSESSMENT (week 5, day 1) (week 6, day 7) ASSESSMENT OF LH OF LH PULSATILITY PULSATILITY (week 1, day 1) (week 7, day 1)

WEEK NUMBER: 1 2 3 4 5 6 7 8

TWICE-WEEKLY BLOOD SAMPLING FOR LH, FSH, E2, AND KISSPEPTIN-IR

ONCE-WEEKLY PELVIC ULTRASOUND SCANS

INJECTIONS NO TWICE-DAILY NO OF SALINE INJECTIONS SALINE OR KP54 INJECTIONS INJECTIONS (weeks 1-2) (weeks 3-4) (weeks 5-6; (weeks 7-8) treatment period)

Baseline period (weeks 1-4) On the first day of the study subjects had an assessment of their LH pulsatility. This involved the subjects attending the clinical investigation unit for 8 hours, a cannula was inserted into a forearm vein and 2.5 ml of blood was taken every 10 minutes into plain red top serum vacutainer tubes (Becton, Dickinson, Oxford, UK). Subjects remained supine throughout the study. After clotting and centrifugation at 3000rpm for 10 minutes, serum was separated and frozen at -200 C, until measurement of LH and FSH and oestradiol. All the eight hour pulsatilty studies were commenced in the morning between

8am and 12pm. Blood pressure and heart rate were monitored every 30 minutes during the pulsatility study.

During weeks 1-2 each volunteer administered twice daily subcutaneous injections of saline.

At this stage only volunteers were blinded to the treatment they were receiving as the aim of this baseline period was to allow volunteers to become acclimatised to the study conditions.

Treatment period (weeks 5-6): each subject self administered either subcutaneous injections of saline (n=5) or kisspeptin-54 (n=5). Both subjects and investigators were blinded to the treatment assigned. The dose of kisspeptin administered was 6.4 nmol/kg.

On the first day of the treatment period i.e. week 5 day 1 subjects underwent a four hour sampling study post injection of saline or kisspeptin. This was conducted in a dedicated clinical investigation unit. After a clinical review and negative pregnancy urine hCG test, subjects had a cannula inserted into a large forearm vein in the antecubital fossa. Blood was sampled on arrival (t = -30 min) and then at t = 0 min, following which a subcutaneous injection of either saline or kisspeptin-54 6.4 nmol/kg was administered by the investigator.

Blood was then sampled at t = 15, 30, 45, 60, 75, 90, 120, 150, 180, 210 and 240 minutes.

3ml of blood was collected into lithium-heparin green top tubes (Becton, Dickinson, Oxford,

UK LIP Ltd. Cambridge, UK) containing 5000 kallikrein inhibitor units (0.2ml) aprotinin

(Trasylol, Bayer, Newbury, UK). After immediate centrifugation, plasma was rapidly separated and stored at -20ºC until measurement of kisspeptin immunoreactivity (IR). An

43 additional 3ml of blood was collected into plain red top serum vacutainer tubes (Becton,

Dickinson, Oxford, UK), these samples were allowed to clot and then centrifuged at 3000 rpm for 10 minutes after which serum was separated and frozen at -200 C until measurement of LH, FSH and oestradiol. Blood pressure and heart rate were measured at every time point.

Subjects remained supine throughout the study and were asked if they felt any nausea or other side effects every 30 min. On the last day of the treatment period subjects received only one injection which was administered in the clinical investigation unit. This was followed by a second 4 hour sampling study (Figure 6).

Figure 6. The four hour study protocol.

Participants remained in a supine position throughout the four study. A cannula was inserted into a vein in the antecubital fossa on arrival. A randomised subcutaneous injection of either saline or kisspeptin was administered at time zero. Blood samples were taken at regular time intervals from -30 to 240 min.

subcutaneous injection of kisspeptin or saline

Time (min) -30-30 0 15 30 45 60 75 90 120 150 180 210 240

cannula blood taken inserted

Post treatment period (weeks 7-8) The day after their final injection (week 7, day 1) subjects had a second assessment of their LH pulsatility over 8 hours. Subjects continued to have twice weekly blood tests taken to measure reproductive hormones and pelvic ultrasounds performed once a week during the two week post treatment period.

Study 2 Effect of chronic kisspeptin administration on GnRH sensitivity in women with hypothalamic amenorrhoea

In order to assess their sensitivity to GnRH pre and post chronic kisspeptin-54 administration; five additional women with hypothalamic amenorrhoea had GnRH stimulation tests performed. These women had a baseline GnRH test conducted seven days prior to commencing twice daily kisspeptin-54 injections for two weeks. These GnRH tests were performed in the clinical investigation unit, women were cannulated and blood was sampled for serum LH, FSH and oestradiol (t=-30), they were then given a 100mcg iv bolus of GnRH

(HRF, Intrapharm Ltd, Kent, UK) at time 0. Serial blood samples were taken at times -30, 0,

15, 30, 45, 60, 90, and 120 minutes and serum gonadotrophins and oestradiol were measured. After two weeks of treatment with kisspeptin-54, they underwent a second GnRH test (Figure 7).

Figure 7. The GnRH study protocol.

Five women with hypothalamic amenorrhoea had a GnRH test performed seven days prior to commencing twice daily injections of kisspeptin-54. After this two week treatment period they had a second GnRH test performed. The aim of this study was to assess sensitivity to GnRH pre and post chronic kisspeptin-54 administration.

Measurement of LH, FSH, oestradiol, progesterone, SHBG

LH, FSH, oestradiol and progesterone were measured using automated chemiluminescent microparticle immunoassays (Abbott Diagnostics, Maidenhead, UK). SHBG was measured using a solid-phase two-site chemiluminescent immunometric assay (DPC Immulite,

Siemens, Llanberis, UK). Manufacturer stated reference ranges for females were LH

(follicular), 2-10 IU/L, (midcycle) 20-60 IU/L (luteal), 4-14; FSH (follicular & luteal) 1.5-8 IU/L, oestradiol (early follicular) less than 300 pmol/l, (luteal) 200-1000 pmol/l; and SHBG 40-80 nmol/l. Interassay coefficients of variation were LH 3.4%, FSH 3.5%, oestradiol 3.4%, progesterone 1.8% and SHBG 5.6%. The limit of detection for oestradiol was 70pmol/l, FSH

0.05 mIU/mL, LH 0.07mIU/ml, progesterone 0.1ng/ml and SHBG 0.1nmol/L.

Kisspeptin radioimmunoassay (RIA)

Antibody GQ2 was raised in a sheep immunised with synthetic human kisspeptin-54

(Bachem UK Ltd.) conjugated to BSA by glutaraldehyde and used at a final dilution of 1:

3,500,000. The antibody cross reacted 100% with human kisspeptin-54, kisspeptin-14, and kisspeptin-10 and less than 0.01% with any other related RFamide peptide including prolactin releasing peptide, RFRP1 (human and rat), RFRP2 (human), RFRP3 (human), neuropeptide FF (human) and neuropeptide AF (human). The 125I-kisspeptin-54 label was prepared using the iodogen method (Salacinski et al. 1981) and purified by HPLC. The specific activity of kisspeptin label was 56 Bq/fmol. The assay was performed in duplicate using dilutions of neat plasma in 0.7 ml of 0.06 M phosphate buffer pH 7.2 containing 0.3%

BSA and incubated for 3 days at 4ºC. Free and antibody bound label were then separated by charcoal adsorption. The assay detected changes of 2 pmol/l of plasma kisspeptin with a

95% confidence limit. The intra- and inter-assay coefficients of variation were 8.3% and

10.2% respectively.

Ultrasound scans

All the pelvic ultrasound scans were performed in the department of Radiology at Charing

Cross Hospital, Imperial College NHS Trust. The scans were transabdominal and performed once a week using Toshiba Apilo XG scanners (Toshiba Medical Systems, Nasu, Japan).

Endometrial thickness, mean ovarian volume, mean number of follicles, and the maximum diameter of the largest follicle was measured and recorded at each scan. Ovulation was determined using the following diagnostic criteria: presence of a dominant follicle of diameter

18mm or greater, subsequent collapse of a preovulatory follicle and an associated rise in serum progesterone levels to above 10nmol/l (Pache et al. 1990).

Statistical analysis

All results are presented as mean ± SEM. Statistical comparisons across multiple means were performed using one way ANOVA with the Bonferroni post-hoc correction. Pairs of means were compared using the unpaired two tailed t test. In all cases P<0.05 was

47 considered to be statistically significant. The GnRH tests and 4 hour studies were analysed using two way ANOVA with the Bonferroni post hoc correction.

Assessment of LH pulses was made using the modified Santen and Bardin method (Adams et al. 1994; Hayes et al. 1999). An LH pulse was defined as consisting of at least two LH points, the first of which was at least 2 IU/l above the preceding nadir and greater than 3 times the CV of the LH assay; and the second point fulfilled one these criteria. LH pulses of three points greater than 1 IU/l above the preceding nadir were also included.

3.Results

Study 1: Effect of chronic kisspeptin administration on reproductive hormone secretion in women with hypothalamic amenorrhoea

The demographics of the women in the kisspeptin and saline control groups is shown in table 2. There were five women with hypothalamic amenorrhea recruited into each group; all the women completed the study. There was no significant difference between the two groups in terms of age, weight, body mass index (BMI), duration of amenorrhoea and baseline gonadotrophin and oestradiol levels. Body weight was monitored throughout the study to ensure that all subjects maintained a constant weight. There were no adverse effects reported by subjects or observed in the 4 hour studies post kisspeptin-54 or saline administration.

Table 2. Characteristics of the women in the kisspeptin and saline control groups.

There were five women randomised to either saline or kisspeptin-54 treatment groups. Data is presented as mean ± SEM.

Saline control group Kisspeptin-54 group P value n=5 n=5

Age (years) 24.8 ± 0.5 26.8 ± 2.4 0.28

Weight (kg) 51.8 ± 3.3 54.5 ± 1.1 0.46

BMI (kg/m2) 19.0 ± 0.7 19.9 ± 0.4 0.25

Amenorrhoea 22.4 ± 9.9 23.2 ± 12.7 0.96 duration (months)

LH (IU/L) 4.5 ± 1.6 2.6 ± 0.9 0.32

FSH (IU/L) 6.6 ± 0.7 6.1 ± 1.0 0.69

Oestradiol (pmol/l) 105 ± 13.9 78 ± 4.9 0.1

Baseline plasma kisspeptin in women with hypothalamic amenorrhoea was <2pmol/L.

Following administration of the first dose of kisspeptin-54 there was an acute rise in plasma kisspeptin immunoreactivity (IR) levels which peaked at 45 minutes post injection (Figure 8).

There was no increase in plasma kisspeptin IR post injection of saline.

Figure 8. Plasma kisspeptin immunoreactivity (IR) levels over 4 hours post injection of kisspeptin-54 (KP54) 6.4 nmol/kg or saline control on the first day of the study.

Injections were administered at time 0 minutes. Data is shown as mean ± SEM. **p<0.01,

***p<0.001.

*** ** 6000

KP54 4000 Saline

2000

Plasma Kisspeptin IR (pmol/L) IR Kisspeptin Plasma 0

0 30 60 90 120 150 180 210 240

Time (minutes)

injection

A similar response in kisspeptin IR was observed following the last saline or kisspeptin injection on the second 4 hour study. This was conducted on the last day of two weeks of treatment, with either twice daily injections of saline or kisspeptin-54 6.4 nmol/kg (Figure 9).

Injections were administered at 0 minutes. Data is shown as mean ± SEM. *p<0.05,

***p<0.001.

*** * 6000

KP54 4000 Saline

2000

Plasma Kisspeptin IR (pmol/L) IR Kisspeptin Plasma 0

0 30 60 90 120 150 180 210 240

Time (minutes)

injection

All of the women with HA underwent a 4 week baseline period. During weeks 1 and 2 of this baseline period women received twice daily subcutaneous injections of saline to acclimatise them to the study conditions. Plasma kisspeptin IR levels during this time were <2 pmol/ L.

During weeks 5 and 6, which was the treatment period, the women were randomised to receive either twice daily subcutaneous injections of kisspeptin-54 6.4 nmol/kg or saline control. During the treatment period women who received kisspeptin-54 had a significant increase in plasma kisspeptin IR levels (Figure 10A) measured twice weekly. Those who received saline control did not have any increase in plasma kisspeptin IR (Figure 10B).

Figure 10. Weekly plasma kisspeptin IR levels in women with HA randomised to receive subcutaneous injections of (A) kisspeptin-54 6.4 nmol/kg or (B) saline.

Plasma kisspeptin immunoreactivity was significantly elevated during treatment weeks 5 and

6 compared to weeks 1-4, in those randomised to receive kisspeptin-54. Data is shown as mean ± SEM. ***p<0.001

A 1200 ***

1000

800

600

400

200

Plasma Kisspeptin IR (pmol/L) 0 1 2 3 4 5 6 7 8

Week number

saline KP54 injections injections

1200

1000

800

600

400

200

Plasma Kisspeptin IR (pmol/L) 0 1 2 3 4 5 6 7 8 Week number

saline saline injections injections

On the first 4 hour study day, which was on the first day of the treatment day, administration of kisspeptin-54 resulted in significant increases in serum gonadotrophins and oestradiol in women with hypothalamic amenorrhoea. There were statistically significant (p<0.001) increases in serum LH levels from baseline from 150 min to 240 minutes post injection of kisspeptin-54. At 240 minutes a maximum increase in LH was noted at 24 ± 3.5 IU/L above baseline. Serum levels of FSH also significantly increased (p<0.001) at 180 to 240 minutes post kisspeptin-54 administration. This increase was not as high as that observed with LH, the maximum rise in FSH was 9.1 ± 2.5 IU/L at 240 minutes. Oestradiol levels showed a late rise, with significant increases only noted after 180 minutes. There were no observed changes in levels of LH, FSH and oestradiol from baseline post injection of saline. (Figure

11).

Figure 11. Changes in serum levels of LH (A), FSH (B) and oestradiol (E2) (C) from baseline after administration of subcutaneous injection of either saline or kisspeptin- 54 6.4 nmol/kg (KP54) on the first day of the treatment period.

Injections were administered at 0 min. Data is shown as mean ± SEM *p<0.05, **p<0.01,

***p<0.001

* *** A 28 24 KP54 saline 20 16 12 8

4 LH increase LH (IU/l) 0 -4 0 30 60 90 120 150 180 210 240

injection Time (minutes)

* *** B 12

0 FSH increase FSH (IU/l)

-4 0 30 60 90 120 150 180 210 240

injection Time (minutes)

C 50 *

10 0

-10

-20

-30 Oestradiolincrease (pmol/l) 0 30 60 90 120 150 180 210 240

Time (minutes) injection

On the last day of the treatment period i.e. after two weeks of twice daily subcutaneous injections of either kisspeptin-54 or saline, saline injection did not result in any change in serum levels of LH, FSH and oestradiol. Although kisspeptin-54 administration did result in a significant increase in serum LH, this was only at 240 minutes post injection. The maximal increase in LH at 240 minutes after an injection of kisspeptin-54 was only 2.48 ± 3.76. There was no significant change in serum FSH and oestradiol levels after kisspeptin-54 administration. All the responses were significantly diminished at each time point compared to those achieved on the first day pre treatment (Figure 12).

Figure 12. Changes in serum levels of LH (A), FSH (B) and oestradiol (E2) (C) from baseline after administration of subcutaneous injection of either saline or kisspeptin- 54 6.4 nmol/kg on the last day of the treatment period.

Data is shown as mean ± SEM *p<0.05, **p<0.01, ***p<0.001

A 28 * 24

20 KP54 16 saline 12

LH LH increase (IU/l) 0

-4 0 30 60 90 120 150 180 210 240

injection Minutes

B 12

0 FSH increase (IU/l) -4 0 30 60 90 120 150 180 210 240

injection Time (minutes)

C 50 40 30

20 10 0 -10 -20 -30 -40

Oestradiol increase (pmol/l) 0 30 60 90 120 150 180 210 240

injection Time (minutes)

A summary of the differences in serum levels of gonadotrophins and oestradiol achieved post injection of kisspeptin-54 on the first and last day of the treatment period is shown in

Figure 13. The area under the curve (AUC) represents the total increase in serum LH, FSH and oestradiol achieved in the four hours post injection of kisspeptin-54 6.4 nmol/kg.

Significantly lower responses in LH, FSH and oestradiol were achieved post injection of kisspeptin-54 6.4 nmol/kg on day 14 compared to day 1 of the treatment period.

*p<0.05, **p<0.01, ***p<0.001

B A 60 * 20 *

15 40

5 Meanincrease LH AUC (h.iU/L) 0 increase MeanFSH AUC (h.iU/L) 0

First injection Last injection First injection Last injection

C * 60

-20

-40

-60 Mean AUC E2 increase MeanE2 AUC (h.pmol/L)

First injection Last injection

The mean levels of serum LH, FSH and oestradiol were calculated for the baseline period

(weeks 1-4), the treatment period (weeks 5 & 6) and post treatment (weeks 7 & 8) in both the kisspeptin-54 and saline treated groups (table 3). The mean levels of LH during the baseline period were lower in the kisspeptin-54 treated group compared to those women in the saline study group (p=0.02). During the treatment period the women in the kisspeptin group had small rises in mean levels of LH (p=0.09) and FSH (p=0.02).

Table 3. Comparison of basal serum reproductive hormone levels at baseline, during and after 2 weeks treatment with either saline or kisspeptin injections.

For each subject mean baseline serum levels of LH, FSH and oestradiol were calculated from the blood tests performed during the baseline period of weeks 1-4. Changes in serum levels of LH, FSH and oestradiol levels during the two week treatment period weeks 5-6, and the post treatment period weeks 7-8 were calculated for each subject. Data shown as mean ± SEM.

Study group P value Saline Kisspeptin54

LH (iU/l) Baseline 3.7 ± 0.6 1.8 ± 0.3 0.02 n=5 n=5 Change during 2 week treatment -0.8 ± 0.7 +1.2 ± 0.7 0.09 period

Change during post treatment period -0.6 ± 0.5 -0.4 ± 0.2 0.51

FSH (iU/l) Baseline 7.0 ± 0.8 5.3 ± 0.2 0.07

Change during 2 week treatment -1.8 ± 0.4 +0.6 ± 0.4 0.02 period

Change during post treatment period 0.0 ± 0.57 -0.6 ± 0.3 0.35

Oestradiol Baseline 109 ± 15 91 ± 6 0.28 (pmol/l) Change during 2 week treatment +33 ± 48 +77 ± 67 0.61 period

Change during post treatment period +16 ± 7 +17 ± 14 0.92

An eight hour LH pulsatility study was performed on day 1 of the baseline period and repeated after two weeks of treatment with either twice daily subcutaneous injections of saline or kisspeptin-54 6.4 nmol/kg. A modified Santen and Bardin method was used to assess LH pulsatility. There were no significant changes in mean LH, number of LH pulses or LH pulse amplitude (Table 4).

Table 4. Comparison of LH pulsatility before and after 2 weeks treatment with saline or kisspeptin-54 6.4 nmol/kg injections in women with HA.

Mean levels of serum LH, number of LH pulses and mean LH pulse amplitude are shown as mean ± SEM.

Characteristic Study group P value

Saline Kisspeptin54

n= 5 n= 5 Mean LH (iU/l) Baseline 2.5 ± 0.5 1.5 ± 0.4 0.16

Post-treatment 2.5 ± 0.8 3.2 ± 2.2 0.77

Change -0.08 ± 0.5 +1.7 ± 1.9 0.40

Number of LH Baseline 0.8 ± 0.5 1.0 ± 0.8 0.24 pulses

Post-treatment 1.6 ± 0.8 0.8 ± 0.6 0.48

Change +0.8 ± 0.5 -0.2 ± 0.2 0.12

Mean pulse Baseline 2.7 ± 0.6 1.1 ± 0.05 0.24 amplitude (iU/l)

Post-treatment 2.4 ± 0.2 2.1 ± 0.1 0.48

Change -0.4 ± 0.4 +1.5 ± 0.3 0.12

There was no observed change in any of the parameters measured in the transabdominal ultrasound scans. Ovulation was determined using the following diagnostic criteria, presence of a dominant follicle of diameter 18mm or greater, subsequent collapse of a preovulatory follicle and an associated rise in serum progesterone levels to above 10nmol/l (Pache et al.

1990). None of the women ovulated during the eight week study (Table 5).

Table 5. Summary of ultrasound parameters at baseline, during and after 2 weeks treatment with saline or kisspeptin injections in women with HA.

Endometrial thickness, ovarian volume, follicle number and maximum follicle diameter are shown as mean ± SEM. Baseline mean values were calculated from the four scans performed during the baseline period, weeks 1-4. The change in value of each ultrasound parameter was calculated from the two scans during the treatment period (weeks 5-6) and the two scans performed in the post treatment period (weeks 7-8).

Characteristic Saline kisspeptin54 P value Ovulation (number 0 0 - of subjects n=5 n= 5

Preovulatory follicle 1 1 - ≥ 18mm (no. of subjects) Dominant follicle ≥ 1 3 - 11mm (no. of subjects) Endometrial Baseline 3.2 ± 0.4 4.3 ± 0.9 0.29 thickness (mm)

Change during treatment +0.4 ± 0.3 -0.5 ± 0.5 0.15

Change post-treatment +0.8 ± 0.6 +0.1 ± 0.5 0.36 Ovarian volume Baseline 5.2 ± 1.2 5.8 ± 0.6 0.67 (cm3)

Change during treatment +0.9 ± 0.4 +1.3 ± 0.8 0.69

Change post-treatment +1.2 ± 0.5 +0.7 ± 0.6 0.50 Follicle number per Baseline 12.4 ± 2.1 11.8 ± 1.2 0.79 ovary

Change during treatment -2.3 ± 1.5 +0.8 ± 1.2 0.13

Change post-treatment -0.6 ± 1.8 -0.2 ± 1.2 0.86 Maximum follicle Baseline 6.9 ± 0.3 8.0 ± 0.5 0.11 diameter (mm)

Change during treatment +0.5 ± 1.3 +0.1 ± 0.6 0.83

Change post-treatment 0.0 ± 0.8 +0.2 ± 0.6 0.84

Study 3.2: Effect of chronic kisspeptin administration on GnRH sensitivity in women with hypothalamic amenorrhoea

GnRH tests were performed in order to ascertain if there was an effect of twice daily kisspeptin-54 6.4 nmol/kg subcutaneous injections on the sensitivity of women with HA to

GnRH. Although there was a reduction in the gonadotrophin response to GnRH this was not statistically significant different prior to or following two weeks of twice daily kisspeptin-54 injections (p = 0.23) (Figure 14).

Figure 14. Mean gonadotrophin response to GnRH 100 mcg bolus pre (A) and post (B) treatment with twice daily injections of kisspeptin-54 (KP54) 6.4 nmol/kg.

Data is shown as mean ± SEM, n= 5 women with hypothalamic amenorrhoea.

A 20

pre KP54 6.4nmol/kg 10 post KP54 6.4nmol/kg

5 LH increase (iU/L) LH increase

0 0 30 60 90 120 Time (minutes)

GnRH injection

B 6

2 FSH increase (iU/L) FSH increase

0 0 30 60 90 120 Time (minutes)

GnRH injection

4. Discussion

This is the first study to investigate the effects of kisspeptin-54 in women with infertility due to hypothalamic amenorrhoea. This study has identified that acute administration of kisspeptin to women with HA results in stimulation of reproductive hormones but chronic administration for two weeks of twice daily injections of kisspeptin at a dose of 6.4 nmol/kg results in tachyphylaxis. The second 4 hour study which was conducted after two weeks of treatment with either saline or kisspeptin-54 twice daily showed much lower responses in gonadotrophin and oestradiol stimulation in the kisspeptin group compared to those achieved in the first four study, which was performed on the first day of the treatment period.

Tachyphylaxis to the effects of kisspeptin on gonadotrophin release occurred despite retained sensitivity to GnRH, suggesting that the desensitisation was occurring at the level of the KISS1R. Thus as seen in animal models the method of administration of kisspeptin appears to determine its effects on the HPG axis.

The second 4 hour study was conducted using an injection of a vial returned from each subject. To ensure that peptide degradation had not caused the lower gonadotrophin response to kisspeptin at the end of the two week treatment period, plasma kisspeptin levels were measured. Plasma kisspeptin immunoreactivity was similar following the first and last injection of kisspeptin-54. Plasma kisspeptin IR levels were raised for 6 hours post injection of kisspeptin-54. Thus the twice daily administration of kisspeptin54 would have led to prolonged exposure to raised kisspeptin levels. Animal model studies have demonstrated that a continuous exposure to kisspeptin fails to sustain gonadotrophin stimulation. Juvenile agonadal rhesus male monkeys given a 98 hour continuous intravenous infusion of kisspeptin-10 resulted in a stimulation of gonadotrophins for only the initial three hours of the infusion (Seminara et al. 2006). Adult male rats given a three day infusion of kisspeptin-54 also demonstrated gonadotrophin stimulation only in the first 24 hours of the infusion, after

70 which LH returned to baseline values (Thompson et al. 2006). These effects appear to be secondary to desensitisation of the KISS1R.

A study in adult male rhesus monkeys (Ramaswamy et al. 2007) demonstrated that a 98 hour continuous intravenous infusion of kisspeptin-10 led to reduced responsiveness to

GnRH. The monkeys all displayed an acute initial dose related stimulation in gonadotrophin release with a bolus intravenous administration of kisspeptin, however a continuous infusion of 200mcg/hour and a second higher dose infusion of 400 mcg/hour of kisspeptin-10 led to a stimulation in gonadotrophin for only the first 3 hours of the infusion; after which LH fell to baseline levels. In their study Ramaswamy et al. report that there was reduced responsiveness to GnRH which was dose dependent and occurred only with the higher

400mcg/hour infusion (Ramaswamy et al. 2007). We conducted a GnRH study in order to ascertain whether women with hypothalamic amenorrhoea maintained responsiveness to

GnRH after two weeks of twice daily kisspeptin-54 6.4 nmol/kg injections. Our results indicate that these women did maintain responsiveness to GnRH, although there was a reduced response in gonadotrophin stimulation, this did not reach statistical significance.

Thus the twice daily administration of kisspeptin appears to have led to desensitisation upstream of the GnRH receptor probably at the level of the kisspeptin receptor. A further study examining the effects of kisspeptin administration on GnRH response in healthy women would be interesting. Weight and serum oestradiol levels appear to influence pituitary responsiveness to GnRH (Aono et al. 1975; Knobil 1980). A study of a larger number of women with hypothalamic amenorrhea and healthy women would allow examination of any correlation between BMI and oestradiol levels and the effects of kisspeptin on responsiveness to GnRH.

The effects of twice daily kisspeptin-54 injections at a dose of 6.4 nmol/kg on LH pulsatility were also assessed in this study. We did not demonstrate any statistically significant change in either LH pulse frequency of the mean amplitude of LH increase in women with hypothalamic amenorrhoea. The study by Ramaswamy et al. on the effects of continuous

71 intravenous infusion of kisspeptin-10 in rhesus male monkeys reported that the high dose of kisspeptin-10 (400mcg/hour) resulted in a reduction in the mean number of LH pulses and

LH pulse amplitude (Ramaswamy et al. 2007). These effects were dose related and did not occur with a lower dose (200mcg/hour) of kisspeptin-10. These studies were performed in healthy monkeys with normal GnRH pulsatility. Women with hypothalamic amenorrhoea have abnormal LH pulsatility (Reame et al. 1985) and thus may not be expected to respond in a similar fashion.

Functional hypothalamic amenorrhoea is often a consequence of undernutrition, due either to decreased calorie intake or increased energy output. Rodent models of undernutrition have demonstrated that kisspeptin-10 stimulates higher gonadotrophin responses in undernourished female rats (Castellano et al. 2005). Seventy-two hours of fasting led to a significant decrease in hypothalamic KiSS-1 mRNA levels and increase in GPR54 mRNA expression in male and female prepubertal rats. Intracerebroventricular injections of kisspeptin-10 stimulated higher LH levels in those rats that were fasted compared to those fed ad libitum (Castellano et al. 2005). Comparing the rise in serum gonadotrophins post administration of kisspeptin in women with HA with those achieved in previous studies in healthy women in the follicular phase of the menstrual cycle (Dhillo et al 2007), it was noted that the women with HA appear to more responsive to kisspeptin. They achieved LH levels which were 4 fold greater than those in healthy women. A comparison with the follicular phase of the menstrual cycle was made because similar to the state in hypothalamic amenorrhoea, the follicular phase is characterised by low gonadotrophin and oestradiol levels. The increased expression of hypothalamic kisspeptin receptor GPR54 in rodent models of under-nutrition, (Castellano et al. 2005) may explain the increased potency of kisspeptin stimulation on gonadotrophin release.

This is the first human study showing the effects of acute and chronic administration of kisspeptin-54 to women with hypothalamic amenorrhoea. The results show that after an initial acute stimulation of gonadotrophin release, two weeks therapy of kisspeptin-54 at a

72 dose of 6.4 nmol/kg led to reduced gonadotrophin response, suggesting desensitisation. The results of this study will have important implications of the use of kisspeptin as a therapy in disorders of reproduction.

Although I did not administer kisspeptin-54 as a continuous infusion, the twice daily injections did lead to raised plasma kisspeptin IR for 6 hours post injection resulting in prolonged exposure to elevated levels of kisspeptin. Animal data has shown that continuous administration of kisspeptin results in desensitisation to its effects on gonadotrophin release

(Ramaswamy et al. 2007; Seminara et al. 2006; Thompson et al. 2006). Therefore a different study protocol of kisspeptin-54 administration i.e. a lower dose or reduced frequency of injections may reduce the desensitisation seen in stimulation of gonadotrophin release.

Chapter 3

The effects of chronic twice weekly kisspeptin administration in women with hypothalamic amenorrhoea

3.1 Introduction

In Chapter 2 it was demonstrated that a single injection of kisspeptin-54 stimulated gonadotrophin release in women with hypothalamic amenorrhoea. However, women with HA treated for two weeks with twice daily kisspeptin-54 injections had a markedly reduced reproductive hormone response to kisspeptin-54 at the end of the study when compared with the first kisspeptin injection, despite maintaining their responsiveness to GnRH injection.

This suggests that desensitisation was occurring at the level of the kisspeptin receptor

(KISS1R).

The kisspeptin receptor also known as the GPR54 (abbreviated to KISS1R in humans) is a

G protein coupled receptor (GPCR). The binding of kisspeptin to its receptor stimulates phospholipase C which in turn activates second messenger systems inositol 1,4,5- trisphosphate and diaclyglycerol resulting in increased intracellular calcium (Kotani et al.

2001; Muir et al. 2001; Stafford et al. 2002). G protein coupled receptors exhibit receptor desensitisation (Lefkowitz 1998; Ritter & Hall 2009). Acutely, desensitisation occurs via loss of signalling to secondary messenger systems due to persistent ligand binding and subsequently receptor internalization. Degradation rather than recycling of these receptors results in chronic desensitisation (Ferguson 2001). Degradation of the kisspeptin receptor appears to be via proteasomes rather than the more common pathway of GPCR degradation which is via lysosomes (Bianco et al. 2011).

Plasma kisspeptin immunoreactivity levels remain elevated for at least four hours following a single subcutaneous injection of kisspeptin-54, therefore twice daily injections of kisspeptin would have led to prolonged exposure to kisspeptin of at least 8 hours a day. Animal models have also shown that chronic administration of kisspeptin-10 to both rats and monkeys results in an acute stimulation of gonadotrophin release followed by a fall in gonadotrophin levels to baseline values when desensitisation occurs.

Acute administration of kisspeptin to women with HA results in a potent stimulation of LH release, but twice daily kisspeptin-54 administration for 2 weeks led to tachyphylaxis

(Jayasena et al. 2009f). These data suggest that kisspeptin-54 could potentially be used to downregulate the hypothalamo-pituitary gonadal axis for the treatment of sex hormone dependant tumours and precocious puberty. In addition it is possible that a modified dosing protocol of kisspeptin-54 administration which prevents tachyphylaxis from occurring could be used to stimulate reproductive hormone release long term in women with HA.

3.2 Hypothesis and Aims

I hypothesised that modification of the protocol of repeated kisspeptin administration would reduce the observed desensitisation to its effects in women with HA.

The aims of this study were to determine in women with hypothalamic amenorrhoea

 The time course over which desensitisation to the effects of kisspeptin-54 occurs

 If varying dosing regimens of kisspeptin-54 administration could reduce the

desensitisation of the effects of kisspeptin on reproductive hormone release.

 The effects of long-term twice-weekly kisspeptin-54 injections on reproductive

hormone release by conducting a randomised, double-blinded placebo-controlled

parallel design study over eight weeks in women with hypothalamic amenorrhoea.

3.3 Methods

3.3.1 Subjects

Ethical approval was granted by the Hammersmith and Queen Charlotte‟s and Chelsea

Hospitals Research Ethics Committee (registration number: 05/Q0406/142). Written informed consent was obtained from all subjects. This study was performed in accordance with the Declaration of Helsinki.

Subjects were recruited through advertisements placed in local newspapers. Responders to adverts were evaluated with a detailed menstrual history, clinical examination and blood tests. Screening blood tests performed were as follows: full blood count; renal profile; liver profile; bone profile; glucose; thyroid profile; LH; FSH; oestradiol; progesterone; androstenedione; dehydroepiandrostenone; testosterone; SHBG; prolactin; 17- hydroxyprogesterone and cortisol. Women were diagnosed with HA and included within the study if they fulfilled the following criteria: body mass index < 25kg/m2; stable body weight over the previous 6 months; age between 18 and 40 years; secondary amenorrhea of at least 6 months duration; absence of oral contraceptive pill therapy for at least one year; absence of co-morbidity; absence of active psychiatric illness; stable body weight; absence of therapeutic or recreational drug use; absence of clinical or biochemical signs of hyperandrogenemia; structurally normal hypothalamus and pituitary region assessed by magnetic resonance imaging; structurally normal female reproductive tract visualised on ultrasound; absence of polycystic ovarian appearances on ultrasound; normal thyroid function tests; normal serum prolactin levels; serum LH:FSH ratio <1.5. Thirty subjects with

HA were recruited to the study.

3.3.2 Study 1: Determination of the time-course of desensitisation to the effects of kisspeptin on gonadotophin release in women with HA

Five subjects with HA received twice-daily injections of 6.4 nmol/kg of kisspeptin-54. On day

1 (the first injection day), days 2, 3, 4, and day 14 (the last injection day) of the study protocol, each subject underwent a 4 hour sampling study post-injection of kisspeptin.

Protocol for 4 hour blood sampling after injection:

Subjects were admitted to our Clinical Investigation unit and asked to lay supine. Kisspeptin-

54 was subcutaneously administered at time 0 minutes by an investigator, and blood was sampled for serum LH, FSH, oestradiol, and SHBG at -30, 0, 15, 30, 45, 60, 75, 90, 120, 150,

180, 210 and 240 minutes (Figure 15).

Figure 15. The four hour study protocol.

Participants remained in a supine position throughout the four studies. A cannula was inserted into a vein in the antecubital fossa on arrival. A randomised subcutaneous injection of either saline or kisspeptin was administered at time zero. Blood samples were taken at regular time intervals from -30 to 240 min as indicated by the arrows.

subcutaneous injection of kisspeptin or saline

Time (min) -30-30 0 15 30 45 60 75 90 120 150 180 210 240

cannula blood taken inserted

3.3.3 Study 2: Effects of reduced dose and increased dose-interval on serum reproductive hormone release in women with HA administered kisspeptin injections for 2 weeks

In order to assess the effects of altering kisspeptin-54 dose and dose interval between kisspeptin-54 injections on reproductive hormone release in women with HA, four different 2 week regimes of subcutaneous kisspeptin-54 injections were investigated: 6.4 nmol/kg twice-daily; 1 nmol/kg once-daily; 0.3 nmol/kg once-daily; 6.4 nmol/kg twice-weekly. For each unblinded 2 week pilot study, 5 subjects with HA (see Table 6 for baseline characteristics) attended our clinical investigation unit twice per week. Basal measurements of reproductive hormone were performed twice weekly. On the first (day 1) and last injection day (day 14), the injection of kisspeptin was administered by a study investigator during a 4 hour sampling study.

3.3.4 Study 3: Effects of twice weekly kisspeptin-54 (6.4nmol/kg) administration for 8 weeks on reproductive hormone release in women with HA

Results from Study 2 showed that administration of kisspeptin-54 6.4 nmol/kg twice weekly significantly reduced desensitisation to the effects of kisspeptin-54 on reproductive hormone release. In order to determine if longer term administration of kisspeptin-54 6.4 nmol/kg twice-weekly would continue to stimulate reproductive hormone release a randomised, double blinded, placebo-controlled, parallel design study was performed. Ten subjects with

HA (see Table 6 for baseline characteristics) were randomised to receive either saline or kisspeptin 6.4 nmol/kg injections (n=5 per group) twice weekly for 8 weeks.

Each subject attended a hospital investigation unit twice per week (Figure 16). Twice-weekly basal measurements of serum LH, FSH, oestradiol, progesterone and SHBG were taken from subjects throughout the 8 week study protocol between 0800 and 1800h. Trans- abdominal ultrasound scans were performed once a week throughout the 8 week study

80 protocol. During each scan the following parameters were measured: endometrial thickness in millimetres (mm); mean ovarian volume in cubic centimetres (cm3); number of mean follicles; maximum diameter of largest follicle in each ovary in mm. Ovulation was confirmed by satisfaction of all of the following criteria: visualisation of a dominant follicle (diameter

11mm or greater); enlargement of dominant follicle into a pre-ovulatory follicle (diameter

18mm or greater); subsequent collapse of pre-ovulatory follicle or appearance of internal echoes on ultrasonography; a rise in serum progesterone to over 10nmol/l (Pache et al.

1990).

Figure 16. Protocol to investigate the effects of twice-weekly kisspeptin-54 administration for 8 weeks in women with HA.

Subjects with HA were randomised to receive twice-weekly injections of saline or kisspeptin- 54 (KP54) 6.4 nmol/kg for 56 days. Four hour blood sampling was performed immediately following saline or kisspeptin injection on days 1, 14, 28, 42 and 56. Once-weekly ultrasound scans and twice-weekly blood sampling for measurement of LH, FSH, oestradiol (E2) and plasma kisspeptin immunoreactivity (IR) were also performed.

During each visit, a double-blinded subcutaneous injection of either saline or kisspeptin was administered to each subject. Following the first injection of saline or kisspeptin a 4 hour sampling study (Figure 15) was performed in order to investigate the acute effects of the treatment on reproductive hormone release. The 4 hour sampling study was repeated at 2, 4 and 6 weeks, and on the final day of the study protocol after 8 weeks.

During each study visit, urine was tested in order to exclude pregnancy (Clearview easy-

HCG, Inverness Medical Innovations Inc. Waltham, MA). Diastolic and systolic blood pressure and heart rate were recorded during 4 hour blood sampling studies performed postinjection of kisspeptin or saline.

3.3.5 Kisspeptin-54

Kisspeptin-54 was synthesised by the Advanced Biotechnology Centre, Imperial College

London and purified by reverse-phase high performance liquid chromatography (HPLC).

Electrospray mass spectroscopy and amino acid analysis confirmed identity of the peptide as previously described (Dhillo et al. 2005; Dhillo et al. 2007). The peptide was tested for bioactivity and toxicity as previously described (Dhillo et al. 2005). The Limulus amebocyte lysate assay (Associates of Cape Cod, Liverpool, UK) was negative for endotoxin, and the peptide was sterile on culture (Department of Microbiology, Hammersmith Hospital, London).

Although kisspeptin-10, -13, -14, and -54 display similar potency in vitro, we used kisspeptin-

54 due to its higher in vivo potency than the other kisspeptin fragments (Thompson et al.

2006; Tovar et al. 2006).

3.3.6 Injections of Kisspeptin-54

Vials of freeze-dried saline or kisspeptin were reconstituted in 0.5 ml of 0.9% saline. Then a

0.5 ml insulin syringe was used to inject a weight-adjusted dose of kisspeptin into the lower anterior abdominal region. Depending on the protocol, subjects received kisspeptin doses of

0.3, 1.0 or 6.4 nmol per kg.

Once- or twice-daily injections of kisspeptin: Due to the frequency of these injections, they were self-administered by subjects at home; except during 4 hour blood sampling studies, when injections were administered in our Clinical investigation Unit by an investigator. Prior to commencement of the study protocol, subjects were trained to perform subcutaneous self- injection. A box containing unlabelled vials of freeze-dried saline or kisspeptin-54, alcohol wipes, saline vials, needles and needle disposal bins was given to each subject. Instructions were given to refrigerate vials stored at home.

Twice-weekly injections of kisspeptin or saline: All injections were administered to subjects by study investigators when the subjects attended our clinical investigation unit twice a week.

3.3.7 Collection and processing of blood samples

Blood samples for serum analysis were collected in plain serum Vacutainer tubes (Beckton

Dickson, Franklin Lakes, NJ, USA). Clotted samples underwent centrifugation using a

Hettich EBA 20 machine (Hettich International, Tuttlingen, Germany) for 10 minutes at

3000rpm. Serum was then separated and stored at -20OC until analysis.

3.3.8 Analytical methods

Serum LH, FSH, oestradiol, and progesterone were measured using automated chemiluminescent immunoassays (Abbott Diagnostics, Maidenhead, UK). SHBG was measured using a solid-phase automated enzyme immunoassay (Immulite; Siemens,

Llanberis, UK). Reference ranges for females were as follows: LH (follicular), 2–10 IU/l; LH

(midcycle), 20–60 IU/l; LH (luteal), 4–14 IU/l; FSH (follicular and luteal), 1.5–8 IU/l; oestradiol

(early follicular), less than 300 pmol/l; oestradiol (midcycle), 400-1500 pmol/l; oestradiol

(luteal), 200-1000 pmol/l; and SHBG 40–80 nmol/l. Interassay coefficients of variation were as follows: LH, 3.4%; FSH, 3.5%; oestradiol, 3.4%; progesterone, 1.8%; and SHBG, 5.6%.

Limits of detection for each assay were as follows: oestradiol 70pmol/l; FSH 0.05mIU/ml; LH

0.07mIU/ml; progesterone 0.1ng/ml; SHBG 1nmol/l.

3.3.9 Data analysis

Data are presented as mean +/- standard error of mean (SEM). Hormone profiles during 4 hour blood sampling studies were analysed using repeated measures 2-way ANOVA with

Bonferonni post hoc correction. Pairs of means were analysed using the unpaired two-tailed t-test. Multiple means were compared using one-way ANOVA with Bonferonni‟s Multiple

Comparison Test. In all cases, P < 0.05 was considered statistically significant.

3.4 Results

Baseline characteristics of age, weight and BMI were similar for all groups of subjects with

HA included in this study and are shown in table 6.

Table 6. Comparison of baseline characteristics of women with hypothalamic amenorrhea

Subjects participating in 2 week pilot studies of kisspeptin administration (n=20 in total), and an 8 week study of saline versus kisspeptin administration (n=5 per group). Data shown as mean +/- SEM.

Protocol 2 week pilot 8 week study P

Treatment studiesKisspeptin 54 Saline Kisspeptin54 value Age (years) 28.1 ± 1.1 27.0 ± 2.6 27.2 ± 2.4 NS

Weight (kg) 52.6 ± 0.7 54.5 ± 3.9 53.4 ± 3.0 NS

Body mass index (kg/m2) 19.1 ± 0.3 19.7 ± 1.1 19.6 ± 0.7 NS

Duration of amenorrhea 22.4 ± 9.9 20.4 ± 5.1 28.8 ± 12.0 NS (months)

Serum LH (iU/l) 2.5 ± 0.6 1.7 ± 0.7 1.4 ± 0.5 NS

Serum FSH (iU/l) 4.8 ± 0.3 3.8 ± 0.9 3.6 ± 0.6 NS

Serum oestradiol (pmol/l) 133 ± 10.0 87 ± 17.0 151 ± 28.0 NS

Study 1: Time course of desensitisation to the effects of kisspeptin on reproductive hormone release in women with HA

Twice-daily sc administration of 6.4 nmol/kg kisspeptin was associated with a progressive reduction in acute LH responses following injection of kisspeptin, in women with HA (Figure

17A). On the first injection day, the mean AUC LH response during the first 4 hours after kisspeptin injection was 59.3  19.0h.IU/l; however mean AUC LH response during the first 4 hours after kisspeptin injection dropped to 36.0  18.8, 14.9  2.9, 12.9  2.4 and 5.3 

1.9h.IU/l on the 2nd, 3rd, 4th and 14th injection days, respectively (Figure 17 A). On the first injection day, the mean AUC FSH response during the first 4 hours after kisspeptin injection was 15.8  3.9h.IU/l; however acute FSH responses following kisspeptin injection were less than 3h.IU/l during the 2nd, 3rd, 4th and 14th injection days (Figure 17 B). Mean oestradiol levels did not significantly change during the study (mean AUC oestradiol response: day 1,

109  119pmol/l; day 14, -65  30pmol/l).

Figure 17. Time course of desensitisation to the effects of kisspeptin-54 (6.4 nmol/kg) on reproductive hormone release administered twice-daily for 2 weeks in women with HA.

Mean area under curve (AUC) LH (A) and FSH responses (B) during the first 4 hours following kisspeptin-54 injections were measured on the 1st, 2nd, 3rd, 4th and 14th days of twice-daily administration of 6.4 nmol/kg sc kisspeptin. Data is shown as mean +/- SEM. * P < 0.05 vs. day 14; *** P < 0.001 vs. day 1.

100 *

80 *

60 40

0 AUC LH increase (iU/L) increase LH AUC

Day 1 Day 2 Day 3 Day 4 Day 14

25 *** 20

0 AUC FSH increase (iU/L) increase FSH AUC

Day 1 Day 2 Day 3 Day 4 Day 14

Study 2: Effects of reduced dose and increased dose-interval on serum reproductive hormones in women with HA, during a 2-week protocol of kisspeptin injections

A: Effect of twice-daily sc injection of kisspeptin-54 (6.4 nmol/kg) on reproductive hormone release.

Twice-daily sc injection of 6.4 nmol/kg kisspeptin-54 to women with HA led to a potent stimulation of gonadotrophin release on the first injection day, with the maximal increase observed 240 minutes after injection (mean maximal increase following injection: LH: 11.2 

4.6 IU/l; FSH: 7.2  2.1 IU/l). However on the last (14th) injection day, reproductive hormone responses were significantly reduced when compared with responses on the first injection day (mean maximal increase following injection on the 14th injection day: LH: 1.0  0.5 IU/l,

P<0.05 when compared with 1st injection day; FSH: 0.2  0.3 IU/l, P<0.001 when compared with 1st injection day) (Figure 18 A, B). Furthermore the oestradiol response was non- significantly lower on the 14th injection day when compared with the first injection day (mean maximal oestradiol increase following injection: first injection day, 48.2  29.7 pmol/l; 14th injection day, -12.7  7.0 pmol/l, P=0.06) (Figure 18 C).

Figure 18. Serum reproductive hormone levels following twice-daily regimens of kisspeptin-54 6.4 nmol/kg injection in 5 women with HA.

Changes in serum LH (A), FSH (B) and oestradiol E2 (C);over 4 hours after sc kisspeptin-54 (KP54) on the first day and 14th day following twice daily sc injection of 6.4 nmol/kg KP54.

A 20 *

15 First injection

10 Last injection

5 LH increase (iU/L) 0 0 30 60 90 120

Time (minutes)

B * *** 10 8 6 4 2

FSH increase (iU/L) 0

0 60 120 180 240

Time (minutes)

C 100 80 60 40 20 0

-20 E2 E2 increase (pmol/l) -40 0 60 120 180 240

Time (minutes)

B: Effect of once-daily sc injection of kisspeptin-54 (1 nmol/kg) on reproductive hormone release.

We then investigated if protocols using lower cumulative doses of kisspeptin treatment would reduce the observed desensitisation in subjects with HA. First, we examined the effects of a protocol of once-daily 1 nmol/kg kisspeptin-54 injections in 5 further subjects with HA. On the first injection day, the maximal LH and FSH responses were 3.7  0.9 pmol/l and 1.9  0.3 pmol/l, respectively (Figure 19 A, B). However on the 14th injection day, LH and FSH responses were significantly reduced when compared with responses on the first injection day (mean maximal increase following injection on the 14th injection day: LH: 1.5  0.2 IU/l,

P<0.05 when compared with 1st injection day; FSH: 0.3  0.2 IU/l, P<0.005 when compared with 1st injection day) (Figure 19 A, B). The oestradiol response was non-significantly lower on the 14th injection day when compared with the first injection day (mean maximal oestradiol increase following injection: first injection day, 48.6  29.3 pmol/l; 14th injection day,

0.8  13 pmol/l, P=0.11) (Figure 19 C).

Figure 19. Serum reproductive hormone levels following once daily dosing regimens of kisspeptin-54 1 nmol/kg injection in 5 women with HA.

Changes in serum LH,(A), FSH (B), and oestradiol E2(C) over 4 hours following once daily sc injection of 1nmol/kg of KP54. Data is shown as mean +/- SEM. * P < 0.05; *** P < 0.001

A 6 * 5 1st injection day 4 14th injection day 3 2 1

LH increase (iU/L) 0

0 60 120 180 240

Time (minutes)

***

B 3

0 FSH increase (iU/L)

0 60 120 180 240

Time (minutes)

C 90 70 50 30 10

-10 E2 E2 increase (pmol/L) -30 0 60 120 180 240 Time (minutes)

C: Effect of once-daily sc injection of kisspeptin-54 (0.3 nmol/kg) on reproductive hormone release.

We then performed a 2 week study of once-daily kisspeptin-54 injections at a lower dose of

0.3 nmol/kg in order to determine if desensitisation would still occur with further reduced exogenous kisspeptin exposure. On the first injection day, following kisspeptin-54 (0.3 nmol/kg) injection the peak LH was only moderately increased, and non-significantly reduced after 14 days of kisspeptin treatment (mean maximal LH increase following injection: first injection day, 5.3  1.8 IU/l; 14th injection day, 1.2  0.4 IU/l, P=0.10). FSH responses followed a similar pattern to LH response (mean maximal FSH increase following injection: first injection day, 0.8  0.6 IU/l; 14th injection day, -0.6  0.5 IU/l, P=0.20) (Figure 20 A, B).

The peak oestradiol response was below 20 pmol/l on the first injection day, and non significantly lower on the 14th injection day (Figure 20 C).

Figure 20. Serum reproductive hormone levels following once daily dosing regimens of kisspeptin-54 0.3 nmol/kg injection in 5 women with HA.

Changes in serum LH (A), FSH (B), and oestradiol (C); over 4 hours following once daily sc injection of 0.3 nmol/kg of KP54.

A 6 1st injection day 4 14th injection day

0 LH increase (iU/L) -2 0 60 120 180 240

Time (minutes)

B 2

-1 FSH increase (iU/L) -2 0 60 120 180 240

Time (minutes)

C 40 30 20 10 0

-10 E2 E2 increase (pmol/L) -20 0 60 120 180 240

Time (minutes)

D: Effect of twice-weekly sc injection of kisspeptin-54 (6.4 nmol/kg) on reproductive hormone release.

I had observed that 1.0 nmol/kg once-daily kisspeptin-54 sc administration led to significant desensitisation of gonadotrophin responses after 2 weeks of administration. Although I did not observe significant desensitisation with 0.3 nmol/kg once-daily kisspeptin-54 sc administration, the absolute rises in reproductive hormone levels at this dose were modest. I therefore studied the effects of further reducing the dose-interval of sc kisspeptin-54 administration to twice-weekly, at a dose of 6.4 nnmol/kg per injection. On the 14th injection day, mean gonadotrophin responses following injection were non-significantly lower on the

14th injection day when compared with the first injection day (mean maximal increase in IU/l following injection: LH: 18.8  6.6 vs. 11.5  4.0, P=0.08; FSH: 5.8  2.0 vs. 4.1  1.1,

P=0.14) (Figure 21 A, B).The mean maximal increase in serum oestradiol following kisspeptin injection was similar on the 1st and 14th injection days (1st day: 44.8  15.7 pmol/l vs. 14th day: 27.5  15.5 pmol/l; P=0.47) (Figure 21 C).

Figure 21. Serum reproductive hormone levels following twice weekly daily dosing regimens of kisspeptin-54 6.4 nmol/kg injection in 5 women with HA.

Changes in serum LH (A), FSH (B) and oestradiol E2 (C); over 4 hours following twice weekly sc injection of 6.4 nmol/kg of KP54.

A 30 1st injection day 14th injection day 20

10 LH increase (iU/L) 0 0 60 120 180 240

Time (minutes)

B 10

2 FSH increase (iU/L) 0 0 60 120 180 240

Minutes

C 80

0 E2 E2 increase (pmol/L) -20 0 60 120 180 240

Minutes

A summary of the effects of sc kisspeptin-54 on the first and last day of each of these different 2 week dosing regimens of kisspeptin-54 is shown as a mean area under the curve for LH, FSH and oestradiol in Figures 22 A-C.

Figure 22. Serum reproductive hormone levels following different 2 week dosing regimens of kisspeptin-54 injection in women with HA.

The effects of sc injection of KP54 on the first day and the 14th day of each of the different 2 week dosing regimens of KP54 is shown as a mean area under the curve for LH, FSH and oestradiol in (A-C). Data is shown as mean +/- SEM. * P < 0.05; ** P < 0.01.

1st injection day A * 80 14th injection day

0 AUC AUC LH increase (h.iU/L)

1nmol/kg once-daily 6.4nmol/kg twice-daily0.3nmol/kg once-daily 6.4nmol/kg twice-weekly

20 **

-5 AUC AUC FSH increase (h.iU/L)

1nmol/kg once-daily 6.4nmol/kg twice-daily0.3nmol/kg once-daily 6.4nmol/kg twice-weekly

200

100

-100 AUC AUC E2 increase (h.pmol/L)

1nmol/kg once-daily 6.4nmol/kg twice-daily0.3nmol/kg once-daily 6.4nmol/kg twice-weekly

Study 3: Effects of twice-weekly kisspeptin-54 administration for 8 weeks on reproductive hormone levels in women with HA.

Baseline age, weight and BMI were not significantly different between kisspeptin and saline study groups (Table 6). Subjects reported no side effects following injection of kisspeptin or saline. No significant acute changes in heart rate, systolic and diastolic blood pressure were observed following kisspeptin administration when compared with saline control.

Saline had no significant effects on reproductive hormone release at any time during the 8 week protocol of twice-weekly injections (Figure 23 A-C).

100

Figure 23. Serum reproductive hormone levels following twice-weekly saline injection for 8 weeks in women with HA.

Changes in serum LH (A), FSH (B) and oestradiol E2 (C) after bolus sc injection of saline (n=5) on first day, and after 2, 4, 6 and 8 weeks of administration. Data is shown as mean +/- SEM. ** P < 0.01 vs. baseline; *** P < 0.001 vs. baseline.

A 3

2 Baseline 2 weeks 1 4 weeks 0 6 weeks 8 weeks

Mean LH increase (iU/L) -1 0 30 60 90 120 150 180 210 240 Minutes

B 1.5

0.5

-0.5

Mean FSH increase-1.5 (iU/L) 0 30 60 90 120 150 180 210 240 Minutes

C 60

-20

-40

Mean E2 increase (pmol/L) 0 30 60 90 120 150 180 210 240 Minutes

101

As expected, kisspeptin-54 stimulated reproductive hormone release following administration on the first injection day. After 2 weeks of twice-weekly injections, LH responses following kisspeptin injection were significantly lower than LH responses following injection on the first injection day (mean maximal LH increase (IU/l): baseline, 21.5  10.7; 2 weeks, 10.0  4.3;

P<0.001) (Figure 24 A). However, no further significant reductions in LH following injection of kisspeptin were observed at 4 weeks (mean maximal LH increase: 9.0  4.1 IU/l; P>0.05 vs. response at 2 weeks), 6 weeks (mean maximal LH increase: 8.9  3.5 IU/l; P>0.05 vs. response at 2 weeks) or 8 weeks (mean maximal LH increase: 7.9  4.5 IU/l; P>0.05 vs. response at 2 weeks) (Figure 24 A). FSH responses following kisspeptin injection changed in a similar manner to LH responses over the 8 week protocol (mean maximal increase in serum FSH following kisspeptin injection (IU/l): baseline, 6.4  3.2; 2 weeks, 2.7  0.7,

P<0.001 vs. baseline response; 4 weeks, 2.6  0.7, P>0.05 vs. response at 2 weeks; 6 weeks, 2.4  0.8, P>0.05 vs. response at 2 weeks; 8 weeks, 2.7  0.8, P>0.05 vs. response at 2 weeks) (Figure 24 B). Oestradiol responses following injection of kisspeptin were similar throughout the 8 week protocol of twice-weekly injections (mean maximal increase in serum oestradiol following kisspeptin injection (pmol/l): baseline, 44.4  19.9; 2 weeks, 39.2  14.8;

4 weeks, 46.2  25.8; 6 weeks, 60.8  17.2; 8 weeks, 20.0  7.5) (Figure 24 C).

102

Figure 24. Serum reproductive hormone levels following twice weekly kisspeptin-54 injection for 8 weeks in women with HA.

Changes in serum LH (A), FSH (B) and oestradiol E2 (C) after bolus sc injection of 6.4 nmol/kg kisspeptin-54 (n=5) on first day, and at after 2, 4, 6 and 8 weeks of administration. Injections were administered at 0 min. Data is shown as mean +/- SEM. ** P < 0.01, 2 weeks vs. day . *** P < 0.001, 2 weeks vs. baseline.

A *** 40 Day1 30 2 weeks 4 weeks 20 6 weeks

10 8 weeks

Mean LH increase (iU/L) 0 0 30 60 90 120 150 180 210 240

Minutes

B 10 ** *** 8

0 Mean FSH increase (iU/L) 30 60 90 120 150 180 210 240

Minutes

C 100

Mean E2 increase (iU/L) -50 0 30 60 90 120 150 180 210 240

Minutes

103

No significant changes in the twice-weekly basal reproductive hormone measurements were observed at any stage during the 8 week protocol, between subjects with HA receiving saline and kisspeptin injections (Table 7). No significant changes in follicle number, maximum follicle size, ovarian volume, or endometrial thickness were observed at any stage of the 8 weeks protocol between subjects with HA who received kisspeptin and saline injections

(Table 8). Although dominant follicles were observed in some subjects of both treatment groups, no preovulatory follicles were observed in any subject during the study. One subject receiving saline treatment developed a preovulatory LH surge after 8 weeks of saline treatment; however no subjects ovulated during the study.

104

Table 7. Comparison of basal serum reproductive hormone levels at baseline, during and 8 weeks of treatment with either saline or kisspeptin injections.

Mean ± SEM basal serum hormone levels are provided for subject groups randomised to receive saline (n=5) or kisspeptin-54 (n=5). For each subject, baseline serum LH, FSH and oestradiol levels were measured at baseline, and calculated as the mean levels from separate blood tests performed during baseline, week 2, weeks 3-4 and weeks 5-8 of the study protocol.

Hormone Study group P value Saline Kisspeptin54 LH (iU/l) Baseline 1.5 ± 0.6 1.8 ± 0.7 NS

Week 2 1.9 ± 0.5 1.7 ± 0.6 NS

Weeks 3-4 1.9 ± 0.5 1.7 ± 0.4 NS

Weeks 5-8 3.7 ± 2.7 1.9 ± 0.5 NS

FSH (iU/l) Baseline 3.7 ± 0.6 3.4 ± 0.5 NS

Week 2 2.8 ± 0.8 4.5 ± 0.9 NS

Weeks 3-4 3.1 ± 1.0 3.1 ± 0.9 NS

Weeks 5-8 3.2 ± 0.8 4.3 ± 0.7 NS

Oestradiol Baseline 97 ± 13.0 78 ± 9.0 NS (pmol/l) Week 2 91 ± 10.0 102 ± 11.0 NS

Weeks 3-4 113 ± 33.0 90 ± 8.0 NS

Weeks 5-8 123 ± 37.0 102 ± 11.0 NS

105

Table 8. Summary of ultrasound parameters at baseline and throughout the 8 week treatment with saline or kisspeptin injections, in women with HA.

Subjects randomised to receive saline (n=5) or kisspeptin-54 (n=5). Baseline mean values were calculated from results of four separate scans performed during weeks 1-4 of the study protocol. The change in value of each ultrasound parameter during weeks 5-6 (during treatment) or 7-8 (post-treatment), was based upon results of two separate scans performed during the respective 2 week period. Data shown as mean ± SEM.

Characteristic Study Group P value Saline kisspeptin-54

Ovulation (number of 0 0 - subjects)

Preovulatory follicle ≥ 0 0 - 18mm (no. subjects)

Dominant follicle ≥ 3 6 - 11mm (no.subjects)

Endometrial thickness Weeks 1-2 2.8 ± 0.1 3.2 ± 0.5 NS (mm) Weeks 3-4 3.2 ± 0.4 3.5 ± 0.4 NS

Weeks 5-6 2.9 ± 0.3 4.0 ± 0.5 NS

Weeks 7-8 3.2 ± 0.7 4.0 ± 0.5 NS Ovarian volume (cm3) Weeks 1-2 5.7 ± 1.8 6.7 ± 2.1 NS

Weeks 3-4 5.2 ± 1.5 5.3 ± 1.0 NS

Weeks 5-6 5.4 ± 1.3 5.2 ± 0.6 NS

Weeks 7-8 6.1 ± 1.9 5.5 ± 0.7 NS Follicle number per Weeks 1-2 12 ± 4.0 13 ± 3.0 NS ovary Weeks 3-4 11 ± 3.0 15 ± 3.0 NS

Weeks 5-6 11 ± 3.0 16 ± 3.0 NS

Weeks 7-8 9.0 ± 4.0 13 ± 4.0 NS Maximum follicle Weeks 1-2 5.0 ± 1.0 7.0 ± 0.9 NS diameter (mm) Weeks 3-4 6.5 ± 1.5 7.8 ± 1.6 NS

Weeks 5-6 6.0 ± 1.1 7.3 ± 0.9 NS

Weeks 7-8 6.3 ± 0.8 7.3 ± 1.2 NS

106

5. Discussion

Intact kisspeptin signalling is pivotal in regulating the HPG axis and essential for reproductive maturation in humans (de Roux N. et al. 2003; Seminara et al. 2003).

Kisspeptin receptor antagonists disrupt the activity of the hypothalamic-pituitary-gonadal

(HPG) axis in adult monkeys, sheep and rodents (Roseweir et al. 2009). Hence there is a need to evaluate the therapeutic potential of kisspeptin. Short-term clinical studies suggest that kisspeptin safely stimulates reproductive hormone release; however chronic administration has until now been shown to cause profound tachyphylaxis in humans

(Jayasena et al. 2009).

The paradoxical effects of kisspeptin on reproductive hormone release has been demonstrated in monkeys; intermittent, hourly intracerebroventicular injections of kisspeptin-

10 to monkeys stimulates pulsatile LH release (Plant, Ramaswamy, & Dipietro 2006), whereas continuous icv infusion of kisspeptin-10 to monkeys leads to desensitisation of gonadotrophin release within 3 hours of commencement of the infusion (Ramaswamy et al.

2007; Seminara et al. 2006). The phenomenon of desensitisation has also been demonstrated in rodents (Ramaswamy et al. 2007; Roa et al. 2008; Thompson et al. 2006). I have presented detailed data of the time-course of desensitisation to the effects of kisspeptin-54 on reproductive hormone release in women with HA. My data suggests that desensitisation of LH response to twice-daily kisspeptin-54 sc administration in women with

HA, occurs gradually over the 14 day injection period. LH responses on the 4th injection day were significantly higher than on the 14th injection day. By contrast, FSH responses dropped to less than 3 h.IU/l after just 24 hours of twice-daily kisspeptin administration. Interestingly,

Roa et al. showed that constant icv infusion of kisspeptin-10 to adult female cycling rats led to desensitisation of LH response by day 3, but desensitisation of the FSH response to kisspeptin took longer (Roa et al. 2008). The reason for this discrepancy between human and rodent FSH responses to kisspeptin is unclear. However, it is possible that icv and

107 peripherally administered kisspeptin have differential effects on gonadotrophin release.

Furthermore, we cannot exclude that humans and rodents respond differently to kisspeptin, or indeed that different kisspeptin peptides (kisspeptin-54 used in our studies compared to kisspeptin-10 used by Roa et al. (Roa et al.2008) have differential effects on FSH release.

In order to reduce the effect of desensitisation associated with repeated administration of kisspeptin (6.4 nmol/kg twice daily); I lowered the dose of sc kisspeptin injection by studying the effects of once-daily administration. Interestingly, once-daily 1 nmol/kg (total dose 14 nmol/kg over 2 weeks) of kisspeptin administration still led to significant desensitisation of gonadotrophin responses in women with HA. I did not observe significant desensitisation with 0.3 nmol/kg (total dose 4.2 nmol/kg over 2 weeks) of kisspeptin. However, even on the first injection day, the stimulation of reproductive hormone release following 1.0 and 0.3 nmol/kg sc kisspeptin injection was only moderate due to the reduced dose of kisspeptin used. Furthermore, on the 14th injection day residual LH and FSH responses following once- daily kisspeptin injection (0.3 or 1.0 nmol/kg) and twice-daily kisspeptin injection (6.4 nmol.kg) were similar. Plasma kisspeptin immunoreactivity following a single subcutaneous injection of kisspeptin-54 persists for up to 6 hours in women with HA (Jayasena et al. 2009). My results therefore suggest that once-daily kisspeptin administration of kisspeptin leads to tachyphylaxis, even at low doses. This data suggests that either once-daily or twice-daily protocol of sc kisspeptin administration leads to tachyphylaxis. This has important implications for the future development of any kisspeptin-based endocrine therapy used to perform medical castration in patients with sex hormone-sensitive tumours.

In view of the tachyphylaxis observed with once-daily administration of kisspeptin, I determined if lengthening the dose interval to twice-weekly would allow recovery from desensitisation to kisspeptin. The dose of 6.4 nmol/kg (total dose 32 nmol/kg over 2 weeks) kisspeptin was chosen, since acute injection of kisspeptin-54 at this dose results in adequate stimulation of gonadotrophin levels in women with HA (Jayasena et al. 2009). I performed a 2 week pilot study which suggested that gonadotrophin responses were only

108 partially diminished after twice-weekly 6.4 nmol/kgkisspeptin administration. This suggests that partial recovery of desensitisation of the kisspeptin receptor may have occurred during the interval between each injection of kisspeptin.

In order to further assess the regime of twice-weekly 6.4 nmol/kgkisspeptin sc injection, I performed a longer 8 week study of its administration. As observed during the pilot study, this regime of kisspeptin administration still lead to partial desensitisation in gonadotrophin responses during the first 2 weeks of administration. However gonadotrophin responses to kisspeptin injection did not significantly diminish further beyond this initial 2 week period. My results therefore suggest that subjects with HA remained partially responsive to kisspeptin injection during the 8 week study period, and that some recovery from desensitisation had occurred between injections. Interestingly oestradiol responses to kisspeptin remained remarkably constant across the entire 8 week period, despite the initial reduction in pituitary responsiveness observed during the first 2 weeks of kisspeptin administration. It is possible that the initial stimulation of oestradiol secretion caused by kisspeptin administration at the beginning of the study, acted to further sensitise the ovaries to further gonadotrophin stimulation. On the other hand, it cannot be excluded that the stimulatory actions of kisspeptin on the ovaries are in part explained by a direct stimulatory action. Kisspeptin receptors are expressed on human ovaries (Gaytan et al. 2009), but no direct stimulatory action of kisspeptin on ovarian tissue has been reported.

During the 8 week study of kisspeptin administration, I did not observe any significant changes in follicle growth when compared with saline administration. This might have reflected that FSH responses were lower than LH responses following kisspeptin administration, which is in agreement with previous data (Dhillo et al. 2005; Dhillo et al. 2007;

Jayasena et al. 2009). Furthermore as discussed, I observed that FSH responses desensitised more rapidly following kisspeptin injection than LH responses. Thus, inadequate amplitude and duration of FSH stimulation might have accounted for the lack of detectable ovarian follicular growth in this study.

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In summary, I have determined the time-course of desensitisation of LH and FSH responses during twice-daily administration of kisspeptin-54 in women with HA. Once or twice-daily administration of kisspeptin are associated with desensitisation, which may be utilised therapeutically in the treatment of hormone-sensitive tumours. I have also conducted the first long-term clinical study of kisspeptin administration. A single injection of kisspeptin still robustly elicits reproductive hormone release in women with HA, even after 2 months of twice-weekly kisspeptin-54 administration. These findings have important therapeutic implications; kisspeptin-54 may be a novel therapeutic tool for chronically inducing reproductive hormone release in HA and other conditions associated with hypogonadotrophic hypogonadism.

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Chapter 4

The Effects of Kisspeptin-10 on Reproductive Hormone Release Show

Sexual Dimorphism in Humans

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4.1 Introduction

The KISS1 gene encodes a 145-amino acid precursor protein, this undergoes proteolytic processing to generate 54, 14, 13 and 10 amino acid sequences (Kotani et al. 2001) . These shorter peptides are named according to their number of constituent amino acids; they all share a common c terminal decapeptide sequence, which is required for in vitro biological activity. There have been a number of studies examining the effects of kisspeptin-10 in animals. These studies have demonstrated that central or peripheral administration of kisspeptin-10 to rodents, sheep, monkeys, hamsters, pigs and cows (Irwig et al. 2004;

Navarro et al. 2004; Gottsch et al 2004; Messager et al. 2005; Caraty et al. 2007; Plant et al.

2006; Ramaswamy et al. 2007; Shahab et al. 2005; (Lents et al. 2008); (Kadokawa et al.

2008), stimulates gonadotrophin release. The stimulatory effects of kisspeptin-10 on gonadotrophin release are inhibited by the central administration of a GnRH antagonist.

Studies examining the effects of kisspeptin-54 in humans have identified that kisspeptin-54 stimulates gonadotrophin release in both healthy men and women (Dhillo et al. 2005; Dhillo et al. 2007). In women these effects are most pronounced in the preovulatory phase of the menstrual cycle (Dhillo et al. 2007). Kisspeptin-54 has also been shown to stimulate gonadotrophin release in women with a model of infertility due to hypothalamic amenorrhoea

(Jayasena et al. 2009; Jayasena et al. 2010). Human male studies have demonstrated that an intravenous bolus injection of kisspeptin-10 potently stimulates LH secretion while a continuous infusion of kisspeptin-10 also increases testosterone, LH pulse frequency and amplitude (George et al. 2011). Administration of kisspeptin-10 appears to reset the GnRH pulsatile clock, by inducing an immediate LH pulse, regardless of the timing of the previous pulse and these pulses were on average of greater amplitude than endogenous pulses

(Chan et al. 2011). The effects of kisspeptin-10 administration on women are not known.

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Compared to kisspeptin-54, kisspeptin-10 has a shorter half-life and faster onset of action after intravenous administration in rodents (Mikkelsen et al. 2009). As kisspeptin-10 is simpler and cheaper to manufacture due to its shorter amino acid sequence, future kisspeptin based reproductive therapies may be based upon kisspeptin-10 rather than kisspeptin-54. It is therefore therapeutically important to determine whether kisspeptin-10 can stimulate reproductive hormone release in healthy men and women.

4.2 Hypothesis and Aim

Animal models suggest that kisspeptin-10 and kisspeptin-54 act similarly to stimulate reproductive hormone release however, kisspeptin-10 is characterised by a shorter half-life.

Recently it has been reported that administration of kisspeptin-10 potently stimulates LH pulse frequency and amplitude in healthy men (George et al. 2011). In addition kisspeptin-10 appears to reset the GnRH pulsatile clock in men (Chan et al. 2011). There have been no previous studies on the effects of kisspeptin-10 on women. I hypothesise that kisspeptin-10 will stimulate reproductive hormone release in healthy men and women in a similar fashion as kisspeptin-54.

This study aimed to determine the effects of kisspeptin-10 administration on reproductive hormone release in healthy men and, for the first time, in healthy women.

4.3 Methods

4.3.1 Subjects

This study was conducted with Ethics Committee approval (reference 08/H0707/95) in accordance with The Declaration of Helsinki. Recruitment was via advertisements in the

113 local press. Full written informed consent was obtained from all subjects, 25 healthy female volunteers and 11 male volunteers were recruited after medical screening (Table 9).

Recruitment involved a clinical history, clinical examination, electrocardiogram, and blood tests (full blood count, renal profile, liver and bone profile, thyroid profile, random glucose, prolactin, gonadotrophins and sex hormone profile). Men and women were included in the study if they fulfilled the following criteria; age 18 to 40 years; no clinical or biochemical evidence of hypogonadism, thyroid dysfunction or hyperprolactinemia; no therapeutic or recreational drug use; no systemic disease co-morbidity. Additional inclusion criteria for women were as follows: regular menstrual cycles; no oral contraceptive pill therapy within the last year; no clinical or biochemical evidence of polycystic ovarian syndrome.

4.3.2 Kisspeptin-10 and -54 peptide synthesis

Human sequence kisspeptin-54 peptide was synthesised by Advanced Biotechnology

Centre, Imperial College London. Human sequence kisspeptin-10 was synthesised by

Bachem Holding AG (Bubendorf, Switzerland). Both peptides were purified by reverse-phase

High Performance Liquid Chromatography (HPLC). Electrospray mass spectroscopy and amino acid analysis confirmed identity of the peptide. Toxicology testing in animals was conducted prior to administration to human volunteers. The Limulus amoebocyte lysate test

(LAL) detected no endotoxin (Associates of Cape Cod, Liverpool, UK), and bacterial culture was sterile (Department of Microbiology, Hammersmith Hospital, London, UK), in samples of kisspeptin-10 and kisspeptin-54 peptide. Vials of freeze-dried kisspeptin-10 and kisspeptin-

54 were stored at minus 20oC and reconstituted in 0.9% saline.

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4.3.3 Study Days

Subjects were admitted to our clinical investigation unit and asked to lay supine for the duration of each study. Urine was tested to exclude pregnancy in women (Clearview Easy-

HCG; Inverness Medical Innovations Inc. Waltham, MA). All blood samples were analysed for measurement of serum LH, FSH, oestradiol, testosterone, and plasma kisspeptin immunoreactivity (IR). Serum and plasma samples were stored at -20oC until analysis. Heart rate, blood pressure, and the presence of any symptoms were recorded at regular intervals

(10-15 minutes). Women in the follicular phase of their menstrual cycle were studied between days 2-10 of their cycle. Women in the preovulatory phase were studied 15-16 days before the start of their next menstrual cycle.

4.3.4 Serum reproductive hormone measurement

Blood samples for serum LH, FSH and oestradiol analysis were collected in plain serum vacutainer tubes (Beckton Dickson, Franklin Lakes, NJ, USA). Samples were allowed to clot prior to centrifugation and separation of serum. Serum samples were stored at -200C until analysis. LH, FSH, oestradiol and total testosterone were measured using automated chemiluminescent immunoassays (Abbott Laboratories, Abbott Park, IL). Reference ranges for males were as follows: LH, 4–14 IU/l; FSH, 1.5–8 IU/l; testosterone, 10–28 nmol/l.

Reference ranges for females were as follows: LH 2–10 IU/L follicular, 20–60 IU/L midcycle,

4–14 IU/L luteal; FSH 10-50 IU/L, mid-cycle, 1.5–8 IU/L, follicular and luteal; oestradiol <300 pmol/L early follicular, 400-1500 pmol/L midcycle, 200-1000 pmol/L luteal. The respective intra- and inter-assay coefficients of variation for each assay were: LH 4.1% and 2.7%; FSH

4.1% and 3.0%; oestradiol 3.3% and 3.0%; total testosterone 4.2% and 2.8%. Analytical sensitivities were: LH 0.5 IU/l, FSH 0.05 IU/l; oestradiol 37 pmol/l; total testosterone 2 nmol/l.

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4.3.5 Kisspeptin measurement

Blood samples for plasma kisspeptin analysis were collected in lithium heparin tubes

(Beckton Dickson, Franklin Lakes, NJ, USA) containing 5000 kallikrein inhibitor units of aprotinin (0.2ml Trasylol; Bayer, Newbury, UK). Samples were immediately centrifuged at room temperature using a Hettich EBA 20 machine (Hettich International, Tuttlingen,

Germany) for 10 minutes at 3000rpm, and then separated. Plasma samples were stored at minus 20OC until analysis. Plasma kisspeptin immunoreactivity (IR) was measured using an in house radioimmunoassay. Antibody GQ2 was raised in sheep immunised with synthetic human kisspeptin-54 (Bachem UK Ltd.) conjugated to bovine serum albumin (BSA) by glutaraldehyde and used at a final dilution of 1:3,500,000. The antibody cross-reacted 100% with human kisspeptin-54, kisspeptin-14, and kisspeptin-10 and less than 0.01% with other related RF amide proteins, including prolactin-releasing peptide, RF amide-related peptide 1

(RFRP1, human and rat), RFRP2 (human), RFRP3 (human), QRFP43 (human), neuropeptide FF (human), and neuropeptide AF (human). The iodogen method was used to prepare the 125I-kisspeptin-54 label, which was subsequently purified by High Performance

Liquid Chromatography (Salacinski et al. 1981). The assay was carried out in duplicate using dilutions of neat plasma in 0.7ml of 0.06M phosphate buffer (pH 7.2) containing 0.3% BSA.

Incubation was for 3 days at 4oC. Subsequently free and antibody-bound label were separated by charcoal adsorption. The limit of detection was 2 pmol/l of plasma kisspeptin with 95% confidence interval, and the intra- and interassay coefficients of variation were 8.3% and 10.2%, respectively.

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4.3.6 Study 1: The effects of intravenous bolus injection of saline or kisspeptin-10 in healthy male volunteers on plasma kisspeptin IR and reproductive hormones

Intravenous bolus injection of 0.9% saline or kisspeptin-10 (at doses 0.3, 1.0, 3.0, or 10 nmol/kg) was administered at time 0 min. For all studies the intravenous bolus was given via a cannulated antecubital vein over 10 seconds and subsequently flushed with 10ml of 0.9% saline. Blood samples were taken at -30, 0, 10, 20, 30, 40, 50, 60, 75, 90, 120, 150, and 180 minutes for measurement of serum reproductive hormones and plasma kisspeptin IR (n=4-5 per group).

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4.3.7 Study 2: Effects of intravenous bolus injection of saline, kisspeptin-10 or kisspeptin-54 in healthy female volunteers

A: Follicular phase of the menstrual cycle

Women between day 2-10 of their menstrual cycle were administered an intravenous bolus injection of 0.9% saline, or kisspeptin-10 (at doses 1.0, 3.0, or 10 nmol/kg), or kisspeptin-54

(1.0 nmol/kg) at time 0 min, and blood samples were taken at -30, 0, 10, 20, 30, 40, 50, 60,

75, 90, 120, 150, and 180 min (n=4-5 per group).

B: Preovulatory phase of the menstrual cycle

Women 15-16 days before their next predicted period received intravenous bolus injection of

10 nmol/kg kisspeptin-10 as described for the follicular phase study above (n=5).

4.3.8 Study 3: Effects of subcutaneous bolus injection of saline or kisspeptin-10 on plasma kisspeptin-IR and serum reproductive hormones in healthy female volunteers in the follicular phase of menstrual cycle

Subjects in the follicular phase of menstrual cycle were admitted to our clinical investigation unit. Kisspeptin-10 (at doses of 2, 4, 8, 16, or 32 nmol/kg) or 0.9% saline were subcutaneously administered at time 0 minutes, and blood samples taken at -30, 0, 15, 30,

45, 60, 75, 90, 120, 150, 180, 210 and 240 minutes for measurement of serum LH, FSH oestradiol and plasma kisspeptin immunoreactivity (IR) (n=4-5 per group).

4.3.9 Study 4: Effects of intravenous infusion of saline or kisspeptin-10 on plasma kisspeptin-IR and serum reproductive hormones in healthy female volunteers

Subjects in the follicular phase of menstrual cycle were admitted to our Clinical Investigation

Unit. Kisspeptin-10 was dissolved in saline containing gelofusine (5% vol/vol) (Braun Medical,

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Sheffield, UK) to minimize peptide adsorption to the infusion system (Kraegen et al. 1975) and was infused over 90 minutes. During the first 30 minutes of infusion, the volunteers were infused with 20, 50, 90, 180, 360 or 720 pmol/kg/min. The infusion rate for each volunteer was then halved for the remaining 60 min of each infusion. This dosing regimen was designed to achieve a steady-state concentration of serum kisspeptin during the infusion period (Dhillo et al. 2005a;Edwards et al. 1999). Blood samples were taken at -30, 0, 15, 30,

45, 60, 75, 90, 120, 150, 180, 210 and 240 minutes for measurement of serum LH, FSH oestradiol and plasma kisspeptin IR.

4.3.9.1 Study 5: Pharmacokinetic profile of kisspeptin-IR during intravenous infusion of kisspeptin-10

To determine the plasma half-life of kisspeptin-10 in men, and in women during the follicular and preovulatory phases of the menstrual cycle, frequent blood sampling was performed during intravenous infusion of 360 pmol/kg/min of kisspeptin-10. Detailed blood sampling was performed at 1 minutely (from 91-100 min) and 2 minutely (from 102-120 min) intervals immediately after stopping the kisspeptin-10 infusion (at time 90 min). Blood samples were assayed for plasma kisspeptin IR. The linear regression line of natural log plasma kisspeptin

IR was used to calculate the half-time of disappearance (t1/2) for infused kisspeptin-10 in healthy males and females.

4.3.9.2 Data Analysis

Data are presented as mean ± SEM. Area under the curve (AUC) was calculated to provide a cumulative measure of kisspeptin/reproductive hormone change throughout the time course of the study. Time profiles of hormone levels were compared using two-way ANOVA with Bonferroni's multiple comparison test. Pairs of means were compared with unpaired t tests, and multiple means of AUC reproductive hormone release were compared using one-

119 way ANOVA with Bonferonni's multiple comparison test. Slopes of linear regression lines were compared using an F test. Half-lives were calculated using natural log 2/gradient of linear regression line and compared using one-way ANOVA with Bonferonni‟s multiple comparison test. For all statistical tests, P < 0.05 was considered statistically significant. All data of serum reproductive hormones during treatment are presented as increases in serum levels after injection when compared with pre-injection level.

4.4 Results

Baseline characteristics of the subjects recruited after medical screening are summarised in table 9 below. There was no significant difference in age or body mass index (BMI) between the male and female volunteers. In accordance with normal physiology circulating levels of

LH, FSH, and oestradiol were significantly higher in the preovulatory phase of the menstrual cycle in females.

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Table 9. Baseline characteristics of healthy male and female volunteers recruited to the study.

Data are shown as mean ± SEM.

Baseline Healthy male Healthy female Age (yr) 28.8 ± 2.1 31.8 ± 1.4 P = 0.23 Characteristic volunteers volunteers BMI (kg/m2) 24.5 ± 0.5 22.6 ± 0.8 P = 0.09 (n=11) (n=15) Length of menstrual 28 ± 0.3 cycle (d)

LH (iU/l) 2.9 ± 0.2

Follicular 3.9 ± 0.4

Preovulatory 28.0 ± 4.5a

FSH (iU/l) 2.6 ± 0.2

Follicular 3.8 ± 0.4

Preovulatory 9.8 ± 0.9a

Testosterone (nmol/l) 21.6 ± 1.5

Oestradiol (pmol/l)

Follicular 228 ± 53

Preovulatory 726 ± 71b

a P < 0.0001 vs. follicular phase of the menstrual cycle b P < 0.001 vs. follicular phase of the menstrual cycle

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4.4.1 Study 1: Effects of IV bolus injection of saline or kisspeptin-10 in healthy male volunteers

Subjects reported no side effects following injection of kisspeptin-10 or saline. No significant changes in heart rate, or blood pressure were observed following kisspeptin-10 administration. Levels of plasma kisspeptin-IR were undetectable following saline injection.

The plasma kisspeptin IR was elevated after intravenous bolus injection of kisspeptin-10 at all doses in healthy male volunteers (Figure 25 A, B). The peak occurred for all doses at 10 minutes after injection and was statistically significant (vs. saline) for 3 and 10 nmol/kg of kisspeptin-10 (Figure 25 A). Subsequently plasma kisspeptin IR returned to undetectable levels 50 minutes after injection. The highest plasma kisspeptin-IR was observed following intravenous bolus injection of 10 nmol/kg kisspeptin-10 (mean AUC kisspeptin-IR: 700  160 h.pmol/l, P<0.001 vs. saline) (figure 25 B). At this dose, mean peak kisspeptin-IR (3350 

725 pmol/l) was observed 10 min post injection.

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Figure 25. Plasma kisspeptin levels post intravenous bolus of saline or kisspeptin-10 in healthy men.

Plasma kisspeptin IR (A) after intravenous bolus injection of saline or 0.3, 1, 3, 10 nmol/kg kisspeptin-10. For 10 nmol/kg vs. saline: P<0.001. For 3 nmol/kg vs. saline: P<0.001. Mean kisspeptin IR AUC (B) post-intravenous injection of saline, 0.3, 1, 3, 10 nmol/kg kisspeptin- 10 over the 180 min timecourse. *, P<0.05; **, P<0.01; ***, P<0.001.

A ***

4000 10nmol/kg

3nmol/kg 3000 *** 1nmol/kg 0.3nmol/kg 2000 Saline

1000 Kisspeptin IR (pmol/l) IR Kisspeptin 0 0 30 60 90 120 150 180

Time after Injection (min)

B *** 1000

800

600 * 400

200

AUC Kisspeptin IR (h.pmol/l) IR Kisspeptin AUC 0 Saline 0.3 1 3 10

Kisspeptin-10 Dose (nmol/kg)

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Serum LH was significantly elevated after administration of each tested dose of intravenous bolus kisspeptin-10 compared with saline (Figure 26 A, B) from 20-120 minutes. Peak stimulation of serum LH was observed 30-50 min after injection which was 20-40 minutes after peak plasma kisspeptin IR. Thereafter serum LH levels returned to baseline 180 min after injection (Figure 26 A). Maximal stimulation of LH was observed after intravenous bolus of 10 nmol/kg kisspeptin-10 (mean AUC LH increase was 5.3 ± 1.2 h.IU/l, P<0.001 vs. saline), although there was significant increase for all doses (Figure 26 B).

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Figure 26. Serum LH levels following intravenous bolus administration of saline or kisspeptin-10 in healthy men.

LH increase (A) after IVB injection of saline or 0.3, 1, 3, 10 nmol/kg kisspeptin-10. For 0.3 nmol/kg vs. saline: α, P<0.05; ααα, P<0.001. For 1 nmol/kg vs. saline: β, P <0.05; ββ, P<0.01; βββ, P<0.001. For 3 nmol/kg vs. saline: γγ, P<0.01; γγγ, P<0.001. For 10 nmol/kg vs. saline: δ, P < 0.05; δδ, P < 0.01, δδδ, P < 0.001. Mean serum LH increase AUC (B) for saline, 0.3, 1, 3, 10 nmol/kg kisspeptin-10 post-intravenous bolus. *, P<0.05; **, P<0.01; ***, P<0.001.

    A  5      Saline 4 0.3nmol/kg 3 1nmol/kg 2 3nmol/kg 10nmol/kg 1

0 LH Increase (iU/l) Increase LH -1 -2 0 30 60 90 120 150 180

Time after Injection (min)

*** B

-2

AUC LH Increase (h.iU/l) LH AUC -4

Saline 0.3 1 3 10

Kisspeptin-10 Dose (nmol/kg)

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Serum FSH was significantly increased compared with saline injection after intravenous bolus injection of 0.3 and 3.0 nmol/kg kisspeptin-10 (Figure 27 A, B) up to 90 minutes after injection. Peak FSH stimulation occurred slightly later than for LH, between 40-150 minutes after injection depending on dose (Figure 27 A). Maximal stimulation of FSH was observed after intravenous bolus of the smallest dose, 0.3 nmol/kg kisspeptin-10 (mean AUC FSH increase was 1.2 ± 0.5 h.iU/l, P<0.05 vs. saline), although significant stimulation was also seen at 3 nmol/kg (Figure 27 B).

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Figure 27. Serum FSH levels following intravenous bolus (IVB) administration of saline or kisspeptin-10 in healthy men.

Serum FSH (A) after IVB of saline or 0.3, 1, 3, 10 nmol/kg kisspeptin-10. For 0.3 nmol/kg vs. saline: α, P<0.05; αα, P<0.01; ααα, P<0.001. For 3 nmol/kg vs. saline: γ, P<0.05; γγ, P<0.01; γγγ, P<0.001. Mean serum FSH increase AUC (B) for saline, 0.3, 1, 3, 10 nmol/kg kisspeptin-10 over the 180 min post-intravenous bolus. *, P<0.05; **, P<0.01; ***, P<0.001.

     A   1.2 Saline 0.8 0.3nmol/kg 1nmol/kg 0.4 3nmol/kg 10nmol/kg 0.0

FSH Increase (iU/l) Increase FSH -0.4

-0.8 0 30 60 90 120 150 180

Time after Injection (min)

B **

2 *

0 AUC FSH Increase (h.iU/l) FSH AUC -1 Saline 0.3 1 3 10

Kisspeptin-10 Dose (nmol/kg)

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Serum testosterone AUC was significantly increased compared with saline injection after intravenous bolus injection of 1.0 nmol/kg kisspeptin-10 (Figures 28 B). Serum levels of testosterone at this dose steadily increased to peak levels 150-180 min after injection

(Figure 28 A).

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Figure 28 Serum testosterone levels following intravenous bolus (IVB) administration of saline or kisspeptin-10 in healthy men.

Serum Testosterone (A) after IVB of saline or 0.3, 1, 3, 10 nmol/kg kisspeptin-10. For 0.3 nmol/kg vs. saline: α, P<0.05. For 1 nmol/kg vs. saline: β, P <0.05; ββ, P<0.01. For 10 nmol/kg vs. saline: δ, P<0.05. Mean serum FSH increase AUC (B) for saline, 0.3, 1, 3, 10 nmol/kg kisspeptin-10 over the 180 min post-intravenous bolus. *, P<0.05; **, P<0.01; ***, P<0.001.

A       10 Saline 0.3nmol/kg 5 1nmol/kg 3nmol/kg 0 10nmol/kg

-5

Testosterone Increase (nmol/l) Increase Testosterone -10 0 30 60 90 120 150 180

Time after Injection (min)

B *

-10

-20

Saline 0.3 1 3 10 AUC Testosterone Increase (h.nmol/l) Increase Testosterone AUC Kisspeptin-10 Dose (nmol/kg)

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4.4.2 Study 2: Effects of intravenous bolus injection of saline, kisspeptin-10 or kisspeptin-54 in healthy female volunteers

A: Follicular phase of the menstrual cycle

The plasma kisspeptin IR was elevated after intravenous bolus injection of kisspeptin-10 at all doses in healthy female volunteers during the follicular phase of the menstrual cycle

(Figure 29 A, B). The highest plasma kisspeptin IR was observed after intravenous bolus injection of 10 nmol/kg kisspeptin-10 (mean AUC kisspeptin in healthy females during the follicular phase was 527 ± 108 h.pmol/l, P < 0.001 vs. saline). Although this was lower when compared with kisspeptin IR after the same dose of kisspeptin-10 to men (700 ± 160 h.pmol/l for men), this difference was not statistically significant (P = 0.42 vs. men). At the dose of 10 nmol/kg kisspeptin-10, mean peak kisspeptin IR (2638 ± 302 pmol/l) was observed 10 min after injection. Plasma kisspeptin IR subsequently returned to undetectable levels 50 min after injection (Fig 29 A).

Kisspeptin-54 was also administered as an IV bolus in this study as a positive control as previous work has demonstrated that it stimulates reproductive hormones in females. Peak kisspeptin IR occurred 30-40 minutes after injection of kisspeptin-54 in contrast to kisspeptin-10 which peaked at 10 minutes after injection (Figure 29 A).

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Figure 29 Plasma kisspeptin levels following intravenous bolus (IVB) administration of saline or kisspeptin-10 in healthy women.

Plasma kisspeptin IR (A) after IVB of saline, 1, 3, 10 nmol/kg kisspeptin-10 and 1 nmol/kg kisspeptin-54 in follicular or preovulatory phase. For 3 nmol/kg vs. saline: φφφ, P<0.001. For 10 nmol/kg follicular vs. saline: λλλ, P<0.001. For 10 nmol/kg preovulatory vs. saline: µµµ, P<0.001. For kisspeptin-54 (1nmol/kg) vs. saline: ϴ, P<0.05; ϴϴϴ, P<0.001. Mean kisspeptin IR AUC (B) for saline, 1, 3, 10 nmol/kg kisspeptin-10 and 1 nmol/kg kisspeptin-54 post- intravenous bolus in follicular and preovulatory phase. *, P<0.05; **, P<0.01; ***, P<0.001.

A  3500     Saline follicular  3000 1nmol/kg KP10 follicular 2500 3nmol/kg KP10 follicular 2000 10nmol/kg KP10 follicular 10nmol/kg KP10 preov 1500 Saline preov 1000 KP54 follicular

500 Kisspeptin IR (pmol/L) IR Kisspeptin 0 0 30 60 90 120 150 180 Time after Injection (min)

B 900 ** *** 800 *** 700 ** 600 500 400 300 200 100

AUC Kisspeptin IR (h.pmol/l) IR Kisspeptin AUC 0 Saline 1 3 10 KP54 Saline 10

FOLLICULAR PREOV

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Unlike in the male study, no significant changes in serum LH, FSH or oestradiol were observed after intravenous bolus injection of kisspeptin-10 at all doses tested in the follicular phase (Figures 30, 31, 32). However mean LH and FSH AUC were significantly elevated after intravenous bolus injection of 1 nmol/kg of kisspeptin-54 (Figures 30, 31).

B: Preovulatory phase of the menstrual cycle

Kisspeptin IR was significantly elevated in women during the preovulatory phase after kisspeptin-10 injection compared with saline. This elevation was not significantly different when compared with kisspeptin IR after the same dose of kisspeptin-10 in follicular phase women or men (mean AUC kisspeptin IR in preovulatory phase was 320 ± 56 h.pmol/l, P =

0.13 vs. follicular phase and P = 0.06 vs. men) (Figure 29).

Serum LH and FSH were both significantly elevated after intravenous bolus injection of 10 nmol/kg kisspeptin-10 in female volunteers during the preovulatory phase of the menstrual cycle (mean AUC increase was 30.4 ± 11.1 h.iU/l (LH), P < 0.05 vs. saline, and 6.9 ± 0.9 h.iU/l (FSH), P < 0.01 vs. saline) (Figures 30, 31). This was in contrast to the lack of response seen in the follicular phase. Peak LH and FSH stimulation occurred 40 minutes after injection of kisspeptin-10 with significant elevation maintained until 75 minutes (Figures

30, 31).

Serum oestradiol, however, was not altered significantly by kisspeptin-10 in the preovulatory phase (mean AUC oestradiol increase was 111 ± 96 h/pmol/l, P =0.13 vs. saline) (Figure 32).

132

Figure 30. Serum LH levels following intravenous bolus (IVB) administration of saline or kisspeptin-10 in healthy women.

Serum LH (A) after intravenous bolus injection of saline, 1, 3, 10 nmol/kg kisspeptin-10 and 1 nmol/kg kisspeptin-54 in follicular or preovulatory phase. For 10 nmol/kg preovulatory vs. saline: µµ, P<0.01; µµµ, P<0.001. Mean serum LH AUC (B) for saline, 1, 3, 10 nmol/kg kisspeptin-10 and 1 nmol/kg kisspeptin-54 over the 180 min timecourse post-intravenous bolus in follicular and preovulatory phase. *, P<0.05; **, P<0.01; ***, P<0.001.

A 35   Saline follicular 30 1nmol/kg KP10 follicular 25 3nmol/kg KP10 follicular 20 10nmol/kg KP10 follicular 15 10nmol/kg KP10 preov 10 Saline preov

5 KP54 follicular LH Increase (iU/l) Increase LH 0 -5 0 30 60 90 120 150 180

Time after Injection (min)

B * * 50 40 30 20 10 0

-10 AUC LH Increase (h.iU/l) Increase LH AUC -20 Saline 1 3 10 KP54 Saline 10

FOLLICULAR PREOV

133

Figure 31. Serum FSH levels following intravenous bolus (IVB) administration of saline or kisspeptin-10 in healthy women.

Serum FSH increase (A) after IVB of saline, 1, 3, 10 nmol/kg kisspeptin-10 and 1 nmol/kg kisspeptin-54 in follicular or preovulatory phase. For 10 nmol/kg preovulatory vs. saline: µ, P<0.05; µµ, P<0.01; µµµ, P<0.001. Mean serum FSH AUC (B) for saline, 1, 3, 10 nmol/kg kisspeptin-10 and 1 nmol/kg kisspeptin-54 post-intravenous bolus in follicular and preovulatory phase. *, P<0.05; **, P<0.01; ***, P<0.001.

A     6 5 4 3 2 1 A 0 35  

FSH Increase (iU/l) Increase FSH Saline follicular 30 -1 1nmol/kg KP10 follicular 25 3nmol/kg KP10 follicular -2 20 0 30 60 90 120 150 180 10nmol/kg KP10 follicular 15 10nmol/kg KP10 preov Time10 after Injection (min) Saline preov 5 KP54 follicular LH Increase (iU/l) Increase LH 0 -5 0 30 60 90 120 150 180 B * Time after Injection** (min) 12 10 8 6 4 2 0

-2 AUC FSH Increase (h.iU/l) FSH AUC -4 Saline 1 3 10 KP54 Saline 10

FOLLICULAR PREOV

134

Figure 32. Serum oestradiol levels following intravenous bolus (IVB) administration of saline or kisspeptin-10 in healthy women.

Serum oestradiol increase (A) after IVB injection of saline, 1, 3, 10 nmol/kg kisspeptin-10 and 1 nmol/kg kisspeptin-54 in follicular or preovulatory phase. Mean serum oestradiol AUC (B) for saline, 1, 3, 10 nmol/kg kisspeptin-10 and 1 nmol/kg kisspeptin-54 post-intravenous bolus in follicular and preovulatory phase.

A 150 100 50 0 -50 -100 A 35   Saline follicular -150 30 1nmol/kg KP10 follicular 25 Oestradiol Increase (pmol/l) Increase Oestradiol -200 3nmol/kg KP10 follicular 20 0 30 60 90 120 150 180 10nmol/kg KP10 follicular 15 10nmol/kg KP10 preov Time10 after Injection (mins) Saline preov 5 KP54 follicular LH Increase (iU/l) Increase LH 0 -5 0 30 60 90 120 150 180 B Time after Injection (min)

300

200

100

-100

-200

-300 AUC Oestradiol Increase (h.iU/l) Increase Oestradiol AUC Saline 1 3 10 KP54 Saline 10

FOLLICULAR PREOV

135

4.4.3 Study 3: Effects of subcutaneous bolus injection of saline or kisspeptin-10 on plasma kisspeptin-IR and serum reproductive hormones in healthy female volunteers

Subjects reported no side effects following subcutaneous injection of kisspeptin-10 or saline.

No significant changes in heart rate, systolic or diastolic blood pressure were observed following kisspeptin-10 administration. Plasma kisspeptin-IR in healthy female volunteers was significantly raised during the 4 hours following subcutaneous (sc) injection of kisspeptin-10 at doses of 4 nmol/kg and higher, when compared with saline (Figure 33 A, B).

Progressively higher plasma kisspeptin-IR was observed with each increment in sc dose of kissppeptin-10 injection. The highest plasma kisspeptin-IR following sc injection was observed following injection of 32 nmol/kg kisspeptin-10 (mean AUC kisspeptin-IR, 201 

16h.pmol/l, P<0.001 vs. saline). No significant changes in serum reproductive hormone levels were observed following sc bolus injection of kisspeptin-10 at any dose (Figure 34 A-

D).

136

Figure 33. Plasma kisspeptin immunoreactivity (IR) following subcutaneous bolus (sc) injection of kisspeptin-10 to healthy women.

Time profiles (A) and area under curve AUC for plasma kisspeptin IR (B) during 4 hours after sc bolus injection of saline or kisspeptin-10 (n=4-5 per group). Data is shown as mean +/- SEM. *P<0.05; **P<0.01; ***P < 0.001

A 300 2 nmol/kg

4 nmol/kg 200 8 nmol/kg

100 16 nmol/kg

32 nmol/kg 0 Kisspeptin IR (pmol/L) IR Kisspeptin 0 60 120 180 240

Time (minutes) Injection

B *** 250 ** 200 * 150

100

AUC Kisspeptin IR (pmol/L) IR Kisspeptin AUC Saline 2 4 8 16 32

Dose of kisspeptin-10 (nmol/kg)

137

Figure 34. Serum reproductive hormone levels following subcutaneous bolus injection of kisspeptin-10 to healthy women.

Time profiles and area under curve (AUC) changes in serum LH (A, B) and FSH (C, D) during 4 hours after sc bolus injection of saline or kisspeptin-10 (n=4-5 per group). Data is shown as mean +/- SEM. *P<0.05; **P<0.01; ***P < 0.001

A 4

-2 LH increase (iU/L) increase LH

-4 0 60 120 180 240 Time (minutes)

Injection

B 8

-4 AUC LH increase (h.iU/L) increase LH AUC -8 Saline 2 4 8 16 32

Dose of kisspeptin-10 (nmol/kg)

138

C 2

-1

-2 (iU/L) increase FSH

-3 0 60 120 180 240 Time (minutes)

Injection

D 8

-4

(h.iU/L) increase FSH AUC -8 Saline 2 4 8 16 32

Dose of kisspeptin-10 (nmol/kg)

139

4.4.4 Study 4: Effects of intravenous infusion of kisspeptin-10 on plasma kisspeptin-IR and serum reproductive hormones in healthy female volunteers

Ninety minute intravenous infusions of kisspeptin-10 were administered to healthy female volunteers in the follicular phase of menstrual cycle at doses 20, 50, 90, 180, 360 or 720 pmol/kg/min. Subjects reported no side effects during infusion of kisspeptin-10. No significant changes in heart rate, systolic or diastolic blood pressure were observed during kisspeptin-10 infusion. Plasma kisspeptin-IR increased during intravenous infusion of kisspeptin-10 at all doses (Figure 35 A). The highest plasma kisspeptin-IR was observed during intravenous infusion of 720 pmol/kg/min kisspeptin-10 (mean AUC kisspeptin-IR,

2518  100 h.pmol/l). All intravenous infusion doses of kisspeptin-10 were associated with a higher mean plasma kisspeptin-IR than the highest studied sc dose of kisspeptin-10 (32 nmol/kg, mean AUC kisspeptin-IR 201  16 h.pmol/l) (figure 35 B). No significant changes in serum reproductive hormone levels were observed for any dose of kisspeptin-10 infusion, in healthy female volunteers in the follicular phase of menstrual cycle (Figure 36 A-C).

140

Figure 35. Plasma kisspeptin immunoreactivity (IR) during a 90 minute intravenous infusion of kisspeptin-10 to healthy women.

Time profiles (A) and area under curve AUC (B) for plasma kisspeptin IR. Data is shown as mean +/- SEM *P<0.05; **P<0.01; ***P < 0.001

A Infusion 2000 20pmol/kg/min 50pmol/kg/min 1500 90pmol/kg/min 180pmol/kg/min 1000 360pmol/kg/min 720pmol/kg/min

500 Kisspeptin IR (pmol/L) IR Kisspeptin 0 0 60 120 180 240 Time (minutes)

3000 ** *** 2000 ***

1000

0 AUC Kisspeptin IR (h.pmol/L) IR Kisspeptin AUC 20 50 90 180 360 720

Dose of kisspeptin-10 (pmol/kg/min)

141

Figure 36. Serum reproductive hormone levels during intravenous infusion of kisspeptin-10 to healthy women.

Area under the curve AUC changes in serum LH (A), FSH (B) and oestradiol E2 (C) during 4 hours after commencement of a 90 minute intravenous infusion of kisspeptin-10 in the follicular phase of menstrual cycle (n=4-5 per group). Data is shown as mean +/- SEM. **P < 0.01; ***P < 0.001.

A 160

120

-40 20 50 90 180 360 720

(h.iU/L) increase LH AUC Dose of kisspeptin-10 (pmol/kg/min)

B 15 20 pmol/kg/min 10 50 pmol/kg/min 90 pmol/kg/min 5 180 pmol/kg/min

0 360 pmol/kg/min 720 pmol/kg/min -5

20 50 90 180 360 720 AUC FSH increase (h.iU/L) increase FSH AUC Dose of kisspeptin-10 (pmol/kg/min)

C 400

200

-200

-400 -600 AUC E2 increase (h.iU/L) increase E2 AUC 20 50 90 180 360 720

Dose of kisspeptin-10 (pmol/kg/min)

142

4.4.5 Study 5: Pharmacokinetic profile of kisspeptin-IR during intravenous infusion of kisspeptin-10

To determine the plasma half-life of kisspeptin-10, detailed blood sampling was performed in men and women during the follicular and preovulatory phases of the menstrual cycle, during the 4 hours after commencing an intravenous infusion of 360 pmol/kg/min of kisspeptin-10.

Peak mean kisspeptin-IR was observed 30 minutes after commencing kisspeptin-10 infusion

(1088  243 pmol/l). Plasma kisspeptin-IR fell rapidly nearly ten-fold within 30 minutes of cessation of the kisspeptin-10 infusion (Figures 37, 38). The plasma half-life of kisspeptin-10 was calculated to be 3.3 minutes. During the infusion males had higher plasma kisspeptin IR than females in both the follicular and preovulatory phases.

143

Figure 37. Plasma kisspeptin IR before, during and after a 90 minute infusion of 360 pmol/kg/min kisspeptin-10.

Blood was sampled every 1 min between 90-100 min and then every 2 min between 100- 120 min. For men vs. women during the follicular phase: ψ, P<0.05; ψψ, P<0.01; ψψψ, P<0.001. For men vs. women during the preovulatory phase: ω, P<0.05; ωωω, P<0.001.

Infusion 2500  Male  2000 Preovulatory    Follicular 1500   

1000

500 Kisspeptin IR (pmol/L) IR Kisspeptin 0 -30 0 30 60 90 120 150 180 Time (min)

144

When plotted on a natural log scale, linear regression slopes were not significantly different among the three groups (F = 0.013; Degrees of Freedom = 2; Degrees of Freedom denominator = 137; P = 0.88). Plasma half-lives of kisspeptin-10 were calculated using these regression slopes and were also statistically similar between all three groups: male, 4.62 ±

0.34 min; female follicular phase 4.72 ± 0.43 min; female preovulatory phase 4.40 ± 0.34 min

(Fig 38).

145

Figure 38. Natural Logarithm kisspeptin IR against time immediately after cessation of a 90 minute infusion of 360 pmol/kg/min kisspeptin-10.

Vertical detached lines indicate calculated half-lives for females in the follicular and preovulatory phase and males.

7.5

Male

7.0 Female: Follicular

Female: Preovulatory 6.5

6.0

5.5 Natural Log (kisspeptin IR pmol/l) IR (kisspeptin Log Natural

5.0 90.0 90.5 91.0 91.5 92.0 92.5 93.0 93.5 94.0 94.5 95.0 95.5 96.0 96.5 97.0 97.5 98.0 98.5 99.0 99.5 100.0 Time (min)

146

5. Discussion

This study reveals a previously unknown sexual dimorphism in responsiveness to kisspeptin-

10, by intravenous bolus injection in healthy men and women. Numerous studies in various male and female animal species have demonstrated that kisspeptin-10 robustly stimulates gonadotrophin release. Thus kisspeptin signalling is a potential therapeutic target for treating reproductive disorders (Arreguin-Arevalo et al. 2007; Caraty et al. 2007; Irwig et al. 2004;

Kadokawa et al. 2008; Lents et al. 2008; Navarro et al. 2004; Plant et al. 2006; Ramaswamy et al. 2007; Shahab et al. 2005). Studies on the effects of kisspeptin 10 on male primate models have demonstrated gonadotrophin stimulation (Plant et al. 2006; Ramaesh et al.

2010; Shahab et al. 2005). Central administration of kisspeptin10 into the stalk median eminence/medial basal hypothalamus stimulates release of GnRH (Keen et al. 2008;

Roseweir et al. 2009). Two studies on the effects of kisspeptin-10 on healthy men have also demonstrated that kisspeptin-10 stimulates gonadotrophin release (Chan et al. 2011;

George et al. 2011). A recent study in pubertal and prepubertal female monkeys has demonstrated that central administration of kisspeptin-10 stimulated GnRH release in both groups in a dose dependent fashion (Guerriero et al. 2012).

I observed that an intravenous bolus injection of kisspeptin-10 significantly stimulated LH release in healthy men at all doses tested. These results are consistent with previous reports on the effects of the administration of kisspeptin-54 (Dhillo et al. 2005) and kisspeptin-10

(Chan et al. 2011; George et al. 2011) on the HPG axis in healthy men.

I noted that intravenous bolus doses of kisspeptin-10 ranging from 0.3 – 10 nmol/kg were associated with similar degrees of gonadotrophin secretion in healthy male subjects. George et al. report that peripheral intravenous administration of kisspeptin-10, stimulates serum LH secretion at doses as low as 0.01 μg/kg with a maximal response seen at 1 μg/kg (George et al. 2011). Therefore, the doses of kisspeptin-10 selected during my study may have

147 stimulated near-maximal levels of gonadotrophin secretion in healthy men. Tachyphylaxis was noted in the George at al study; with increases in serum LH at higher kisspeptin doses of 3 μg/kg being lower than those stimulated by 1 μg/kg of kisspeptin-10 (George et al. 2011).

This is consistent with previous reports of tachyphylaxis occurring in women after chronic kisspeptin-54 injections (Jayasena et al. 2009). Thus, the similarity between LH responses at all doses between 0.3 and 10 nmol/kg kisspeptin-10 in men might be explained in part by tachyphylaxis to kisspeptin-10 at the higher tested doses. Additional studies are required to investigate these observations.

Surprisingly, intravenous bolus injection of kisspeptin-10 failed to stimulate reproductive hormone release in healthy female volunteers during the follicular phase of menstrual cycle.

Plasma kisspeptin IR was raised for only 40 min following intravenous bolus injection of kisspeptin-10 to male and female volunteers. Our previous clinical studies suggest that sc bolus injection of kisspeptin-54 raises kisspeptin IR for a longer period of time when compared with intravenous administration of kisspeptin-54. I therefore studied the effects of sc bolus injection of kisspeptin-10 in order to determine if a more sustained exposure to exogenous kisspeptin-10 would stimulate reproductive hormone release in healthy women.

Despite elevations in plasma kisspeptin IR for up to 90 min post-injection, serum gonadotrophin levels were not elevated following sc bolus injection of kisspeptin-10 in healthy women in the follicular phase of menstrual cycle.

In order to study the effects of sustained, high dose kisspeptin-10 administration on reproductive hormone release, I then administered intravenous infusions of kisspeptin-10 to healthy women. Despite markedly raised plasma levels of kisspeptin IR up to 2000pmol/l, serum gonadotrophin levels were not elevated during intravenous infusion of kisspeptin-10 to healthy female volunteers in the follicular phase of menstrual cycle. Our data therefore suggest that intravenous bolus injection, subcutaneous bolus injection, or intravenous infusion of kisspeptin-10 fails to stimulate gonadotrophin release in healthy women during the follicular phase of menstrual cycle, at the doses tested in this study. This is the first study

148 to investigate the effects of kisspeptin-10 in women. This study demonstrates that women in the follicular phase of menstrual cycle are less responsive to kisspeptin-10 administration than men.

Only a marginal elevation of plasma kisspeptin IR (approximately 10 h.pmol/l) was necessary to stimulate significant LH secretion in men after injection of kisspeptin-10 (0.3 nmol/kg iv bolus); by contrast, a 50-fold greater elevation in plasma kisspeptin IR failed to stimulate LH release in women given the high dose 10 nmol/kg kisspeptin-10 during the follicular phase. Furthermore the doses used in the study produced similar cumulative plasma kisspeptin-IR in both males and females (P = 0.42 for 10 nmol/kg kisspeptin-10 in men vs. women). It may be expected, therefore that levels of kisspeptin-10 may have been modified by factors known to differ between the sexes, such as body fat content or clearance of the peptide from the plasma (Soldin, Chung, & Mattison 2011). However this study also demonstrated no differences in plasma half-life and linear regression gradient for kisspeptin-

10 in males and females.

Consistent with our previous studies, (Dhillo 2007, Jayasena et al. 2009, Jayasena 2010) kisspeptin-54 was able to significantly stimulate LH and FSH increases in women. Previous studies have suggested that kisspeptin 54 has a higher in vivo potency for stimulating the kisspeptin receptor. Jayasena et al. reported an increased potency of kisspeptin-54 over kisspeptin-10 (Jayasena et al. 2011) for stimulating LH in female adult rats. It has also been previously described that kisspeptin-52 (Irwig et al. 2004) and kisspeptin-54 (Thompson et al.

2004) stimulate LH more potently when compared with kisspeptin10 in male rats. Therefore our findings may be explained by an increased potency of kisspeptin 54 compared to kisspeptin 10. However, a ten-fold increase in molar dose for kisspeptin-10 compared to kisspeptin-54 still failed to stimulate reproductive hormone release in women in the follicular phase on the menstrual cycle in this study.

149

This difference between kisspeptin-10 and kisspeptin-54 on gonadotrophin release may be a consequence of rapid breakdown of kisspeptin-10 in the circulation. Indeed the in vivo plasma half-life of iv kisspeptin-10 in this study was calculated as approximately 4 minutes, which is 7-fold shorter than the calculated in vivo plasma half-life of kisspeptin-54; which is

28 minutes (Dhillo et al. 2005). It is possible that the kisspeptin receptor requires a prolonged exposure to raised plasma kisspeptin levels for activation. However, the administration of a subcutaneous dose of kisspeptin 10 or a 90 minute infusion of kisspeptin-

10; which resulted in a similar time-profile of plasma kisspeptin IR as that seen with kisspeptin-54, also failed to cause reproductive hormone stimulation.

Women in the preovulatory phase of the menstrual cycle are known to be significantly more sensitive to the effects of kisspeptin-54 on gonadotrophin release when compared with women in the follicular phase of the menstrual cycle (Dhillo et al. 2007). In keeping with this observation, an intravenous bolus injection of kisspeptin-10 significantly stimulated LH and

FSH release in women during the preovulatory phase of the menstrual cycle in this study. As the pharmacokinetic profiles of plasma kisspeptin IR in both the follicular and preovulatory phases were similar, these results suggest that as with kisspeptin-54, women have heightened sensitivity to kisspeptin-10 during the preovulatory phase of the menstrual cycle.

This suggests that the different background hormonal milieu with higher oestradiol levels in the pre-ovulatory phase may influence the responsiveness to kisspeptin-10.

A recent study, published after the completion of my work, examined the effects of kisspeptin-10 in women at different stages of the menstrual cycle and supports my findings.

In this study the authors also demonstrate a difference in response to an intravenous bolus of kisspeptin-10 across the menstrual cycle, with stimulation in gonadotrophin release in the luteal and preovulatory phase and a minimal response seen in the follicular phase (Chan et al. 2012). In this study the authors report that an intravenous bolus dose of 0.24 nmol/kg of kisspeptin-10 consistently stimulated a significant LH pulse in the preovulatory and luteal phase, with the response in the preovulatory phase being significantly higher. However,

150 despite increasing the administered dose of kisspeptin-10 to 0.72 nmol/kg no consistent response in LH stimulation was noted in the follicular phase (Chan et al. 2012). The authors suggest that GnRH neurones may have varied responsiveness to exogenous kisspeptin stimulation across the menstrual cycle. They postulate a theory that GnRH neurones are maximally stimulated by endogenous kisspeptin in the follicular phase and thus the additional ability to respond to exogenous kisspeptin is limited. Further studies are required to investigate this hypothesis.

Studies in rodents have identified two distinct kisspeptin neuronal populations that have different responses to oestradiol. In female rodents, c-fos expression within kisspeptin neurones and levels of KISS1 expression are increased within the AVPV nucleus of the hypothalamus immediately preceding ovulation as a result of positive feedback on this nucleus by oestradiol (Smith et al. 2006). This contrasts against the negative feedback by oestradiol on a different distinct population of kisspeptin neurones in the ARC as evidenced by reduced levels of ARC KISS1r expression in rat hypothalamic fragments at proestrus when compared with diestrus (Roa et al. 2006). This suggests a complex interplay between

AVPV and ARC neurones with GnRH neurones in rodents.

KISS1 expressing neurones have only been identified in humans in the infundibular nucleus and to a lesser extent the medial preoptic area (Rometo et al. 2007). These neurones express the oestrogen receptor α (ERα). GnRH neurones express ERβ, but this is not involved in feedback mechanisms and hence it is thought that gonadal steroid feedback on the hypothalamus in the reproductive axis is via the kisspeptin neurones. In the low oestrogen state of menopause there is significant increase in KISS1 expression in the infundibular nucleus together with hypertrophy and increased numbers of these KISS1- expressing neurones when compared to pre-menopausal specimens (Rance, 2009). Hence this suggests that higher oestogen states may downregulate KISS1 expression in humans.

However, to explain the preovulatory surge of gonadotrophins there is likely to be a currently unidentified subpopulation of kisspeptin neurones in women that are stimulated by increased

151 oestrogen levels. To explain the results of the current study it is possible that there is a complex interaction dependent on background oestradiol levels between these kisspeptin neurones, exogenous kisspeptin-10 and the kisspeptin receptor of the GnRH neurones.

Guerriero et al. report that in female rhesus monkeys low doses of kisspeptin 10 (10nM) stimulated larger GnRH responses in pubertal monkeys compared to prepubertal monkeys

(Guerriero, Keen, & Terasawa 2012). The authors note that circulating oestradiol levels are higher in pubertal monkeys and that ovariectomy in these animals, which reduces oestradiol levels, eliminated the GnRH response to low and high doses of kisspeptin-10. Thus the difference in response to kisspeptin appears to be related to oestradiol levels. However, the replacement of oestradiol in pubertal monkeys who had undergone ovariectomy resulted in only a partial restoration of the GnRH response to kisspeptin-10. The authors postulate whether this may be due to absence of other ovarian hormones, or failure in adequate amount and length of oestradiol replacement or a change in properties of the oestrogen receptor post ovariectomy (Guerriero et al. 2012).

In this study I noted that in both men and preovulatory women, kisspeptin-10 stimulated LH secretion more potently than FSH. This is consistent with previous studies of the effects of kissepptin-10 and kisspeptin-54 in healthy men and women (Chan et al. 2011; Dhillo et al.

2005; Dhillo et al. 2007; George et al. 2011). In addition, in men the peak FSH increase occurred slightly later than the peak LH increase following iv bolus of kissepptin-10 (40-150 min for FSH vs. 30-50 min for LH). This difference has previously been observed for both males and females (Dhillo et al. 2005; Dhillo et al. 2007). This study therefore suggests that both kisspeptin-10 and kisspeptin-54 stimulate LH secretion more potently and more rapidly than FSH. Administration of GnRH demonstrates a similar pattern of differing LH/FSH potency and rapidity of effect. Combining this with the additional similarity that LH and FSH responses to GnRH are also heightened in the pre-ovulatory phase, provides further evidence to suggest that GnRH is the intermediary messenger for kisspeptin to stimulate the anterior pituitary production of LH and FSH.

152

I did not observe any consistent stimulation of testosterone secretion after iv bolus kisspeptin-10 injection in healthy men when compared with the robust increases in serum LH and FSH observed at all tested doses. Significant increases in serum testosterone were observed only at 1.0 nmol/kg iv bolus kisspeptin-10, but these rises were marginal (no more than 10 h.nmol/l above baseline). Similarly, iv bolus injection of kisspeptin-10 stimulated gonadotropin release in women during the preovulatory phase of the menstrual cycle but did not increase serum oestradiol during 3 h after injection. Previous data suggest that at least 4 hours are required for serum levels of sex steroids to peak after a sc bolus injection of kisspeptin-54 (Dhillo et al. 2005; Dhillo et al. 2007; Jayasena et al. 2009). A longer period of blood sampling after injection may have revealed more pronounced alterations in sex steroid secretion in subjects after injection of kisspeptin-10. Alternatively it is possible that an iv bolus injection of kisspeptin-10 and kisspeptin-54 have a duration of action inadequate to stimulate significant gonadal sex steroid release.

In summary, this is the first clinical study to compare the effects of kisspeptin-10 administration on reproductive hormone release in healthy men and women. Kisspeptin-10 robustly stimulates gonadotrophin release in men and women in the preovulatory phase but fails to stimulate gonadotrophin release in the follicular phase. This sexual dimorphism as well as the different responses between the follicular and preovulatory phase have important clinical implications for the potential therapeutic use of kisspeptin-10 or kisspeptin-54 to treat disorders of reproduction and suggest that kisspeptin-54 may offer a more potent alternative.

153

Chapter 5

General Discussion

154

Since 2003 and the discovery of the role of kisspeptin in hypothalamo-pituitary-gonadal function our understanding of reproductive physiology has been revolutionised. Inactivating mutations of KISS1R in humans and rodents results in hypogonadotrophic hypogonadism and pubertal failure (de Roux et al. 2003; Seminara et al. 2003). Activating mutations of the

KISS1R result in the clinical phenotype of central precocious puberty (Teles et al. 2008).

The novel finding that administration of kisspeptin to healthy men and women stimulates gonadotrophin hormone release (Dhillo et al. 2005; Dhillo et al. 2007), has revealed the potential of kisspeptin as a therapeutic agent for those patients with disorders of reproduction. In this thesis I report the first clinical studies examining the acute effects of administration of kisspeptin-54 on the reproductive axis in women with infertility due to hypothalamic amenorrhoea; as well as the effects of chronic administration of kisspeptin-54.

I also report on the original discovery of sexual dimorphism in gonadotrophin response to exogenous administration of kisspeptin-10.

Hypothalamic amenorrhoea is a common cause of infertility, accounting for 30% of cases of amenorrhoea (Reindollar 1986). Hypothalamic amenorrhoea results from a failure of pulsatile GnRH secretion from the hypothalamus and may be triggered by energy deficits as well as psychological stress (Berga et al. 1989). In this thesis I report that exogenous administration of kisspeptin-54 results in an acute stimulation of serum levels of LH and FSH in women with hypothalamic amenorrhoea. Women with hypothalamic amenorrhoea appeared to be more sensitive to the effects of kisspeptin on gonadotrophin stimulation, with a 4 fold greater response, compared to previously published data on kisspeptin administration to healthy women. Castellano et al. reported that negative energy balance due to short term fasting in rats results in a decrease in hypothalamic expression of Kiss-1 mRNA and an increased hypothalamic expression of kisspeptin receptor GPR54 mRNA

(Castellano et al. 2005). In vitro and rodent in vivo experiments have demonstrated an increased sensitivity to the effects of kisspeptin-10 administration on GnRH secretion and gonadotrophin stimulation in fasted animals (Castellano et al. 2005). Our finding of increased

155 sensitivity to the effects of kisspeptin on the HPG axis, in women with hypothalamic amenorrhoea, is consistent with these animal model data.

Although acute administration of kisspeptin has been shown to stimulate gonadotrophin release in healthy men and women (Dhillo et al. 2005; Dhillo et al. 2007), there have been no previous studies conducted on the effects of long-term chronic administration of kisspeptin in humans. I report that twice daily injections of kisspeptin-54 6.4 nmol/kg to women with hypothalamic amenorrhoea for two weeks results in tachyphylaxis. The reproductive hormone response to kisspeptin was much lower following the last kisspeptin injection compared to the first kisspeptin injection. In order to investigate whether the desensitisation to the effects of kisspeptin was due to desensitisation at the level of the hypothalamus on GnRH secretion or whether there was a decrease in sensitivity of the pituitary gonadotrophs to GnRH; I conducted a second experiment in which I investigated the effects of exogenous GnRH administration on gonadotrophin release following chronic kisspeptin administration. Despite losing sensitivity to the effects of kisspeptin, women with hypothalamic amenorrhoea treated with twice daily injections of kisspeptin-54, remained responsive to the gonadotrophin stimulation with GnRH. Thus, tachyphylaxis to the effects of kisspeptin administration appears to be upstream of the GnRH receptor probably at the level of hypothalamic KISS1R rather than at the pituitary gonadotrophs.

Continuous administration of kisspeptin has been shown to result in desensitisation of the effects of kisspeptin on gonadotrophin stimulation in monkeys and rodents (Seminara et al.

2006; Thompson et al. 2006; Roa et al. 2008). Delayed desensitisation of the KISS1R due to an Arg386Pro mutation, results in the clinical phenotype of precocious puberty (Teles et al.

2008). In vitro studies measuring total inositol phosphate production as a marker of KISS1R signalling, have demonstrated that the KISS1R is desensitised in a time dependent manner

(Bianco et al. 2011). A proportion of internalised KISS1R is recycled and the remainder degraded by proteasomes, which results in long term desensitisation of the KISS1R (Bianco et al. 2011). The Arg386Pro mutation associated with precocious puberty results in reduced

156 degradation of the KISS1R resulting in an increased responsiveness to kisspeptin (Bianco et al. 2011).

Although I did not administer kisspeptin-54 as a continuous infusion, the twice daily injections did lead to raised plasma kisspeptin IR for 6 hours post injection resulting in prolonged exposure to elevated levels of kisspeptin. Therefore a different study protocol of kisspeptin-54 administration i.e. a lower dose or reduced frequency of injections may reduce the desensitisation seen in stimulation of gonadotrophin release. My subsequent studies went on to investigate the time course of the desensitisation of gonadotrophin response and the effects of different dosing regimens of kisspeptin to women with hypothalamic amenorrhoea. I found that tachyphylaxis of LH response to twice-daily sc kisspeptin-54 6.4 nmol/kg occurred gradually over the 14 day injection period, but FSH responsiveness reduced much more rapidly.

The studies comparing different dosing regimens of kisspeptin-54 showed that once- and twice-daily administration of kisspeptin-54 led to significant desensitisation of gonadotrophin responses in women with HA. The tachyphylaxis in response to chronic administration of kisspeptin-54 may be utilised for its clinical effects on down regulating the HPG axis. It has been suggested that chronic kisspeptin administration may be a useful a therapy for sex hormone responsive cancers (Seminara et al. 2006). In addition, pharmacological blockade of kisspeptin signalling may suppress LH pulsatility rather than basal LH levels (Roseweir et al. 2009), posing the potential development of a contraceptive therapy.

My studies demonstrated that twice-weekly administration of 6.4 nmol/kg kisspeptin-54 was only associated with partial desensitisation of gonadotrophin responses during a 2 week pilot study. I therefore conducted a randomised placebo controlled double blind study of twice weekly kisspeptin-54 6.4 nmol/kg administration to women with hypothalamic amenorrhoea.

The findings from this study showed that over the study period of eight weeks participants remained responsive to kisspeptin with a partially sustained gonadotrophin response. There

157 were no adverse effects noted. Although there was stimulation of gonadotrophin release, on review of pelvic ultrasounds there was no significant changes in follicle growth in those women who received kisspeptin-54. This failure in stimulating follicle growth may be secondary to a reduced FSH response to kisspeptin compared to LH. Previous studies in healthy men and women have demonstrated that kisspeptin-54 administration does stimulate LH and FSH, but that the FSH reponse is lower (Dhillo et al. 2005; Dhillo et al.

2007). In addition my results demonstrate that the FSH responses to kisspeptin-54 desensitised more rapidly following twice-daily kisspeptin-54 injection than LH responses.

Thus, FSH stimulation on the gonads may not have been high enough in amplitude or pulsatility to stimulate follicle growth.

Kisspeptin-10 is a shorter peptide produced by protelytic processing of a 145-amino acid precursor protein encoded by the KISS1 gene (Kotani et al. 2001). Previous studies have demonstrated that central or peripheral administration of kisspeptin-10 to a number of mammalian species stimulates gonadotrophin release (Irwig et al. 2004; Navarro et al. 2004;

Gottsch et al 2004; Messager et al. 2005; Caraty et al. 2007; Plant et al. 2006; Ramaswamy et al. 2007; Shahab et al. 2005; Grieves et al. 2007; Lents et al. 2008; Kadokawa et al. 2008).

The stimulatory effects of kisspeptin-10 on gonadotrophin release are inhibited by the central administration of a GnRH antagonist. Compared to kisspeptin-54, kisspeptin-10 has a shorter half-life and faster onset of action after intravenous administration in rodents

(Mikkelsen et al. 2008;Mikkelsen et al. 2009). As kisspeptin-10 is simpler and thus cheaper to manufacture, future kisspeptin-based reproductive therapies may be based upon kisspeptin-10 rather than kisspeptin-54. Human male studies have demonstrated that an intravenous bolus injection of kisspeptin-10 potently stimulates LH secretion while a continuous infusion of kisspeptin-10 also increases testosterone, LH pulse frequency and amplitude (George et al. 2011, Chan et al. 2011). I carried out the first study comparing the effects of kisspeptin-10 administration in women versus men.

158

I report a previously unknown sexual dimorphism in responsiveness to kisspeptin-10 in healthy men and women. I observed that an intravenous bolus injection of kisspeptin-10 significantly stimulated LH release in healthy men. These results are consistent with previous reports on the effects of the administration of kisspeptin-54 (Dhillo et al. 2005) and kisspeptin-10 (Chan et al. 2011; George et al. 2011) on the HPG axis in healthy men.

However, an intravenous bolus injection of kisspeptin-10 failed to stimulate reproductive hormone release in healthy female volunteers during the follicular phase of menstrual cycle.

In order to assess whether a more sustained exposure to exogenous kisspeptin-10 would stimulate reproductive hormone release in healthy women I studied the effects of subcutaneous bolus administration of kisspeptin-10 in addition to an intravenous bolus administration. Despite elevations in plasma kisspeptin IR for up to 90 min post-injection, serum gonadotrophin levels were not elevated by sc bolus injection of kisspeptin-10 in healthy women in the follicular phase of menstrual cycle.

I then went on to investigate the effects of administering intravenous infusion of kisspeptin-

10 to healthy women to study the effects of sustained, high dose kisspeptin-10 administration on reproductive hormone release. Despite markedly raised plasma levels of kisspeptin IR up to 2000pmol/l, serum gonadotrophin levels were not elevated during intravenous infusion of kisspeptin-10 to healthy female volunteers in the follicular phase of menstrual cycle.

My data therefore demonstrates that intravenous bolus injection, subcutaneous bolus injection, or intravenous infusion of kisspeptin-10 fails to stimulate gonadotrophin release in healthy women during the follicular phase of menstrual cycle. This study demonstrates that women in the follicular phase of menstrual cycle are less responsive to kisspeptin-10 administration than men.

Women in the preovulatory phase of the menstrual cycle are known to be significantly more sensitive to the effects of kisspeptin-54 on gonadotrophin release when compared with

159 women in the follicular phase of the menstrual cycle (Dhillo et al. 2007). In keeping with this observation, I found an intravenous bolus injection of kisspeptin-10 significantly stimulated

LH and FSH release in women during the preovulatory phase of the menstrual cycle.

The pharmacokinetic profiles of plasma kisspeptin IR in both the follicular and preovulatory phases were similar; these results suggest that as with kisspeptin-54, women have heightened sensitivity to kisspeptin-10 during the preovulatory phase of the menstrual cycle.

A recently published study also demonstrated a difference in response to kisspeptin-10 across the menstrual cycle, with stimulation in gonadotrophin release in the luteal and preovulatory phase and an inconsistent response seen in the follicular phase (Chan et al.

2012). The authors postulate that GnRH neurones may have varied responsiveness to exogenous kisspeptin stimulation across the menstrual cycle.

Studies in rodents have identified two distinct kisspeptin neuronal populations that have different responses to oestradiol. In female rodents, c-fos expression within kisspeptin neurones and levels of KISS1 expression are increased within the AVPV nucleus of the hypothalamus immediately preceding ovulation as a result of positive feedback on this nucleus by oestradiol (Smith et al. 2006). This contrasts against the negative feedback by oestradiol on kisspeptin neurones in the ARC, as evidenced by reduced levels of ARC

KISS1r expression in rat hypothalamic fragments at proestrus when compared with diestrus

(Roa et al. 2006).

In humans KISS1 expressing neurones have only been identified in the infundibular nucleus and to a lesser extent the medial preoptic area (Rometo et al. 2007). In the low oestrogen state of menopause there is significant increase in KISS1 expression in the infundibular nucleus together with hypertrophy and increased numbers of KISS1-expressing neurones when compared to pre-menopausal specimens (Rance 2009).

Guerriero et al. reported that in female rhesus monkeys low doses of kisspeptin-10 stimulated larger GnRH responses in pubertal monkeys compared to prepubertal monkeys

160

(Guerriero et al. 2012). Oestradiol levels are higher in pubertal monkeys and ovariectomy in these animals, which reduces oestradiol levels, eliminated the GnRH response to low and high doses of kisspeptin-10. However, the replacement of oestradiol in pubertal monkeys who had undergone ovariectomy resulted in only a partial restoration of the GnRH response to kisspeptin-10; suggesting an effect of another ovarian factor in addition to oestradiol on the response to kisspeptin (Guerriero et al. 2012).

In summary, in the course of this thesis I have reported on the first studies examining the effects of kisspeptin-54 in a model of infertility due to hypothalamic amenorrhoea and I have conducted the first clinical study to compare the effects of kisspeptin-10 administration on reproductive hormone release in healthy men and women. These studies suggest several important implications for the potential therapeutic use of kisspeptin-10 or kisspeptin-54 to treat disorders of reproduction.

My future work will investigate the effects of kisspeptin to improve in invitro fertilisation (IVF) treatment for infertility. Infertility is commonly defined as the inability to conceive after two years of regular unprotected sexual intercourse (HFEA Fertility Facts and Figures 2008).

Infertility has a high prevalence; it is estimated that around one in six UK couples have difficulty conceiving i.e. approximately 3.5 million couples (HFEA Fertility Facts and Figures

2008). The inability to have children can be devastating, and has important implications for mental, social, and reproductive health. IVF treatment is widely and successfully used to allow infertile couples to conceive and is now approved by the National Institute for Health and Clinical Excellence. However, the most common serious complication of IVF is ovarian hyperstimulation syndrome (OHSS). This is a potentially life threatening condition which has significant morbidity and mortality (RCOG guideline 5, 2006). The major cause of OHSS is the use of human chorionic gonadotrophin (hCG) in current IVF protocols for oocyte maturation (Golan et al. 1989). Human chorionic gonadotrophin binds to the LH receptor and therefore mimics the actions of endogenous LH. However, hCG has a much longer circulating half life than LH; this results in overstimulation of the corpus luteum which is the

161 underlying cause of OHSS (Damewood et al. 1989). Human chorionic gonadotrophin circulates for up to a week after injection (Fauser et al. 2002), whereas the physiological stimulus, the LH surge, only lasts for 48 hours (Hoff et al. 1983). This overstimulation of the corpus luteum also results in an environment more hostile to embryo implantation (Macklon et al. 2006). A more physiological method for oocyte maturation in IVF treatment should prevent overstimulation of the corpus luteum and thus the subsequent development of

OHSS (Macklon et al. 2006). Kisspeptin offers a novel approach in IVF therapy to improve pregnancy rates and avoid the complication of OHSS. Since kisspeptin stimulates the release of physiological levels of GnRH and the consequent release of endogenous gonadotrophins (Irwig et al. 2004; Thompson et al. 2004; Messager et al. 2005) it should lead to a physiological LH surge. A more physiological approach for oocyte maturation in IVF treatment would prevent overstimulation of the corpus luteum and OHSS in IVF treatment

(Macklon et al. 2006). The significant advantage of kisspeptin over current treatments is that its effects would depend on the sensitivity of an individual‟s hypothalamic-pituitary gonadal

(HPG) axis. This would result in a more physiological LH surge and oocyte maturation during

IVF treatment, thereby improving safety and efficacy of IVF treatment.

I am planning a future study in which I hypothesise that the use of kisspeptin in place of hCG in IVF protocols will result in the physiological release of the endogenous releasable pool of

GnRH and subsequent gonadotrophin secretion; this in turn will lead to oocyte maturation.

The aim of this study will be to provide proof of concept that administration of kisspeptin can induce oocyte maturation. Thus kisspeptin may have an exciting role in the treatment of infertility in the future.

162

References

Adachi, S., Yamada, S., Takatsu, Y., Matsui, H., Kinoshita, M., Takase, K., Sugiura, H., Ohtaki, T., Matsumoto, H., Uenoyama, Y., Tsukamura, H., Inoue, K., & Maeda, K. 2007, "Involvement of anteroventral periventricular metastin/kisspeptin neurons in estrogen positive feedback action on luteinizing hormone release in female rats", J Reprod.Dev., vol. 53, no. 2, pp. 367-378.

Adams, J. M., Taylor, A. E., Schoenfeld, D. A., Crowley, W. F., Jr., & Hall, J. E. 1994, "The midcycle gonadotropin surge in normal women occurs in the face of an unchanging gonadotropin-releasing hormone pulse frequency", J.Clin.Endocrinol.Metab, vol. 79, no. 3, pp. 858-864.

Ahima, R. S., Prabakaran, D., Mantzoros, C., Qu, D., Lowell, B., Maratos-Flier, E., & Flier, J. S. 1996, "Role of leptin in the neuroendocrine response to fasting", Nature, vol. 382, no. 6588, pp. 250-252.

Aitken, R. J., Baker, M. A., Doncel, G. F., Matzuk, M. M., Mauck, C. K., & Harper, M. J. 2008, "As the world grows: contraception in the 21st century", J.Clin.Invest, vol. 118, no. 4, pp. 1330-1343.

Aono, T., Kinugasa, T., Yamamoto, T., Miyake, A., & Kurachi, K. 1975, "Assessment of gonadotrophin secretion in women with anorexia nervosa", Acta Endocrinol.(Copenh), vol. 80, no. 4, pp. 630-641.

Arreguin-Arevalo, J. A., Lents, C. A., Farmerie, T. A., Nett, T. M., & Clay, C. M. 2007, "KiSS- 1 peptide induces release of LH by a direct effect on the hypothalamus of ovariectomized ewes", Anim Reprod.Sci., vol. 101, no. 3-4, pp. 265-275.

Belchetz, P. E., Plant, T. M., Nakai, Y., Keogh, E. J., & Knobil, E. 1978, "Hypophysial responses to continuous and intermittent delivery of hypopthalamic gonadotropin-releasing hormone", Science, vol. 202, no. 4368, pp. 631-633.

Bianco, S. D., Vandepas, L., Correa-Medina, M., Gereben, B., Mukherjee, A., Kuohung, W., Carroll, R., Teles, M. G., Latronico, A. C., & Kaiser, U. B. 2011, "KISS1R intracellular trafficking and degradation: effect of the Arg386Pro disease-associated mutation", Endocrinology, vol. 152, no. 4, pp. 1616-1626.

Boyar, R. M., Katz, J., Finkelstein, J. W., Kapen, S., Weiner, H., Weitzman, E. D., & Hellman, L. 1974, "Anorexia nervosa. Immaturity of the 24-hour luteinizing hormone secretory pattern", N.Engl.J.Med., vol. 291, no. 17, pp. 861-865.

Caraty, A., Smith, J. T., Lomet, D., Ben, S. S., Morrissey, A., Cognie, J., Doughton, B., Baril, G., Briant, C., & Clarke, I. J. 2007, "Kisspeptin synchronizes preovulatory surges in cyclical ewes and causes ovulation in seasonally acyclic ewes", Endocrinology, vol. 148, no. 11, pp. 5258-5267.

Caronia, L. M., Martin, C., Welt, C. K., Sykiotis, G. P., Quinton, R., Thambundit, A., Avbelj, M., Dhruvakumar, S., Plummer, L., Hughes, V. A., Seminara, S. B., Boepple, P. A., Sidis, Y., Crowley, W. F., Jr., Martin, K. A., Hall, J. E., & Pitteloud, N. 2011, "A genetic basis for functional hypothalamic amenorrhea", N.Engl.J.Med., vol. 364, no. 3, pp. 215-225.

Castellano, J. M., Navarro, V. M., Fernandez-Fernandez, R., Nogueiras, R., Tovar, S., Roa, J., Vazquez, M. J., Vigo, E., Casanueva, F. F., Aguilar, E., Pinilla, L., Dieguez, C., & Tena- Sempere, M. 2005, "Changes in hypothalamic KiSS-1 system and restoration of pubertal

163 activation of the reproductive axis by kisspeptin in undernutrition", Endocrinology, vol. 146, no. 9, pp. 3917-3925.

Cerrato, F., Shagoury, J., Kralickova, M., Dwyer, A., Falardeau, J., Ozata, M., Van, V. G., Bouloux, P., Hall, J. E., Hayes, F. J., Pitteloud, N., Martin, K. A., Welt, C., & Seminara, S. B. 2006, "Coding sequence analysis of GNRHR and GPR54 in patients with congenital and adult-onset forms of hypogonadotropic hypogonadism", Eur.J Endocrinol., vol. 155 Suppl 1, p. S3-S10.

Chan, J. L., Heist, K., DePaoli, A. M., Veldhuis, J. D., & Mantzoros, C. S. 2003, "The role of falling leptin levels in the neuroendocrine and metabolic adaptation to short-term starvation in healthy men", J.Clin.Invest, vol. 111, no. 9, pp. 1409-1421.

Chan, Y. M., Butler, J. P., Sidhoum, V. F., Pinnell, N. E., & Seminara, S. B. 2012, "Kisspeptin Administration to Women: A Window into Endogenous Kisspeptin Secretion and GnRH Responsiveness across the Menstrual Cycle", J.Clin.Endocrinol.Metab.

Chehab, F. F., Lim, M. E., & Lu, R. 1996, "Correction of the sterility defect in homozygous obese female mice by treatment with the human recombinant leptin", Nat.Genet., vol. 12, no. 3, pp. 318-320.

Chou, S. H., Chamberland, J. P., Liu, X., Matarese, G., Gao, C., Stefanakis, R., Brinkoetter, M. T., Gong, H., Arampatzi, K., & Mantzoros, C. S. 2011, "Leptin is an effective treatment for hypothalamic amenorrhea", Proc.Natl.Acad.Sci.U.S.A, vol. 108, no. 16, pp. 6585-6590.

Clarke, I., Moore, L., & Veldhuis, J. 2002, "Intensive direct cavernous sinus sampling identifies high-frequency, nearly random patterns of FSH secretion in ovariectomized ewes: combined appraisal by RIA and bioassay", Endocrinology, vol. 143, no. 1, pp. 117-129.

Clarke, I. J., Findlay, J. K., Cummins, J. T., & Ewens, W. J. 1986, "Effects of ovine follicular fluid on plasma LH and FSH secretion in ovariectomized ewes to indicate the site of action of inhibin", J.Reprod.Fertil., vol. 77, no. 2, pp. 575-585.

Clarkson, J., d'Anglemont, d. T., X, Moreno, A. S., Colledge, W. H., & Herbison, A. E. 2008, "Kisspeptin-GPR54 signaling is essential for preovulatory gonadotropin-releasing hormone neuron activation and the luteinizing hormone surge", J Neurosci., vol. 28, no. 35, pp. 8691- 8697.

Clarkson, J. & Herbison, A. E. 2006, "Postnatal development of kisspeptin neurons in mouse hypothalamus; sexual dimorphism and projections to gonadotropin-releasing hormone neurons", Endocrinology, vol. 147, no. 12, pp. 5817-5825.

Clements, M. K., McDonald, T. P., Wang, R., Xie, G., O'Dowd, B. F., George, S. R., Austin, C. P., & Liu, Q. 2001, "FMRFamide-related neuropeptides are agonists of the orphan G- protein-coupled receptor GPR54", Biochem.Biophys.Res.Commun., vol. 284, no. 5, pp. 1189-1193.

Conn, P. M. & Ulloa-Aguirre, A. 2010, "Trafficking of G-protein-coupled receptors to the plasma membrane: insights for pharmacoperone drugs", Trends Endocrinol.Metab, vol. 21, no. 3, pp. 190-197.

Damewood MD, Shen W, Zacur HA, Schlaff WD, Rock JA, Wallach EE. Disappearance of exogenously administered human chorionic gonadotropin. Fertil Steril. 1989; 52(3):398-400.

164 de Roux N., Genin, E., Carel, J. C., Matsuda, F., Chaussain, J. L., & Milgrom, E. 2003, "Hypogonadotropic hypogonadism due to loss of function of the KiSS1-derived peptide receptor GPR54", Proc.Natl.Acad.Sci.U.S.A, vol. 100, no. 19, pp. 10972-10976.

Dhar, D. K., Naora, H., Kubota, H., Maruyama, R., Yoshimura, H., Tonomoto, Y., Tachibana, M., Ono, T., Otani, H., & Nagasue, N. 2004, "Downregulation of KiSS-1 expression is responsible for tumor invasion and worse prognosis in gastric carcinoma", Int.J.Cancer, vol. 111, no. 6, pp. 868-872.

Dhillo, W. S., Chaudhri, O. B., Patterson, M., Thompson, E. L., Murphy, K. G., Badman, M. K., McGowan, B. M., Amber, V., Patel, S., Ghatei, M. A., & Bloom, S. R. 2005, "Kisspeptin- 54 stimulates the hypothalamic-pituitary gonadal axis in human males", J.Clin.Endocrinol.Metab, vol. 90, no. 12, pp. 6609-6615.

Dhillo, W. S., Chaudhri, O. B., Thompson, E. L., Murphy, K. G., Patterson, M., Ramachandran, R., Nijher, G. K., Amber, V., Kokkinos, A., Donaldson, M., Ghatei, M. A., & Bloom, S. R. 2007, "Kisspeptin-54 stimulates gonadotropin release most potently during the preovulatory phase of the menstrual cycle in women", J Clin.Endocrinol.Metab, vol. 92, no. 10, pp. 3958-3966.

Dhillo, W. S., Savage, P., Murphy, K. G., Chaudhri, O. B., Patterson, M., Nijher, G. M., Foggo, V. M., Dancey, G. S., Mitchell, H., Seckl, M. J., Ghatei, M. A., & Bloom, S. R. 2006, "Plasma kisspeptin is raised in patients with gestational trophoblastic neoplasia and falls during treatment", Am J Physiol Endocrinol.Metab, vol. 291, no. 5, p. E878-E884.

Dierschke, D. J., Bhattacharya, A. N., Atkinson, L. E., & Knobil, E. 1970, "Circhoral oscillations of plasma LH levels in the ovariectomized rhesus monkey", Endocrinology, vol. 87, no. 5, pp. 850-853.

Donato, J., Jr., Cravo, R. M., Frazao, R., Gautron, L., Scott, M. M., Lachey, J., Castro, I. A., Margatho, L. O., Lee, S., Lee, C., Richardson, J. A., Friedman, J., Chua S Jr, Coppari, R., Zigman, J. M., Elmquist, J. K., & Elias, C. F. 2011, "Leptin's effect on puberty in mice is relayed by the ventral premammillary nucleus and does not require signaling in Kiss1 neurons", J.Clin.Invest, vol. 121, no. 1, pp. 355-368.

Dungan, H. M., Gottsch, M. L., Zeng, H., Gragerov, A., Bergmann, J. E., Vassilatis, D. K., Clifton, D. K., & Steiner, R. A. 2007, "The role of kisspeptin-GPR54 signaling in the tonic regulation and surge release of gonadotropin-releasing hormone/luteinizing hormone", J Neurosci., vol. 27, no. 44, pp. 12088-12095.

Edwards, C. M., Todd, J. F., Mahmoudi, M., Wang, Z., Wang, R. M., Ghatei, M. A., & Bloom, S. R. 1999, "Glucagon-like peptide 1 has a physiological role in the control of postprandial glucose in humans: studies with the antagonist exendin 9-39", Diabetes, vol. 48, no. 1, pp. 86-93.

Estrada, K. M., Clay, C. M., Pompolo, S., Smith, J. T., & Clarke, I. J. 2006, "Elevated KiSS-1 expression in the arcuate nucleus prior to the cyclic preovulatory gonadotrophin-releasing hormone/lutenising hormone surge in the ewe suggests a stimulatory role for kisspeptin in oestrogen-positive feedback", J.Neuroendocrinol., vol. 18, no. 10, pp. 806-809.

Evans, J. J. 1999, "Modulation of gonadotropin levels by peptides acting at the anterior pituitary gland", Endocr.Rev., vol. 20, no. 1, pp. 46-67.

Farooqi, I. S. & O'rahilly, S. 2004, "Monogenic human obesity syndromes", Recent Prog.Horm.Res., vol. 59, pp. 409-424.

165

Fauser BC, de Jong D, Olivennes F, Wramsby H, Tay C, Itskovitz-Eldor J et al. Endocrine profiles after triggering of final oocyte maturation with GnRH agonist after cotreatment with the GnRH antagonist ganirelix during ovarian hyperstimulation for in vitro fertilization. J Clin Endocrinol Metab. 2002 Feb;87(2):709-15.

Ferguson, S. S. 2001, "Evolving concepts in G protein-coupled receptor endocytosis: the role in receptor desensitization and signaling", Pharmacol.Rev., vol. 53, no. 1, pp. 1-24.

Finn, P. D., Cunningham, M. J., Pau, K. Y., Spies, H. G., Clifton, D. K., & Steiner, R. A. 1998, "The stimulatory effect of leptin on the neuroendocrine reproductive axis of the monkey", Endocrinology, vol. 139, no. 11, pp. 4652-4662.

Frisch, R. E. 1996, "The right weight: body fat, menarche, and fertility", Nutrition, vol. 12, no. 6, pp. 452-453.

Frisch, R. E. & McArthur, J. W. 1974, "Menstrual cycles: fatness as a determinant of minimum weight for height necessary for their maintenance or onset", Science, vol. 185, no. 4155, pp. 949-951.

Funabashi, T., Daikoku, S., Shinohara, K., & Kimura, F. 2000, "Pulsatile gonadotropin- releasing hormone (GnRH) secretion is an inherent function of GnRH neurons, as revealed by the culture of medial olfactory placode obtained from embryonic rats", Neuroendocrinology, vol. 71, no. 2, pp. 138-144.

Funes, S., Hedrick, J. A., Vassileva, G., Markowitz, L., Abbondanzo, S., Golovko, A., Yang, S., Monsma, F. J., & Gustafson, E. L. 2003, "The KiSS-1 receptor GPR54 is essential for the development of the murine reproductive system", Biochem.Biophys.Res.Commun., vol. 312, no. 4, pp. 1357-1363.

Gaytan, F., Gaytan, M., Castellano, J. M., Romero, M., Roa, J., Aparicio, B., Garrido, N., Sanchez-Criado, J. E., Millar, R. P., Pellicer, A., Fraser, H. M., & Tena-Sempere, M. 2009, "KiSS-1 in the mammalian ovary: distribution of kisspeptin in human and marmoset and alterations in KiSS-1 mRNA levels in a rat model of ovulatory dysfunction", Am J.Physiol Endocrinol.Metab, vol. 296, no. 3, p. E520-E531.

Giles, D. E. & Berga, S. L. 1993, "Cognitive and psychiatric correlates of functional hypothalamic amenorrhea: a controlled comparison", Fertil.Steril., vol. 60, no. 3, pp. 486-492.

Glidewell-Kenney, C., Hurley, L. A., Pfaff, L., Weiss, J., Levine, J. E., & Jameson, J. L. 2007, "Nonclassical estrogen receptor alpha signaling mediates negative feedback in the female mouse reproductive axis", Proc.Natl.Acad.Sci.U.S.A, vol. 104, no. 19, pp. 8173-8177.

Gordon, C. M. 2010, "Clinical practice. Functional hypothalamic amenorrhea", N.Engl.J.Med., vol. 363, no. 4, pp. 365-371.

Gore, A. C. 2002, GnRH The Master Molecule of Reproduction.

Gottsch, M. L., Cunningham, M. J., Smith, J. T., Popa, S. M., Acohido, B. V., Crowley, W. F., Seminara, S., Clifton, D. K., & Steiner, R. A. 2004, "A role for kisspeptins in the regulation of gonadotropin secretion in the mouse", Endocrinology, vol. 145, no. 9, pp. 4073-4077.

Guerriero, K. A., Keen, K. L., & Terasawa, E. 2012, "Developmental increase in kisspeptin- 54 release in vivo is independent of the pubertal increase in estradiol in female rhesus monkeys (Macaca mulatta)", Endocrinology, vol. 153, no. 4, pp. 1887-1897.

166

Gutierrez-Pascual, E., Leprince, J., Martinez-Fuentes, A. J., Segalas-Milazzo, I., Pineda, R., Roa, J., Duran-Prado, M., Guilhaudis, L., Desperrois, E., Lebreton, A., Pinilla, L., Tonon, M. C., Malagon, M. M., Vaudry, H., Tena-Sempere, M., & Castano, J. P. 2009, "In vivo and in vitro structure-activity relationships and structural conformation of Kisspeptin-10-related peptides", Mol.Pharmacol., vol. 76, no. 1, pp. 58-67.

Hayes, F. J., McNicholl, D. J., Schoenfeld, D., Marsh, E. E., & Hall, J. E. 1999, "Free alpha- subunit is superior to luteinizing hormone as a marker of gonadotropin-releasing hormone despite desensitization at fast pulse frequencies", J.Clin.Endocrinol.Metab, vol. 84, no. 3, pp. 1028-1036.

Herbison, A. E. 2008, "Estrogen positive feedback to gonadotropin-releasing hormone (GnRH) neurons in the rodent: the case for the rostral periventricular area of the third ventricle (RP3V)", Brain Res.Rev., vol. 57, no. 2, pp. 277-287.

HFEA Fertility Facts & Figures 2008, HFEA Fertility Facts & Figures 2008.

Irwig, M. S., Fraley, G. S., Smith, J. T., Acohido, B. V., Popa, S. M., Cunningham, M. J., Gottsch, M. L., Clifton, D. K., & Steiner, R. A. 2004, "Kisspeptin activation of gonadotropin releasing hormone neurons and regulation of KiSS-1 mRNA in the male rat", Neuroendocrinology, vol. 80, no. 4, pp. 264-272.

Jayasena, C. N., Nijher, G. M., Abbara, A., Murphy, K. G., Lim, A., Patel, D., Mehta, A., Todd, C., Donaldson, M., Trew, G. H., Ghatei, M. A., Bloom, S. R., & Dhillo, W. S. 2010, "Twice- weekly administration of kisspeptin-54 for 8 weeks stimulates release of reproductive hormones in women with hypothalamic amenorrhea", Clin.Pharmacol.Ther., vol. 88, no. 6, pp. 840-847.

Jayasena, C. N., Nijher, G. M., Chaudhri, O. B., Murphy, K. G., Ranger, A., Lim, A., Patel, D., Mehta, A., Todd, C., Ramachandran, R., Salem, V., Stamp, G. W., Donaldson, M., Ghatei, M. A., Bloom, S. R., & Dhillo, W. S. 2009, "Subcutaneous injection of kisspeptin-54 acutely stimulates gonadotropin secretion in women with hypothalamic amenorrhea, but chronic administration causes tachyphylaxis", J.Clin.Endocrinol.Metab, vol. 94, no. 11, pp. 4315- 4323.

Jayasena, C. N., Nijher, G. M., Comninos, A. N., Abbara, A., Januszewki, A., Vaal, M. L., Sriskandarajah, L., Murphy, K. G., Farzad, Z., Ghatei, M. A., Bloom, S. R., & Dhillo, W. S. 2011, "The effects of kisspeptin-10 on reproductive hormone release show sexual dimorphism in humans", J.Clin.Endocrinol.Metab, vol. 96, no. 12, p. E1963-E1972.

Kadokawa, H., Matsui, M., Hayashi, K., Matsunaga, N., Kawashima, C., Shimizu, T., Kida, K., & Miyamoto, A. 2008, "Peripheral administration of kisspeptin-10 increases plasma concentrations of GH as well as LH in prepubertal Holstein heifers", J.Endocrinol., vol. 196, no. 2, pp. 331-334.

Kauffman, A. S., Gottsch, M. L., Roa, J., Byquist, A. C., Crown, A., Clifton, D. K., Hoffman, G. E., Steiner, R. A., & Tena-Sempere, M. 2007, "Sexual differentiation of Kiss1 gene expression in the brain of the rat", Endocrinology, vol. 148, no. 4, pp. 1774-1783.

Keen, K. L., Wegner, F. H., Bloom, S. R., Ghatei, M. A., & Terasawa, E. 2008, "An increase in kisspeptin-54 release occurs with the pubertal increase in luteinizing hormone-releasing hormone-1 release in the stalk-median eminence of female rhesus monkeys in vivo", Endocrinology, vol. 149, no. 8, pp. 4151-4157.

167

Kinoshita, M., Tsukamura, H., Adachi, S., Matsui, H., Uenoyama, Y., Iwata, K., Yamada, S., Inoue, K., Ohtaki, T., Matsumoto, H., & Maeda, K. 2005, "Involvement of central metastin in the regulation of preovulatory luteinizing hormone surge and estrous cyclicity in female rats", Endocrinology, vol. 146, no. 10, pp. 4431-4436.

Knobil, E. 1980, "The neuroendocrine control of the menstrual cycle", Recent Prog.Horm.Res., vol. 36, pp. 53-88.

Kotani, M., Detheux, M., Vandenbogaerde, A., Communi, D., Vanderwinden, J. M., Le, P. E., Brezillon, S., Tyldesley, R., Suarez-Huerta, N., Vandeput, F., Blanpain, C., Schiffmann, S. N., Vassart, G., & Parmentier, M. 2001, "The metastasis suppressor gene KiSS-1 encodes kisspeptins, the natural ligands of the orphan G protein-coupled receptor GPR54", J Biol.Chem., vol. 276, no. 37, pp. 34631-34636.

Kraegen, E. W., Lazarus, L., Meler, H., Campbell, L., & Chia, Y. O. 1975, "Carrier solutions for low-level intravenous insulin infusion", Br.Med.J., vol. 3, no. 5981, pp. 464-466.

Lanfranco, F., Gromoll, J., von, E. S., Herding, E. M., Nieschlag, E., & Simoni, M. 2005, "Role of sequence variations of the GnRH receptor and G protein-coupled receptor 54 gene in male idiopathic hypogonadotropic hypogonadism", Eur.J Endocrinol., vol. 153, no. 6, pp. 845-852.

Lee, D. K., Nguyen, T., O'Neill, G. P., Cheng, R., Liu, Y., Howard, A. D., Coulombe, N., Tan, C. P., Tang-Nguyen, A. T., George, S. R., & O'Dowd, B. F. 1999, "Discovery of a receptor related to the galanin receptors", FEBS Lett., vol. 446, no. 1, pp. 103-107.

Lee, J. H. & Welch, D. R. 1997, "Suppression of metastasis in human breast carcinoma MDA-MB-435 cells after transfection with the metastasis suppressor gene, KiSS-1", Cancer Res., vol. 57, no. 12, pp. 2384-2387.

Lefkowitz, R. J. 1998, "G protein-coupled receptors. III. New roles for receptor kinases and beta-arrestins in receptor signaling and desensitization", J.Biol.Chem., vol. 273, no. 30, pp. 18677-18680.

Lents, C. A., Heidorn, N. L., Barb, C. R., & Ford, J. J. 2008, "Central and peripheral administration of kisspeptin activates gonadotropin but not somatotropin secretion in prepubertal gilts", Reproduction., vol. 135, no. 6, pp. 879-887.

Levi-Setti, P. E., Cavagna, M., Baggiani, A., Zannoni, E., Colombo, G. V., & Liprandi, V. 2004, "FSH and LH together in ovarian stimulation", Eur.J.Obstet.Gynecol.Reprod.Biol., vol. 115 Suppl 1, p. S34-S39.

Luan, X., Yu, H., Wei, X., Zhou, Y., Wang, W., Li, P., Gan, X., Wei, D., & Xiao, J. 2007a, "GPR54 polymorphisms in Chinese girls with central precocious puberty", Neuroendocrinology, vol. 86, no. 2, pp. 77-83.

Luan, X., Zhou, Y., Wang, W., Yu, H., Li, P., Gan, X., Wei, D., & Xiao, J. 2007b, "Association study of the polymorphisms in the KISS1 gene with central precocious puberty in Chinese girls", Eur.J Endocrinol., vol. 157, no. 1, pp. 113-118.

Macklon NS, Stouffer RL, Giudice LC, Fauser BC. The science behind 25 years of ovarian stimulation for in vitro fertilization. Endocr Rev. 2006; 27(2):170-207.

Martin, K. A., Hall, J. E., Adams, J. M., & Crowley, W. F., Jr. 1993, "Comparison of exogenous gonadotropins and pulsatile gonadotropin-releasing hormone for induction of

168 ovulation in hypogonadotropic amenorrhea", J.Clin.Endocrinol.Metab, vol. 77, no. 1, pp. 125- 129.

Masui, T., Doi, R., Mori, T., Toyoda, E., Koizumi, M., Kami, K., Ito, D., Peiper, S. C., Broach, J. R., Oishi, S., Niida, A., Fujii, N., & Imamura, M. 2004, "Metastin and its variant forms suppress migration of pancreatic cancer cells", Biochem.Biophys.Res.Commun., vol. 315, no. 1, pp. 85-92.

Matsui, H., Takatsu, Y., Kumano, S., Matsumoto, H., & Ohtaki, T. 2004, "Peripheral administration of metastin induces marked gonadotropin release and ovulation in the rat", Biochem.Biophys.Res.Commun., vol. 320, no. 2, pp. 383-388.

Mead, E. J., Maguire, J. J., Kuc, R. E., & Davenport, A. P. 2007, "Kisspeptins are novel potent vasoconstrictors in humans, with a discrete localization of their receptor, G protein- coupled receptor 54, to atherosclerosis-prone vessels", Endocrinology, vol. 148, no. 1, pp. 140-147.

Meczekalski, B., Podfigurna-Stopa, A., Warenik-Szymankiewicz, A., & Genazzani, A. R. 2008, "Functional hypothalamic amenorrhea: current view on neuroendocrine aberrations", Gynecol.Endocrinol., vol. 24, no. 1, pp. 4-11.

Messager, S., Chatzidaki, E. E., Ma, D., Hendrick, A. G., Zahn, D., Dixon, J., Thresher, R. R., Malinge, I., Lomet, D., Carlton, M. B., Colledge, W. H., Caraty, A., & Aparicio, S. A. 2005, "Kisspeptin directly stimulates gonadotropin-releasing hormone release via G protein- coupled receptor 54", Proc.Natl.Acad.Sci.U.S.A, vol. 102, no. 5, pp. 1761-1766.

Mikkelsen, J. D., Bentsen, A. H., Ansel, L., Simonneaux, V., & Juul, A. 2009, "Comparison of the effects of peripherally administered kisspeptins", Regul.Pept., vol. 152, no. 1-3, pp. 95- 100.

Miller, K. K., Parulekar, M. S., Schoenfeld, E., Anderson, E., Hubbard, J., Klibanski, A., & Grinspoon, S. K. 1998, "Decreased leptin levels in normal weight women with hypothalamic amenorrhea: the effects of body composition and nutritional intake", J.Clin.Endocrinol.Metab, vol. 83, no. 7, pp. 2309-2312.

Muir, A. I., Chamberlain, L., Elshourbagy, N. A., Michalovich, D., Moore, D. J., Calamari, A., Szekeres, P. G., Sarau, H. M., Chambers, J. K., Murdock, P., Steplewski, K., Shabon, U., Miller, J. E., Middleton, S. E., Darker, J. G., Larminie, C. G., Wilson, S., Bergsma, D. J., Emson, P., Faull, R., Philpott, K. L., & Harrison, D. C. 2001a, "AXOR12, a novel human G protein-coupled receptor, activated by the peptide KiSS-1", J Biol.Chem., vol. 276, no. 31, pp. 28969-28975.

Nagatani, S., Guthikonda, P., Thompson, R. C., Tsukamura, H., Maeda, K. I., & Foster, D. L. 1998, "Evidence for GnRH regulation by leptin: leptin administration prevents reduced pulsatile LH secretion during fasting", Neuroendocrinology, vol. 67, no. 6, pp. 370-376.

Navarro, V. M., Castellano, J. M., Fernandez-Fernandez, R., Barreiro, M. L., Roa, J., Sanchez-Criado, J. E., Aguilar, E., Dieguez, C., Pinilla, L., & Tena-Sempere, M. 2004, "Developmental and hormonally regulated messenger ribonucleic acid expression of KiSS-1 and its putative receptor, GPR54, in rat hypothalamus and potent luteinizing hormone- releasing activity of KiSS-1 peptide", Endocrinology, vol. 145, no. 10, pp. 4565-4574.

Navarro, V. M., Fernandez-Fernandez, R., Castellano, J. M., Roa, J., Mayen, A., Barreiro, M. L., Gaytan, F., Aguilar, E., Pinilla, L., Dieguez, C., & Tena-Sempere, M. 2004, "Advanced

169 vaginal opening and precocious activation of the reproductive axis by KiSS-1 peptide, the endogenous ligand of GPR54", J Physiol, vol. 561, no. Pt 2, pp. 379-386.

Neill J.D 2006, Knobil and Neill's physiology of reproduction St Louis: Academic Press.

Niida, A., Wang, Z., Tomita, K., Oishi, S., Tamamura, H., Otaka, A., Navenot, J. M., Broach, J. R., Peiper, S. C., & Fujii, N. 2006, "Design and synthesis of downsized metastin (45-54) analogs with maintenance of high GPR54 agonistic activity", Bioorg.Med.Chem.Lett., vol. 16, no. 1, pp. 134-137.

Nijher, G. M., Chaudhri, O. B., Ramachandran, R., Murphy, K. G., Zac-Varghese, S. E., Fowler, A., Chinthapalli, K., Patterson, M., Thompson, E. L., Williamson, C., Kumar, S., Ghatei, M. A., Bloom, S. R., & Dhillo, W. S. 2010, "The effects of kisspeptin-54 on blood pressure in humans and plasma kisspeptin concentrations in hypertensive diseases of pregnancy", Br.J.Clin.Pharmacol., vol. 70, no. 5, pp. 674-681.

Ohtaki, T., Shintani, Y., Honda, S., Matsumoto, H., Hori, A., Kanehashi, K., Terao, Y., Kumano, S., Takatsu, Y., Masuda, Y., Ishibashi, Y., Watanabe, T., Asada, M., Yamada, T., Suenaga, M., Kitada, C., Usuki, S., Kurokawa, T., Onda, H., Nishimura, O., & Fujino, M. 2001d, "Metastasis suppressor gene KiSS-1 encodes peptide ligand of a G-protein-coupled receptor", Nature, vol. 411, no. 6837, pp. 613-617.

Orsini, M. J., Klein, M. A., Beavers, M. P., Connolly, P. J., Middleton, S. A., & Mayo, K. H. 2007, "Metastin (KiSS-1) mimetics identified from peptide structure-activity relationship- derived pharmacophores and directed small molecule database screening", J Med.Chem., vol. 50, no. 3, pp. 462-471.

Pache, T. D., Wladimiroff, J. W., de Jong, F. H., Hop, W. C., & Fauser, B. C. 1990, "Growth patterns of nondominant ovarian follicles during the normal menstrual cycle", Fertil.Steril., vol. 54, no. 4, pp. 638-642.

Pallais, J. C., Bo-Abbas, Y., Pitteloud, N., Crowley, W. F., Jr., & Seminara, S. B. 2006, "Neuroendocrine, gonadal, placental, and obstetric phenotypes in patients with IHH and mutations in the G-protein coupled receptor, GPR54", Mol.Cell Endocrinol., vol. 254-255, pp. 70-77.

Plant, T. M., Ramaswamy, S., & Dipietro, M. J. 2006, "Repetitive activation of hypothalamic G protein-coupled receptor 54 with intravenous pulses of kisspeptin in the juvenile monkey (Macaca mulatta) elicits a sustained train of gonadotropin-releasing hormone discharges", Endocrinology, vol. 147, no. 2, pp. 1007-1013.

Quennell, J. H., Howell, C. S., Roa, J., Augustine, R. A., Grattan, D. R., & Anderson, G. M. 2011, "Leptin deficiency and diet-induced obesity reduce hypothalamic kisspeptin expression in mice", Endocrinology, vol. 152, no. 4, pp. 1541-1550.

Quennell, J. H., Mulligan, A. C., Tups, A., Liu, X., Phipps, S. J., Kemp, C. J., Herbison, A. E., Grattan, D. R., & Anderson, G. M. 2009, "Leptin indirectly regulates gonadotropin-releasing hormone neuronal function", Endocrinology, vol. 150, no. 6, pp. 2805-2812.

Ramaswamy, S., Seminara, S. B., Pohl, C. R., Dipietro, M. J., Crowley, W. F., Jr., & Plant, T. M. 2007, "Effect of continuous intravenous administration of human metastin 45-54 on the neuroendocrine activity of the hypothalamic-pituitary-testicular axis in the adult male rhesus monkey (Macaca mulatta)", Endocrinology, vol. 148, no. 7, pp. 3364-3370.

170

Rance, N. E. 2009, "Menopause and the human hypothalamus: evidence for the role of kisspeptin/neurokinin B neurons in the regulation of estrogen negative feedback", Peptides, vol. 30, no. 1, pp. 111-122.

Reame, N. E., Sauder, S. E., Case, G. D., Kelch, R. P., & Marshall, J. C. 1985, "Pulsatile gonadotropin secretion in women with hypothalamic amenorrhea: evidence that reduced frequency of gonadotropin-releasing hormone secretion is the mechanism of persistent anovulation", J.Clin.Endocrinol.Metab, vol. 61, no. 5, pp. 851-858.

Reindollar, R. H., Novak, M., Tho, S. P., & McDonough, P. G. 1986, "Adult-onset amenorrhea: a study of 262 patients", Am.J.Obstet.Gynecol., vol. 155, no. 3, pp. 531-543.

Ritter, S. L. & Hall, R. A. 2009, "Fine-tuning of GPCR activity by receptor-interacting proteins", Nat.Rev.Mol.Cell Biol., vol. 10, no. 12, pp. 819-830.

Roa, J., Vigo, E., Garcia-Galiano, D., Castellano, J. M., Navarro, V. M., Pineda, R., Dieguez, C., Aguilar, E., Pinilla, L., & Tena-Sempere, M. 2008, "Desensitization of gonadotropin responses to kisspeptin in the female rat: analyses of LH and FSH secretion at different developmental and metabolic states", Am J.Physiol Endocrinol.Metab, vol. 294, no. 6, p. E1088-E1096.

Rometo, A. M., Krajewski, S. J., Voytko, M. L., & Rance, N. E. 2007, "Hypertrophy and increased kisspeptin gene expression in the hypothalamic infundibular nucleus of postmenopausal women and ovariectomized monkeys", J.Clin.Endocrinol.Metab, vol. 92, no. 7, pp. 2744-2750.

Roseweir, A. K., Kauffman, A. S., Smith, J. T., Guerriero, K. A., Morgan, K., Pielecka- Fortuna, J., Pineda, R., Gottsch, M. L., Tena-Sempere, M., Moenter, S. M., Terasawa, E., Clarke, I. J., Steiner, R. A., & Millar, R. P. 2009a, "Discovery of potent kisspeptin antagonists delineate physiological mechanisms of gonadotropin regulation", J.Neurosci., vol. 29, no. 12, pp. 3920-3929.

RCOG Guideline No.5. The Management of Ovarian Hyperstimulation Syndrome. Sept 2006

Salacinski, P. R., McLean, C., Sykes, J. E., Clement-Jones, V. V., & Lowry, P. J. 1981, "Iodination of proteins, glycoproteins, and peptides using a solid-phase oxidizing agent, 1,3,4,6-tetrachloro-3 alpha,6 alpha-diphenyl glycoluril (Iodogen)", Anal.Biochem., vol. 117, no. 1, pp. 136-146.

Schneider, J. E. 2004, "Energy balance and reproduction", Physiol Behav., vol. 81, no. 2, pp. 289-317.

Schurgin, S., Canavan, B., Koutkia, P., DePaoli, A. M., & Grinspoon, S. 2004, "Endocrine and metabolic effects of physiologic r-metHuLeptin administration during acute caloric deprivation in normal-weight women", J.Clin.Endocrinol.Metab, vol. 89, no. 11, pp. 5402- 5409.

Seminara, S. B., Dipietro, M. J., Ramaswamy, S., Crowley, W. F., Jr., & Plant, T. M. 2006, "Continuous human metastin 45-54 infusion desensitizes G protein-coupled receptor 54- induced gonadotropin-releasing hormone release monitored indirectly in the juvenile male Rhesus monkey (Macaca mulatta): a finding with therapeutic implications", Endocrinology, vol. 147, no. 5, pp. 2122-2126.

Seminara, S. B., Messager, S., Chatzidaki, E. E., Thresher, R. R., Acierno, J. S., Jr., Shagoury, J. K., Bo-Abbas, Y., Kuohung, W., Schwinof, K. M., Hendrick, A. G., Zahn, D.,

171

Dixon, J., Kaiser, U. B., Slaugenhaupt, S. A., Gusella, J. F., O'Rahilly, S., Carlton, M. B., Crowley, W. F., Jr., Aparicio, S. A., & Colledge, W. H. 2003, "The GPR54 gene as a regulator of puberty", N.Engl.J.Med., vol. 349, no. 17, pp. 1614-1627.

Semple, R. K., Achermann, J. C., Ellery, J., Farooqi, I. S., Karet, F. E., Stanhope, R. G., O'rahilly, S., & Aparicio, S. A. 2005, "Two novel missense mutations in g protein-coupled receptor 54 in a patient with hypogonadotropic hypogonadism", J Clin.Endocrinol.Metab, vol. 90, no. 3, pp. 1849-1855.

Shahab, M., Mastronardi, C., Seminara, S. B., Crowley, W. F., Ojeda, S. R., & Plant, T. M. 2005, "Increased hypothalamic GPR54 signaling: a potential mechanism for initiation of puberty in primates", Proc.Natl.Acad.Sci.U.S.A, vol. 102, no. 6, pp. 2129-2134.

Shibata, M., Friedman, R. L., Ramaswamy, S., & Plant, T. M. 2007, "Evidence that down regulation of hypothalamic KiSS-1 expression is involved in the negative feedback action of testosterone to regulate luteinising hormone secretion in the adult male rhesus monkey (Macaca mulatta)", J.Neuroendocrinol., vol. 19, no. 6, pp. 432-438.

Shirasaki, F., Takata, M., Hatta, N., & Takehara, K. 2001, "Loss of expression of the metastasis suppressor gene KiSS1 during melanoma progression and its association with LOH of chromosome 6q16.3-q23", Cancer Res., vol. 61, no. 20, pp. 7422-7425.

Silveira, L. G., Noel, S. D., Silveira-Neto, A. P., Abreu, A. P., Brito, V. N., Santos, M. G., Bianco, S. D., Kuohung, W., Xu, S., Gryngarten, M., Escobar, M. E., Arnhold, I. J., Mendonca, B. B., Kaiser, U. B., & Latronico, A. C. 2010, "Mutations of the KISS1 gene in disorders of puberty", J.Clin.Endocrinol.Metab, vol. 95, no. 5, pp. 2276-2280.

Smith, J. T., Clay, C. M., Caraty, A., & Clarke, I. J. 2007, "KiSS-1 messenger ribonucleic acid expression in the hypothalamus of the ewe is regulated by sex steroids and season", Endocrinology, vol. 148, no. 3, pp. 1150-1157.

Smith, J. T., Cunningham, M. J., Rissman, E. F., Clifton, D. K., & Steiner, R. A. 2005a, "Regulation of Kiss1 gene expression in the brain of the female mouse", Endocrinology, vol. 146, no. 9, pp. 3686-3692.

Smith, J. T., Dungan, H. M., Stoll, E. A., Gottsch, M. L., Braun, R. E., Eacker, S. M., Clifton, D. K., & Steiner, R. A. 2005b, "Differential regulation of KiSS-1 mRNA expression by sex steroids in the brain of the male mouse", Endocrinology, vol. 146, no. 7, pp. 2976-2984.

Smith, J. T., Popa, S. M., Clifton, D. K., Hoffman, G. E., & Steiner, R. A. 2006, "Kiss1 neurons in the forebrain as central processors for generating the preovulatory luteinizing hormone surge", J.Neurosci., vol. 26, no. 25, pp. 6687-6694.

Soldin, O. P., Chung, S. H., & Mattison, D. R. 2011, "Sex differences in drug disposition", J.Biomed.Biotechnol., vol. 2011, p. 187103.

Stafford, L. J., Xia, C., Ma, W., Cai, Y., & Liu, M. 2002, "Identification and characterization of mouse metastasis-suppressor KiSS1 and its G-protein-coupled receptor", Cancer Res., vol. 62, no. 19, pp. 5399-5404.

Teles, M. G., Bianco, S. D., Brito, V. N., Trarbach, E. B., Kuohung, W., Xu, S., Seminara, S. B., Mendonca, B. B., Kaiser, U. B., & Latronico, A. C. 2008, "A GPR54-activating mutation in a patient with central precocious puberty", N.Engl.J.Med., vol. 358, no. 7, pp. 709-715.

172

Tenenbaum-Rakover, Y., Commenges-Ducos, M., Iovane, A., Aumas, C., Admoni, O., & de, R. N. 2007, "Neuroendocrine phenotype analysis in five patients with isolated hypogonadotropic hypogonadism due to a L102P inactivating mutation of GPR54", J.Clin.Endocrinol.Metab, vol. 92, no. 3, pp. 1137-1144.

Terasawa, E. 2001, "Luteinizing hormone-releasing hormone (LHRH) neurons: mechanism of pulsatile LHRH release", Vitam.Horm., vol. 63, pp. 91-129.

Terasawa, E., Keen, K. L., Mogi, K., & Claude, P. 1999, "Pulsatile release of luteinizing hormone-releasing hormone (LHRH) in cultured LHRH neurons derived from the embryonic olfactory placode of the rhesus monkey", Endocrinology, vol. 140, no. 3, pp. 1432-1441.

Thompson, E. L., Murphy, K. G., Patterson, M., Bewick, G. A., Stamp, G. W., Curtis, A. E., Cooke, J. H., Jethwa, P. H., Todd, J. F., Ghatei, M. A., & Bloom, S. R. 2006, "Chronic subcutaneous administration of kisspeptin-54 causes testicular degeneration in adult male rats", Am.J Physiol Endocrinol.Metab, vol. 291, no. 5, p. E1074-E1082.

Thompson, E. L., Patterson, M., Murphy, K. G., Smith, K. L., Dhillo, W. S., Todd, J. F., Ghatei, M. A., & Bloom, S. R. 2004, "Central and peripheral administration of kisspeptin-10 stimulates the hypothalamic-pituitary-gonadal axis", J.Neuroendocrinol., vol. 16, no. 10, pp. 850-858.

Tomita, K., Narumi, T., Niida, A., Oishi, S., Ohno, H., & Fujii, N. 2007a, "Fmoc-based solid- phase synthesis of GPR54-agonistic pentapeptide derivatives containing alkene- and fluoroalkene-dipeptide isosteres", Biopolymers, vol. 88, no. 2, pp. 272-278.

Tomita, K., Niida, A., Oishi, S., Ohno, H., Cluzeau, J., Navenot, J. M., Wang, Z. X., Peiper, S. C., & Fujii, N. 2006, "Structure-activity relationship study on small peptidic GPR54 agonists", Bioorg.Med.Chem., vol. 14, no. 22, pp. 7595-7603.

Tomita, K., Oishi, S., Cluzeau, J., Ohno, H., Navenot, J. M., Wang, Z. X., Peiper, S. C., Akamatsu, M., & Fujii, N. 2007b, "SAR and QSAR studies on the N-terminally acylated pentapeptide agonists for GPR54", J.Med.Chem., vol. 50, no. 14, pp. 3222-3228.

Tomita, K., Oishi, S., Ohno, H., & Fujii, N. 2008, "Structure-activity relationship study and NMR analysis of fluorobenzoyl pentapeptide GPR54 agonists", Biopolymers, vol. 90, no. 4, pp. 503-511.

Tovar, S., Vazquez, M. J., Navarro, V. M., Fernandez-Fernandez, R., Castellano, J. M., Vigo, E., Roa, J., Casanueva, F. F., Aguilar, E., Pinilla, L., Dieguez, C., & Tena-Sempere, M. 2006, "Effects of single or repeated intravenous administration of kisspeptin upon dynamic LH secretion in conscious male rats", Endocrinology, vol. 147, no. 6, pp. 2696-2704.

Warren, M. P. 1980, "The effects of exercise on pubertal progression and reproductive function in girls", J.Clin.Endocrinol.Metab, vol. 51, no. 5, pp. 1150-1157.

Welt, C. K., Chan, J. L., Bullen, J., Murphy, R., Smith, P., DePaoli, A. M., Karalis, A., & Mantzoros, C. S. 2004, "Recombinant human leptin in women with hypothalamic amenorrhea", N.Engl.J.Med., vol. 351, no. 10, pp. 987-997.

Wetsel, W. C., Valenca, M. M., Merchenthaler, I., Liposits, Z., Lopez, F. J., Weiner, R. I., Mellon, P. L., & Negro-Vilar, A. 1992, "Intrinsic pulsatile secretory activity of immortalized luteinizing hormone-releasing hormone-secreting neurons", Proc.Natl.Acad.Sci.U.S.A, vol. 89, no. 9, pp. 4149-4153.

173

Wintermantel, T. M., Campbell, R. E., Porteous, R., Bock, D., Grone, H. J., Todman, M. G., Korach, K. S., Greiner, E., Perez, C. A., Schutz, G., & Herbison, A. E. 2006, "Definition of estrogen receptor pathway critical for estrogen positive feedback to gonadotropin-releasing hormone neurons and fertility", Neuron, vol. 52, no. 2, pp. 271-280.

Zhang, Y., Proenca, R., Maffei, M., Barone, M., Leopold, L., & Friedman, J. M. 1994, "Positional cloning of the mouse obese gene and its human homologue", Nature, vol. 372, no. 6505, pp. 425-432.

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Appendix 1 Principle of radioimmunoassay

Radioimmunoasasy (RIA) is a technique in which unlabelled and radioactively labelled antigen (peptide) compete for an antibody binding site. Specificity is dependent upon the ability of the antibody to recognise subtle structural features of the antigen molecule. The most commonly used radiolabel is I-125, and antibodies are often obtained by immunising rabbits with antigen (peptide) plus adjuvant (though polyclonal antibodies from other species and monoclonal antibodies can be used). In the RIA both the labelled peptide and antibody are at specific, constant concentrations. The concentration of antibody is limiting, so antigen binding will be finite. This allows determination of the amount of unlabelled peptide in a sample. Unlabelled peptide in a sample will compete with labelled peptide for antibody binding. Thus the amount of radiolabelled antigen bound to the antibody is inversely proportional to the amount of unlabelled antigen in the sample being assayed. A „standard curve‟ is determined using known concentrations of pure peptide to calculate the percentage of labelled peptide bound for each concentration of unlabelled pure peptide. Separation of the unbound radiolabelled antigen from the radiolabelled antigen-antibody complex and counting the proportion of radiolabel present in the two fractions enables direct measurement of the amount of unlabelled antigen that has bound to the antibody. By reference to the standard curve, the unknown concentrations of peptide in the sample can be obtained by interpolation.

Schematic representation of the principle of RIA:

Ag* + Ab + Ag ↔ Ag*Ab + AgAb

Ag* = radiolabelled antigen

Ag = unlabelled antigen

Ab = antibody

175

Various techniques may be used in order to distinguish antibody-bound antigen from free antigen, and to determine the distribution of radioactive antigen between the two fractions.

The methods used in this work are separation by adsorption with charcoal (free radiolabelled antigen is contained in the charcoal pellet following centrifugation), or using a primary antibody / secondary antibody complex (free label is contained in the supernatant following centrifugation).

With charcoal separation, dextran is added to a charcoal suspension to block the larger holes in porous charcoal. The suspension is then added to the RIA tubes, where it traps the free (unbound) radiolabelled antigen. The tubes are then centrifuged and the supernatant

(containing antibody-antigen complex) and charcoal pellet (free radiolabelled antigen) are separated by aspiration with a Pasteur pipette. The bound and free labels are counted in a gamma counter. With the secondary antibody separation method, the secondary antibody is derived from an animal species different from the species used to generate primary (antigen- binding) antibody. For example, the leptin primary antiserum is raised in a rabbit, and separation is achieved by using a goat anti-rabbit secondary antibody. Free radiolabelled antigen is in the supernatant, and the pellet contains antigen-antibody complexes. As with charcoal separation, bound and free label are counted in a gamma counter after incubation, centrifugation and separation.

Each RIA has optimum reaction conditions with respect to buffer medium, assay volume, antibody titre, incubation time, temperature, and separation method used. Phosphate buffers are used for a wide range of RIAs. The in-house departmental assays used here are incubated for 3-5 days.

All samples are added in duplicate. “Non-specific binding” tubes, minus primary antibody, are added at the beginning of the assay. Tubes containing half and twice the standard

176 volume of added labelled antigen are also included in order to confirm the quality of the label.

Tubes with no samples („zero‟ tubes) are placed at regular intervals throughout the assay, and standard curves (containing known quantities of unlabelled antigen) are performed at the beginning and end of each assay, in order to calibrate the assay. The analytical sensitivity of an assay is determined by the smallest change in hormone concentration that can be reliably detected. This is essentially governed by the steepness of the standard curve, and the error of a known value on the curve. The analytical sensitivity is calculated as two standard deviations from the mean concentration of antigen measured in the zero standards

(which contains no unlabelled peptide)

Kisspeptin radioimmunoassay:

Measurement of plasma kisspeptin immunoreactivity (IR) was performed using an established radioimmunoassay (RIA) (Dhillo et al. 2005; Dhillo et al. 2007). A rabbit antiserum was raised against kisspeptin. The antibody cross-reacted 100% with human kisspeptin-54, kisspeptin-14, and kisspeptin-10 and less than 0.01% with other related RF amide proteins, including prolactin-releasing peptide, RF amide-related peptide 1 (RFRP1),

RFRP2, RFRP3, QRFP43, neuropeptide FF, and neuropeptide AF. The limit of detectability was 2pmol/l, and the intra- and interassay coefficients of variation were 8.3 and 10.2%, respectively.

All kisspeptin RIAs were performed using 0.06M phosphate buffer (0.05M Na2HPO4.2H2O,

0.006M KH2PO4, 0.01M disodium-EDTA.2H2O, 0.008M NaN3) (pH 7.4). A standard concentration of 0.5 pmol/mL kisspeptin-54 was used. Radiolabelled kisspeptin with a radioactivity 25-30 Bq/mL was used. Antiserum was used at a final dilution of 1:3,000,000.

Assay tubes were set up in duplicate according to the volumes described in Table 10.

Assays were incubated for 3-5 days at 4OC then separated using charcoal adsorption.

177

Table 10. Volumes of reactants for addition to kisspeptin radioimmunoassay.

NSB, non-specific binding,1/2 X, addition of half-volume of labelled kisspeptin; 2 X, addition of double volume of labelled kisspeptin; XS, addition of excess volume of antibody.

TUBE BUFFER LABEL ANTIBODY SAMPLE / TOTAL IDENTITY VOLUME VOLUME VOLUME STANDARD VOLUME (µL) (µL) (µL) (µL) (µL)

NSB 600 100 0 0 700

1/2 X 550 50 100 0 700

2 X 400 200 100 0 700

ZERO 500 100 100 0 700

STD 1-15 500 100 100 1-15 701-715

STD 20 480 100 100 10 700

STD 30 470 100 100 30 700

50 450 100 100 50 700

100 400 100 100 100 700

XS 0 100 600 0 700

178

Appendix 2 Original Publications

179

ORIGINAL ARTICLE

Endocrine Care

Subcutaneous Injection of Kisspeptin-54 Acutely Stimulates Gonadotropin Secretion in Women with Hypothalamic Amenorrhea, But Chronic Administration Causes Tachyphylaxis

Channa N. Jayasena, Gurjinder M. K. Nijher, Owais B. Chaudhri, Kevin G. Murphy, Amita Ranger, Adrian Lim, Daksha Patel, Amrish Mehta, Catriona Todd, Radha Ramachandran, Victoria Salem, Gordon W. Stamp, Mandy Donaldson, Mohammad A. Ghatei, Stephen R. Bloom, and Waljit S. Dhillo

Department of Investigative Medicine (C.N.J., G.M.K.N., O.B.C., K.G.M., A.R., R.R., V.S., M.A.G., S.R.B., W.S.D.), Imperial College London, Hammersmith Hospital, London W12 ONN, United Kingdom; Imaging Department (A.L., D.P., A.M., C.T.), Imperial College Healthcare NHS Trust, Charing Cross Hospital, London W6 8RF, United Kingdom; Department of Histopathology (G.W.S.), Imperial College London, Hammersmith Hospital, London W12 ONN, United Kingdom; and Department of Clinical Biochemistry (M.D.), Imperial College Healthcare NHS Trust, Charing Cross Hospital, London W6 8RF, United Kingdom

Background: Kisspeptin is a critical regulator of normal reproductive function. A single injection of kisspeptin in healthy human volunteers potently stimulates gonadotropin release. However, the effects of kisspeptin on gonadotropin release in women with hypothalamic amenorrhea (HA) and the effects of repeated administration of kisspeptin to humans are unknown.

Aim: The aim of this study was to determine the effects of acute and chronic kisspeptin administration on gonadotropin release in women with HA.

Methods: We performed a prospective, randomized, double-blinded, parallel design study. Women with HA received twice-daily sc injections of kisspeptin (6.4 nmol/kg) or 0.9% saline (n ϭ 5 per group) for 2 wk. Changes in serum gonadotropin and estradiol levels, LH pulsatility, and ultrasound measurements of reproductive activity were assessed.

Results: On the first injection day, potent increases in serum LH and FSH were observed after sc kisspeptin injection in women with HA (mean maximal increment from baseline within 4 h after injection: LH, 24.0 Ϯ 3.5 IU/liter; FSH, 9.1 Ϯ 2.5 IU/liter). These responses were significantly reduced on the 14th injection day (mean maximal increment from baseline within 4 h postinjection: LH, 2.5 Ϯ 2.2 IU/liter, P Ͻ 0.05; FSH, 0.5 Ϯ 0.5 IU/liter, P Ͻ 0.05). Subjects remained responsive to GnRH after kisspeptin treatment. No significant changes in LH pulsatility or ultrasound measurements of reproductive activity were observed.

Conclusion: Acute administration of kisspeptin to women with infertility due to HA potently stimulates gonadotropin release, but chronic administration of kisspeptin results in desensitization to its effects on gonadotropin release. These data have important implications for the development of kisspeptin as a novel therapy for reproductive disorders in humans. (J Clin Endocrinol Metab 94: 4315–4323, 2009)

ISSN Print 0021-972X ISSN Online 1945-7197 Abbreviations: HA, Hypothalamic amenorrhea; IR, immunoreactivity; KISS1R, kisspeptin Printed in U.S.A. receptor; RFRP, RF amide-related peptide. Copyright © 2009 by The Endocrine Society doi: 10.1210/jc.2009-0406 Received February 23, 2009. Accepted August 17, 2009. First Published Online October 9, 2009

J Clin Endocrinol Metab, November 2009, 94(11):4315–4323 jcem.endojournals.org 4315 4316 Jayasena et al. Effects of Kisspeptin in Women with HA J Clin Endocrinol Metab, November 2009, 94(11):4315–4323

ypothalamic amenorrhea (HA) is defined as the ces- fects of administration of kisspeptin in humans have not H sation of menstruation due to abnormal signaling been studied to date. between the hypothalamus and the pituitary gland (1), and In prepubertal female rats, caloric restriction leads to it accounts for approximately 30% of cases of amenorrhea reduced gonadotropin levels, delayed vaginal opening, in women of reproductive age (2). Functional HA is de- and low hypothalamic kiss1 expression (32). Twice-daily fined as HA occurring in the absence of a structural lesion administration of kisspeptin-10 to these animals restores and often results from a relative energy deficit within the vaginal opening and gonadotropin secretion (32). Based body (low body weight or weight loss) (3–7). Although on these data, we hypothesized that repetitive administra- current treatments for women with HA such as clomi- tion of kisspeptin would restore gonadotropin secretion in phene, gonadotropin injections, and GnRH pump therapy human female subjects with HA. are efficacious, each has associated limitations (8, 9). A randomized, double-blinded, placebo-controlled, The kisspeptins are a group of arginine-phenylalanine parallel design study was conducted to determine whether (RF) amide peptides, encoded by the KISS1 gene, that have twice-daily administration of kisspeptin to human female been identified as potential novel agents for treating re- subjects with HA would sustainably stimulate gonadotro- productive disorders. They act as endogenous ligands for pin release. the kisspeptin receptor (KISS1R, alternatively known as G protein-coupled receptor 54) (10–12). KISS1 and KISS1R are expressed in the hypothalamus, pituitary, and placenta Subjects and Methods (10, 12–14). Kisspeptin signaling exerts powerful effects on the mammalian reproductive system. Mice lacking Kisspeptin-54 kisspeptin or the kisspeptin receptor fail to undergo pu- Kisspeptin-54 was synthesized by the Advanced Biotechnol- ogy Centre, Imperial College London, and purified by reverse- berty and are infertile (15, 16). In humans, inactivating phase HPLC. Electrospray mass spectroscopy and amino acid mutations of KISS1R cause pubertal failure (16, 17), and analysis confirmed identity of the peptide as previously described activating mutations lead to precocious puberty (18). Fur- (26, 27). The peptide was tested for bioactivity and toxicity as thermore, central or peripheral administration of kisspep- previously described (26). The Limulus amebocyte lysate assay tin induces gonadotropin and sex steroid release in all (Associates of Cape Cod, Liverpool, UK) was negative for endotoxin, and the peptide was sterile on culture (Department of mammalian species investigated, including rats (19–21), Microbiology, Hammersmith Hospital, London). Although mice (22, 23), monkeys (24) and sheep (23, 25). We kisspeptin-10, -13, -14, and -54 display similar potency in vitro, have previously demonstrated that iv infusion or sc bo- we used kisspeptin-54 due to its higher in vivo potency than the lus injection of kisspeptin-54 stimulates gonadotropin other kisspeptin fragments (31, 33). secretion in healthy human male and female subjects, respectively (26, 27). Kisspeptin may therefore be a po- Subjects tential novel therapy for treating reproductive disorders Ethical approval was granted by the Hammersmith and in humans. However, the effects of kisspeptin admin- Queen Charlotte’s and Chelsea Hospitals Research Ethics Com- mittee (registration number: 05/Q0406/142). Written informed istration in patients with infertility have not been pre- consent was obtained from all subjects. This study was per- viously investigated. formed in accordance with the Declaration of Helsinki. Although it has been consistently demonstrated that Subjects were recruited through advertisements placed in lo- acute administration of kisspeptin stimulates gonadotro- cal newspapers. Responders to advertisements were evaluated pin release (19–27), the effects of chronic administration with a detailed menstrual history, clinical examination, electrocardiogram, and blood tests. Screening blood tests performed of kisspeptin on reproductive function are less clear. were as follows: full blood count, renal profile, liver profile, bone Chronic administration of kisspeptin to nonhuman mam- profile, glucose, thyroid profile, LH, FSH, estradiol, progester- mals causes either sustained or nonsustained stimulation one, androstenedione, dehydroepiandrosterone, testosterone, of reproductive function, depending on the mode of ad- SHBG, prolactin, 17-hydroxyprogesterone, and cortisol. Women ministration used. Intermittent administration of kisspep- were diagnosed with functional HA and included within the study if they fulfilled the following criteria: body mass index tin-10 to juvenile female rats (twice-daily injections) for below 25 kg/m2; stable body weight over the previous 6 months; 5 d and juvenile male monkeys (hourly injections) for 2 d age between 18 and 40 yr; secondary amenorrhea of at least 6 induces precocious reproductive maturation (28, 29). In months duration; absence of oral contraceptive pill therapy for contrast, continuous peripheral infusion of kisspeptin-10 at least 1 yr; absence of systemic disease comorbidity; absence of to monkeys or rats increases LH release only during the active psychiatric illness; stable body weight; absence of therapeutic or recreational drug use; absence of clinical or biochemical first3horthefirst day of administration, respectively; LH hyperandrogenemia; structurally normal hypothalamopituitary concentrations subsequently return to levels observed be- region assessed by magnetic resonance imaging; structurally nor- fore infusion of kisspeptin-10 (30, 31). The long-term ef- mal female reproductive tract visualized on ultrasound; absence J Clin Endocrinol Metab, November 2009, 94(11):4315–4323 jcem.endojournals.org 4317

TABLE 1. Comparison of baseline characteristics of Kisspeptin injections women with HA randomized to saline vs. kisspeptin-54 All subjects were trained in self-administration of sc injections by an investigator at the start of the study protocol. At the beginning of each week when injections were to be performed, a Study group P box containing unlabeled vials of freeze-dried saline or kisspep- Characteristic Saline Kisspeptin-54 value tin-54, alcohol wipes, saline vials, needles, and needle disposal Ϯ Ϯ Age (yr) 24.8 0.5 26.8 2.4 0.28 bins was given to each subject. For injection, vial contents were Weight (kg) 51.8 Ϯ 3.3 54.5 Ϯ 1.1 0.46 reconstituted in 0.5 ml of 0.9% saline. Then a 0.5-ml insulin Body mass index 19.0 Ϯ 0.7 19.9 Ϯ 0.4 0.25 syringe was used to inject a weight-adjusted volume of dissolved (kg/m2) Duration of 22.4 Ϯ 9.9 23.2 Ϯ 12.7 0.96 vial contents into the lower anterior abdominal region. Subjects amenorrhea (months) were instructed to refrigerate vials stored at home. Serum LH (IU/liter) 4.5 Ϯ 1.6 2.6 Ϯ 0.9 0.32 Serum FSH (IU/liter) 6.6 Ϯ 0.7 6.1 Ϯ 1.0 0.69 4-h blood sampling after injection of saline or Ϯ Ϯ Serum estradiol 105 13.9 78 4.9 0.10 kisspeptin (pmol/liter) All subjects underwent blood sampling in the 4-h period im- Values are provided for subjects randomized to receive saline (n ϭ 5) mediately after the first (wk 5, d 1) and final (wk 6, d 7) injection or kisspeptin-54 (n ϭ 5). Data are shown as mean Ϯ SEM. of saline (five subjects) or kisspeptin-54 (five subjects) of treatment period. These studies were done in an investigation unit. An of polycystic ovarian appearances on ultrasound; normal thy- unused vial returned by each subject from home storage was used roid function tests; normal serum prolactin levels; and serum for their final injection of kisspeptin or saline. Saline or kisspep- LH:FSH ratio less than 1.5. Ten subjects with HA were recruited tin-54 (6.4 nmol/kg) was sc administered at 0 min by the inves- to the study. We have previously published baseline clinical and tigator, and blood was sampled for serum LH, FSH, estradiol, biochemical data for healthy women in the follicular phase of the and SHBG, and plasma kisspeptin-immunoreactivity (IR) at menstrual cycle and for their responses to kisspeptin-54 admin- Ϫ30, 0, 15, 30, 45, 60, 75, 90, 120, 150, 180, 210, and 240 min. istration (27). In one subject, the study was extended to include blood sampling at 270, 300, 330, 360, 390, 420, 450, and 480 min postinjection. Protocol A randomized, double-blinded, placebo-controlled, parallel Assessments of LH pulsatility design study was performed. Ten subjects with HA (see Table 1 Subjects underwent assessment of LH pulsatility on the first for baseline characteristics) were randomized to either saline or study day (wk 1, d 1) and approximately 24 h after the final kisspeptin treatment groups (five subjects per group). Before injection of the treatment period (wk 7, d 1). Blood was sampled commencement of the 8-wk study protocol, subjects were taught sequentially every 10 min for serum LH over an 8-h period. These how to self administer sc injections of saline. studies were commenced between the hours of 0800 h and 1200 h. Baseline period This initial 4-wk control period (wk 1–4) allowed the mea- Basal measurement of reproductive hormones surement of baseline values of reproductive hormones and ul- Twice-weekly basal measurements of serum LH, FSH, estra- trasound markers and the acclimatization of subjects to study diol, progesterone, SHBG, and plasma kisspeptin-IR were taken conditions. During wk 1–2 of the protocol, all women with HA from subjects throughout the 8-wk study protocol between 0800 self-administered twice-daily sc injections of saline (blinded to and 1800 h. During weeks when injections were self-adminis- subjects only). No injections were administered during wk 3–4 tered by volunteers (wk 1, 2, 5, and 6), these blood tests were of the protocol. performed a mean of 4.5 Ϯ 0.4 h after the previous injection, depending on the availability of volunteers. These twice-weekly Treatment period basal measurements were used to calculate mean values for se- During wk 5–6 of the protocol, women with HA either self- rum LH, FSH, and estradiol during the baseline period (wk 1–4), administered twice-daily, double-blinded sc injections of saline treatment period (wk 5–6) and posttreatment period (wk 7–8) (five subjects) or kisspeptin-54 (five subjects), depending on the of the study protocol. Kisspeptin-IR was measured to confirm treatment group to which they had been assigned. The dose of subject compliance to kisspeptin injections. kisspeptin administered was 6.4 nmol/kg [equivalent to 37 ␮g/kg (26)]. Twice-daily injections were self-administered at home by Ultrasound scans subjects during the treatment period, except on the last day (wk Transabdominal ultrasound scans were performed once a 6, d 7) when just one injection was administered in the morn- week throughout the 8-wk study period. During each scan, the ing. This final injection was administered within the investi- following parameters were measured: endometrial thickness in gation unit as part of a 4-h sampling study (see 4-h blood millimeters; mean ovarian volume in cubic centimeters; mean sampling after injection of saline or kisspeptin). follicles number; and maximum diameter of largest follicle in each ovary in millimeters. Ovulation was confirmed by satisfac- Posttreatment period tion of all of the following criteria: visualization of a dominant During wk 7–8, subjects underwent a posttreatment obser- follicle (diameter, 11 mm or greater); enlargement of dominant vation period to measure reproductive hormones and ultrasound follicle into a preovulatory follicle (diameter, 18 mm or greater); markers. No injections were administered during this period. subsequent collapse of preovulatory follicle or appearance of 4318 Jayasena et al. Effects of Kisspeptin in Women with HA J Clin Endocrinol Metab, November 2009, 94(11):4315–4323 internal echoes on ultrasonography; and a rise in serum proges- Measurement of plasma kisspeptin immunoreactivity (IR) terone to over 10 nmol/liter. was performed using an established RIA (26, 27). The antibody cross-reacted 100% with human kisspeptin-54, kisspeptin-14, Other measurements and kisspeptin-10 and less than 0.01% with other related RF amide proteins, including prolactin-releasing peptide, RF amide- Weight was measured on the first study day (wk 1, d 1) and related peptide 1 (RFRP1), RFRP2, RFRP3, QRFP43, neuropep- subsequently every 2 wk during the 8-wk protocol. During each tide FF, and neuropeptide AF. The limit of detectability was 2 study visit, urine was tested to exclude pregnancy (Clearview pmol/liter, and the intra- and interassay coefficients of variation easy-HCG; Inverness Medical Innovations Inc., Waltham, MA). were 8.3 and 10.2%, respectively. Diastolic and systolic blood pressure and heart rate were recorded every 30 min during the LH pulsatility studies, to compare mean values for each parameter before and after the treat- Data analysis ment period (wk 5–6). Blood pressure and heart rate were also Data are presented as mean Ϯ SEM. Hormone profiles during recorded during 4-h blood sampling studies performed after in- 4-h blood sampling studies and GnRH tests were analyzed using jection of kisspeptin or saline. repeated measures two-way ANOVA with Bonferonni post hoc correction. Pairs of means were analyzed using the unpaired two- Response of HA subjects to GnRH before and after tailed t test. Multiple means were compared using one-way injections of kisspeptin ANOVA with Bonferonni’s Multiple Comparison Test. A previously described modified Santen and Bardin method was used A second group of five female subjects was recruited using to assess LH pulsatility (34, 35). In all cases, P Ͻ 0.05 was con- identical inclusion criteria for HA described in this study, to sidered statistically significant. determine whether sensitivity to the effects of GnRH was retained after desensitization to the effects of kisspeptin. A baseline GnRH test was performed in all subjects in the investigation unit. In brief, subjects were cannulated and given a 100-␮g iv bolus Results injection of GnRH (HRF; Intrapharm Ltd., Kent, UK) at 0 min. Blood was sampled for measurement of serum LH, FSH, and Characteristics of subjects recruited to the study estradiol at Ϫ30, 0, 15, 30, 45, 60, 90, and 120 min. Seven days Baseline age, weight, and body mass index were not after the GnRH test, all five women self-administered twice-daily kisspeptin injections (6.4 nmol/kg) for 2 wk. The GnRH test was significantly different between kisspeptin and saline study repeated in each subject 8–12 h after their final kisspeptin groups (Table 1). Weight remained stable in both treat- injection. ment groups during the study (mean weight change from beginning to end of study: saline, Ϫ0.5 kg; kisspeptin, Collection and processing of blood samples Ϫ0.1 kg; P ϭ 0.09). Subjects reported no increased inci- Blood samples for serum analysis were collected in plain se- dence of nausea or other side effects after injection of rum Vacutainer tubes (Becton Dickinson, Franklin Lakes, NJ). kisspeptin or saline. Mean heart rate and systolic and di- Samples were allowed to clot before centrifugation and separa- astolic blood pressure were similar before and after the tion of serum. Blood samples for plasma kisspeptin analysis were collected in lithium heparin tubes (Becton Dickinson) containing treatment period (wk 5–6) in all participants (data not 5000 kallikrein inhibitor units of aprotinin (0.2 ml Trasylol; shown). Furthermore, no significant acute changes in Bayer, Newbury, UK). Samples were immediately centrifuged at heart rate or systolic and diastolic blood pressure were room temperature using a Hettich EBA 20 machine (Hettich observed after kisspeptin administration when compared International, Tuttlingen, Germany) for 10 min at 3000 rpm and with saline control (data not shown). then separated. Serum and plasma samples were stored at Ϫ20 C until analysis. Kisspeptin immunoreactivity in plasma was raised Analytical methods after injection of kisspeptin Serum LH, FSH, estradiol, and progesterone were measured Baseline plasma kisspeptin-IR was below 2 pmol/liter using automated chemiluminescent immunoassays (Abbott Di- and remained unchanged during the 4-h period after in- agnostics, Maidenhead, UK). SHBG was measured using a solid- jection of saline (Fig. 1, A and B). Kisspeptin injection phase automated enzyme immunoassay (Immulite; Siemans, resulted in a rise in plasma kisspeptin-IR, with peak mean Llanberis, UK). Reference ranges for females were as follows: LH kisspeptin-IR of approximately 5000 pmol/liter at 45 min (follicular), 2–10 IU/liter; LH (midcycle), 20–60 IU/liter; LH (luteal), 4–14 IU/liter; FSH (follicular and luteal), 1.5–8 IU/liter; after injection (Fig. 1, A and B). Similar patterns of estradiol (early follicular), less than 300 pmol/liter; estradiol kisspeptin-IR were observed after injection of kisspeptin (midcycle), 400–1500 pmol/liter; estradiol (luteal), 200–1000 on the first and last injection days (Fig. 1, A and B). pmol/liter; and SHBG, 40–80 nmol/liter. Interassay coefficients In subjects randomized to receive saline injections, of variation were as follows: LH, 3.4%; FSH, 3.5%; estradiol, plasma kisspeptin-IR remained less than 2 pmol/liter dur- 3.4%; progesterone, 1.8%; and SHBG, 5.6%. Limits of detectability for each assay were as follows: estradiol, 70 pmol/liter; ing basal measurements taken throughout the 8-wk study FSH, 0.05 mIU/ml; LH, 0.07 mIU/ml; progesterone, 0.1 ng/ml; protocol (Fig. 1D). In subjects randomized to receive and SHBG, 0.1 nmol/liter. kisspeptin injections, plasma kisspeptin-IR was raised J Clin Endocrinol Metab, November 2009, 94(11):4315–4323 jcem.endojournals.org 4319

A *** ** C 6000 1200 ***

1000 KP54 Saline 4000 800

600

2000 400

200

Plasma Kisspeptin IR (pmol/L) IR Kisspeptin Plasma 0 Plasma Kisspeptin IR (pmol/L) 0 0 30 60 90 120 150 180 210 240 1 2 3 4 5 6 7 8 Week number Time (minutes)

injection saline KP54 injections injections *** * B 6000 D 1200

1000 KP54 Saline 4000 800

600

2000 400

200 Plasma Kisspeptin IR (pmol/L) 0 (pmol/L) IR Kisspeptin Plasma 0 0 30 60 90 120 150 180 210 240 1 2 3 4 5 6 7 8 Week number Time (minutes) saline saline injection injections injections

FIG. 1. Effect of injections of saline or kisspeptin-54 on plasma kisspeptin-IR in women with HA. A and B, Mean Ϯ SEM plasma kisspeptin-IR after bolus sc injection of saline or kisspeptin-54 (KP54) 6.4 nmol/kg (n ϭ 5 per group) on the first day (A) or last (14th) day (B). Injections were administered at 0 min. C and D, Mean Ϯ SEM basal plasma kisspeptin-IR during each week of the 8-wk study protocol in subjects randomized to receive kisspeptin-54 (C) or saline (D) injections during wk 5–6. Data are shown as mean Ϯ SEM.*,P Ͻ 0.05; **, P Ͻ 0.01; ***, P Ͻ 0.001. during the 2-wk kisspeptin treatment period (wk 5–6) but tently increased serum FSH levels compared with saline remained below 2 pmol/liter for the remainder of the study (P Ͻ 0.001 at time points 180 to 240 min; Fig. 2B). After protocol (Fig. 1C). These twice-weekly blood samples kisspeptin injection, the maximal FSH rise was observed at were taken at various times of the day (determined by 240 min and was 9.1 Ϯ 2.5 IU/liter above baseline. Estra- subject availability) between the twice-daily injections. diol levels after kisspeptin injection were initially similar Accordingly, the mean kisspeptin-IR values during the to those following saline. However, estradiol levels sig- treatment period (wk 5, mean plasma kisspeptin-IR, nificantly increased above baseline between 180 and 240 416 Ϯ 217 pmol/liter; and wk 6, mean plasma kisspeptin- min after kisspeptin administration (P Ͻ 0.05; Fig. 2C). IR, 751 Ϯ 252 pmol/liter) were lower than the peak kisspeptin-IR observed 45 min after kisspeptin injection. Effects of last injection of saline or kisspeptin on serum reproductive hormones in women with HA Effects of first injection of saline or kisspeptin on On the last injection day of the treatment period (wk 6, serum reproductive hormones in women with HA d 7), saline injection did not change serum LH, FSH, or On the first injection day of the treatment period (wk 5, estradiol levels compared with baseline (Fig. 2, D–F). d 1), saline injection did not change serum LH, FSH, or Kisspeptin administration resulted in a significant rise estradiol levels compared with baseline (Fig. 2, A–C). in LH only at 240 min after injection and no significant Kisspeptin-54 injection acutely and potently increased se- rises in FSH or estradiol. Responses of LH, FSH, and rum LH levels in subjects with HA in comparison to saline estradiol to the kisspeptin administration were all sig- (P Ͻ 0.001 at time points 150 to 240 min; Fig. 2A). The nificantly reduced after the last injection (wk 6, d 7) mean maximal increase in LH from baseline after kisspep- when compared with the responses after the first tin injection was observed at 240 min and was 24.0 Ϯ 3.5 kisspeptin injection (wk 5, d 1) (P Ͻ 0.05 for LH, FSH, IU/liter above baseline. Kisspeptin-54 injection also po- and estradiol responses). 4320 Jayasena et al. Effects of Kisspeptin in Women with HA J Clin Endocrinol Metab, November 2009, 94(11):4315–4323

ABC * *** * *** * 28 12 50

24 KP54 KP54 40 KP54 Saline Saline Saline 20 8 30

16 20

12 4 10

8 0

4 0 -10

0 -20

-4 -4 -30

Change in serum LH (IU/l) LH serum baseline from in Change 0 30 60 90 120 150 180 210 240 0 30 60 90 120 150 180 210 240 0 30 60 90 120 150 180 210 240 Change serum in FSH (IU/l) from baseline Change in serum E2 (pmol/l) from baseline from (pmol/l) E2 serum in Change Time (minutes) Time (minutes) Time (minutes)

injection injection injection DEF 28 * 12 50 24 KP54 KP54 40 KP54 Saline Saline 30 Saline 20 8 20 16 10 12 4 0 8 -10 4 0 -20 0 -30

-4 -4 -40 0 30 60 90 120 150 180 210 240 0 30 60 90 120 150 180 210 240

Change serum in LH (IU/l) from baseline 0 30 60 90 120 150 180 210 240 Change serum in FSH (IU/l) from baseline Change in serum E2 (pmol/l) from baseline

Time (minutes) Time (minutes) Time (minutes)

injection injection injection FIG. 2. Effects of the first and last injections of saline or kisspeptin-54 on serum reproductive hormones in women with HA. A–C, Changes in serum LH (A), FSH (B), and estradiol (C) after bolus sc injection of saline (n ϭ 5) or 6.4 nmol/kg kisspeptin-54 (KP54, n ϭ 5) on first day (wk 5, d 1) of treatment period are shown. D–F, Changes in serum LH (D), FSH (E), and estradiol (F) after bolus sc injection of saline or kisspeptin-54 on the last day (wk 6, d 7) of the treatment period are shown. Injections were administered at 0 min. Data are shown as mean Ϯ SEM.*,P Ͻ 0.05; ***, P Ͻ 0.001. E2, Estradiol.

After the observation of significantly reduced gonado- plementary Table 1, published as supplemental data on tropin responses to kisspeptin on the last injection day, we The Endocrine Society’s Journals Online web site at http:// decided to assess further the duration of response to jcem.endojournals.org]. Small rises in mean basal LH and kisspeptin injection in one volunteer in whom blood sam- FSH levels were detected during wk 5 to 6 (the treatment pling was extended to 8 h after injection. In this volunteer, period) in women randomized to kisspeptin vs. saline kisspeptin-IR was raised until 6 h after injection (data not (mean basal LH, saline Ϫ0.8 Ϯ 0.7 vs. kisspeptin ϩ1.2 Ϯ shown). Furthermore, serum LH, FSH, and estradiol were 0.7; P ϭ 0.09; mean basal FSH, saline Ϫ1.8 Ϯ 0.4 vs. still raised above baseline by the end of the 8-h sampling kisspeptin ϩ0.6 Ϯ 0.4; P Ͻ 0.05). Basal serum reproduc- period (data not shown). Baseline response We also examined responsiveness to iv GnRH in five to GnRH Post-treatment additional subjects with HA, both before and after 20 response to GnRH

kisspeptin treatment. We observed LH responses to 15 GnRH in all subjects before commencing kisspeptin treat- 10 ment (mean peak LH increase during first 2 h after GnRH 5 injection, 14.4 Ϯ 4.6 IU/liter) (Fig. 3). Furthermore these LH increase (iU/L) LH increase 0 subjects remained responsive to GnRH injection 8–12 h 0 30 60 90 120 ϭ after their final kisspeptin injection (P 0.23 vs. baseline Time (minutes) LH response, using two-way ANOVA) (Fig. 3). Injection FIG. 3. Comparison of LH responses to GnRH administration before Reproductive hormones, LH pulsatility pattern, and after kisspeptin-54 injections in women with HA. Intravenous ␮ and radiological findings after saline or kisspeptin GnRH (100 g) was administered 7 d before commencement of a 14-d, twice-daily regime of sc kisspeptin injections (6.4 nmol/kg) treatment (baseline response to GnRH) (n ϭ 5). The GnRH test was repeated 8– Mean LH levels from twice-weekly basal blood tests 12 h after the last injection of kisspeptin (posttreatment response to during wk 1 to 4 (the baseline period) were slightly lower GnRH). When comparing baseline and posttreatment LH responses to GnRH injection, overall responses were similar (P ϭ 0.23), as were LH in the kisspeptin group than the saline group [mean LH, changes at each time-point after GnRH injection. Injections were saline 3.7 Ϯ 0.6 vs. kisspeptin 1.8 Ϯ 0.3; P Ͻ 0.05; Sup- administered at 0 min. Data are shown as mean Ϯ SEM. J Clin Endocrinol Metab, November 2009, 94(11):4315–4323 jcem.endojournals.org 4321 tive hormone levels were otherwise similar between kisspeptin-10 pulses were sufficient to induce a train of kisspeptin and saline groups throughout the 8-wk study GnRH discharges characteristic of puberty in juvenile period. monkeys. We were therefore surprised to observe that LH, There was no significant change in mean LH, number of FSH, and estradiol responses to the last kisspeptin injec- LH pulses, or mean pulse amplitude observed in patients tion were markedly lower than responses to the first in- receiving saline or kisspeptin (Supplementary Table 2). jection. In addition, reproductive ultrasound and basal There was no significant change in mean values for reproductive hormone parameters were similar between endometrial thickness, ovarian volume, follicle number, the two treatment groups. The last kisspeptin injection, or maximum follicle diameter observed after kisspeptin vs. which was reconstituted using peptide returned from saline treatment (Supplementary Table 3). One subject home storage by each volunteer, led to similarly elevated receiving kisspeptin developed radiological changes sug- plasma kisspeptin-IR to that observed after the first injec- gesting possible ovulation, with subsequent symptoms of tion. This suggests that peptide degradation caused by premenstruation. Ultrasound scans of the subject revealed home storage of kisspeptin did not account for the mark- the rupture of a preovulatory follicle (19-mm diameter) edly reduced gonadotropin responses to kisspeptin on the together with subsequent appearance of a corpus luteum. last injection day. However, this subject had no detectable rise in serum pro- Kisspeptin-10 was used during the animal studies of gesterone level and reported no menstrual bleeding. repetitive kisspeptin administration (28, 29), whereas the 54-amino acid form of kisspeptin was administered during this study. Our results reveal that plasma kisspeptin-IR is Discussion raised for up to 6 h after each sc injection of kisspeptin-54. We report the first study of kisspeptin administration in a Sustained exposure of monkeys and rodents to kisspeptin human model of infertility and the first investigation of the also leads to desensitization to its effects. Seminara et al. effects of chronic administration of kisspeptin in humans. (30) demonstrated that continuous iv kisspeptin-10 ad- Our results show that acute administration of kisspep- ministration to male rhesus monkeys for 98 h led to in- tin-54 increased serum gonadotropin levels in women creased LH release lasting only 3 h, followed by a return with HA but repeated injections lead to reduced effect. of gonadotropin concentrations to levels similar to those Subjects with HA display a reproductive hormone pro- observed before the kisspeptin-10 infusion. Similarly, file that resembles the follicular phase of the menstrual Thompson et al. (31) observed increased LH levels only cycle (low circulating gonadotropin and estradiol levels) during the first day of a continuous 3-d sc kisspeptin-54 more closely than the other phases. Acute LH response to infusion to adult male rats. A recent publication by Keen kisspeptin injection was approximately 4-fold greater in et al. (36) demonstrates the pattern of kisspeptin release patients with HA than in previously studied healthy fe- within the monkey hypothalamic median eminence to be males in the follicular phase of the menstrual cycle given pulsatile. Animal data suggest that a protocol using inter- an identical weight-adjusted dose (mean area under curve mittent administration of kisspeptin (28–29, 32) may be LH increase during first 4 h post-kisspeptin injection in less likely to result in desensitization than a protocol using h.iU/liter: women with HA in the current study, 40.2; continuous administration (30, 31). However, given the healthy females in follicular phase, 9.8; P Ͻ 0.01) (27). prolonged action of sc kisspeptin-54 injection on plasma This is consistent with the observation that LH responses kisspeptin-IR and reproductive hormone levels, our pro- to kisspeptin-10 may be higher in undernourished female tocol of twice-daily kisspeptin-54 injections may have re- rats when compared with those fed ad libitum (32). Fur- sulted in desensitization through prolonged and nonpul- ther work in a single study comparing women with HA satile kisspeptin exposure. An intermittent, iv method of and women with normal menstrual cycles is required to kisspeptin administration might minimize or prevent the confirm the observation made in this study. If confirmed, desensitization of gonadotropin responses observed in this it would be interesting to determine whether increased study. responsiveness of women with HA to kisspeptin is attrib- We observed GnRH administration to stimulate LH utable to factors such as increased sensitivity to kisspeptin secretion in HA subjects even after 2 wk of kisspeptin itself or increased pituitary sensitivity to GnRH. treatment. A study by Ramaswamy et al. (37) demon- In juvenile female rats, twice-daily intracerebroventric- strated that responsiveness to GnRH bolus was main- ular administration of kisspeptin-10 induces precocious tained in adult male rhesus monkeys during an infusion of vaginal opening in ad libitum fed animals (28) and restores kisspeptin-10 delivered at 200 ␮g/h but was reduced at a vaginal opening under conditions of caloric restriction higher infusion rate of 400 ␮g/h. In our study, a lower LH (32). Furthermore, Plant et al. (29) found that hourly iv response to GnRH administration was observed after 4322 Jayasena et al. Effects of Kisspeptin in Women with HA J Clin Endocrinol Metab, November 2009, 94(11):4315–4323 kisspeptin treatment when compared with the baseline References response; however, this difference was not significant. Our 1. Yen SS 1993 Female hypogonadotropic hypogonadism. Hypotha- results therefore suggest that the protocol of kisspeptin lamic amenorrhea syndrome. Endocrinol Metab Clin North Am administration used during this study led to desensitiza- 22:29–58 tion upstream of the pituitary gland. It is possible that our 2. Reindollar RH, Novak M, Tho SP, McDonough PG 1986 Adult- onset amenorrhea: a study of 262 patients. Am J Obstet Gynecol observations are explained by KISS1R down-regulation, 155:531–543 which has been previously demonstrated in vitro (38). 3. Frisch RE, Revelle R 1970 Height and weight at menarche and a Kisspeptin antagonism has been shown to inhibit pul- hypothesis of critical body weights and adolescent events. Science 169:397–399 satile GnRH release in pubertal female rhesus monkeys 4. Laughlin GA, Dominguez CE, Yen SS 1998 Nutritional and endo- and pulsatile LH release in adult female sheep (39). Fur- crine-metabolic aberrations in women with functional hypotha- thermore, Ramaswamy et al. (37) observed that LH pulse lamic amenorrhea. J Clin Endocrinol Metab 83:25–32 5. Loucks AB, Verdun M, Heath EM 1998 Low energy availability, not amplitude and frequency was reduced by infusion of stress of exercise, alters LH pulsatility in exercising women. J Appl kisspeptin-10 at 400 ␮g/h (but not 200 ␮g/h) in adult male Physiol 84:37–46 monkeys. In the current study, neither LH pulse amplitude 6. Marcus MD, Loucks TL, Berga SL 2001 Psychological correlates of functional hypothalamic amenorrhea. Fertil Steril 76:310–316 nor LH pulse frequency was significantly altered after 7. Reame NE, Sauder SE, Case GD, Kelch RP, Marshall JC 1985 Pul- kisspeptin treatment in women with HA. Our results satile gonadotropin secretion in women with hypothalamic amen- might be explained by rapid recovery from kisspeptin ex- orrhea: evidence that reduced frequency of gonadotropin-releasing hormone secretion is the mechanism of persistent anovulation. J Clin posure during the 24-h period between the final kisspeptin Endocrinol Metab 61:851–858 injection and the second assessment of LH pulsatility 8. Martin K, Santoro N, Hall J, Filicori M, Wierman M, Crowley Jr WF study. It is also plausible that a protocol using higher or 1990 Clinical review 15: management of ovulatory disorders with pulsatile gonadotropin-releasing hormone. J Clin Endocrinol Metab more frequent doses of kisspeptin injections would have 71:1081A–1081G significantly altered LH pulsatility. 9. Rossing MA, Daling JR, Weiss NS, Moore DE, Self SG 1994 Ovarian This study demonstrates that acute sc administration of tumors in a cohort of infertile women. N Engl J Med 331:771–776 10. Kotani M, Detheux M, Vandenbogaerde A, Communi D, Vanderwinden kisspeptin-54 potently stimulates pituitary-gonadal func- JM, Le Poul E, Bre´zillon S, Tyldesley R, Suarez-Huerta N, Vandeput tion in human females with HA. However, significantly F, Blanpain C, Schiffmann SN, Vassart G, Parmentier M 2001 The reduced gonadotropin responses to kisspeptin-54 admin- metastasis suppressor gene KiSS-1 encodes kisspeptins, the natural ligands of the orphan G protein-coupled receptor GPR54. J Biol istration were observed after 2 wk of twice-daily kisspep- Chem 276:34631–34636 tin-54 injections, suggesting desensitization. These results 11. Lee JH, Miele ME, Hicks DJ, Phillips KK, Trent JM, Weissman BE, have important implications for the therapeutic potential Welch DR 1996 KiSS-1, a novel human malignant melanoma metastasis-suppressor gene. J Natl Cancer Inst 88:1731–1737 of kisspeptin to treat patients with reproductive disorders. 12. Ohtaki T, Shintani Y, Honda S, Matsumoto H, Hori A, Kanehashi K, Terao Y, Kumano S, Takatsu Y, Masuda Y, Ishibashi Y, Watanabe T, Asada M, Yamada T, Suenaga M, Kitada C, Usuki S, Kurokawa T, Onda H, Nishimura O, Fujino M 2001 Metastasis Acknowledgments suppressor gene KiSS-1 encodes peptide ligand of a G-protein-coupled receptor. Nature 411:613–617 We are grateful to the Wellcome Trust and Sir John McMichael 13. Lee DK, Nguyen T, O’Neill GP, Cheng R, Liu Y, Howard AD, Coulombe N, Tan CP, Tang-Nguyen AT, George SR, O’Dowd BF Centre for providing infrastructure for this study. 1999 Discovery of a receptor related to the galanin receptors. FEBS Lett 446:103–107 Address all correspondence and requests for reprints to: Prof. 14. Muir AI, Chamberlain L, Elshourbagy NA, Michalovich D, Moore Stephen R. Bloom, Department of Investigative Medicine, Im- DJ, Calamari A, Szekeres PG, Sarau HM, Chambers JK, Murdock perial College London, Sixth Floor, Commonwealth Building, P, Steplewski K, Shabon U, Miller JE, Middleton SE, Darker JG, Hammersmith Hospital, Du Cane Road, London W12 ONN, Larminie CG, Wilson S, Bergsma DJ, Emson P, Faull R, Philpott KL, United Kingdom. E-mail: [email protected]. Harrison DC 2001 AXOR12, a novel human G protein-coupled receptor, activated by the peptide KiSS-1. J Biol Chem 276:28969– C.N.J. and G.M.K.N. are supported by Research Training 28975 Fellowships from the Wellcome Trust. R.R. is supported by a 15. Lapatto R, Pallais JC, Zhang D, Chan YM, Mahan A, Cerrato F, Le National Institute for Health Research Fellowship. V.S. is sup- WW, Hoffman GE, Seminara SB 2007 Kiss1Ϫ/Ϫ mice exhibit more ported by a Medical Research Council Research Training Fel- variable hypogonadism than Gpr54Ϫ/Ϫ mice. Endocrinology 148: lowship. W.S.D. is supported by an NIHR Clinician Scientist 4927–4936 Award and a Wellcome Trust Value in People Award. K.G.M. 16. Seminara SB, Messager S, Chatzidaki EE, Thresher RR, Acierno Jr holds a Biotechnology and Biological Sciences Research Council JS, Shagoury JK, Bo-Abbas Y, Kuohung W, Schwinof KM, Hendrick New Investigator Award. This work is funded by an MRC Ex- AG, Zahn D, Dixon J, Kaiser UB, Slaugenhaupt SA, Gusella JF, O’Rahilly S, Carlton MB, Crowley Jr WF, Aparicio SA, Colledge perimental Medicine project grant. The department is funded by WH 2003 The GPR54 gene as a regulator of puberty. N Engl J Med an Integrative Mammalian Biology Capacity Building Award 349:1614–1627 and the NIHR Biomedical Research Centre Funding Scheme. 17. de Roux N, Genin E, Carel JC, Matsuda F, Chaussain JL, Milgrom Disclosure Summary: The authors have nothing to disclose. E 2003 Hypogonadotropic hypogonadism due to loss of function of J Clin Endocrinol Metab, November 2009, 94(11):4315–4323 jcem.endojournals.org 4323

the KiSS1-derived peptide receptor GPR54. Proc Natl Acad Sci USA of hypothalamic G protein-coupled receptor 54 with intravenous 100:10972–10976 pulses of kisspeptin in the juvenile monkey (Macaca mulatta) elicits 18. Teles MG, Bianco SD, Brito VN, Trarbach EB, Kuohung W, Xu S, a sustained train of gonadotropin-releasing hormone discharges. Seminara SB, Mendonca BB, Kaiser UB, Latronico AC 2008 A Endocrinology 147:1007–1013 GPR54-activating mutation in a patient with central precocious pu- 30. Seminara SB, Dipietro MJ, Ramaswamy S, Crowley Jr WF, Plant berty. N Engl J Med 358:709–715 TM 2006 Continuous human metastin 45-54 infusion desensitizes 19. Irwig MS, Fraley GS, Smith JT, Acohido BV, Popa SM, Cunningham G protein-coupled receptor 54-induced gonadotropin-releasing MJ, Gottsch ML, Clifton DK, Steiner RA 2004 Kisspeptin activation hormone release monitored indirectly in the juvenile male Rhesus of gonadotropin releasing hormone neurons and regulation of monkey (Macaca mulatta): a finding with therapeutic implications. KiSS-1 mRNA in the male rat. Neuroendocrinology 80:264–272 Endocrinology 147:2122–2126 20. Navarro VM, Castellano JM, Fernańdez-Fernańdez R, Barreiro 31. Thompson EL, Murphy KG, Patterson M, Bewick GA, Stamp GW, ML, Roa J, Sanchez-Criado JE, Aguilar E, Dieguez C, Pinilla L, Curtis AE, Cooke JH, Jethwa PH, Todd JF, Ghatei MA, Bloom SR Tena-Sempere M 2004 Developmental and hormonally regulated 2006 Chronic subcutaneous administration of kisspeptin-54 causes messenger ribonucleic acid expression of KiSS-1 and its putative testicular degeneration in adult male rats. Am J Physiol Endocrinol receptor, GPR54, in rat hypothalamus and potent luteinizing hor- Metab 291:E1074–E1082 mone-releasing activity of KiSS-1 peptide. Endocrinology 145: 32. Castellano JM, Navarro VM, Fernańdez-Fernańdez R, Nogueiras 4565–4574 R, Tovar S, Roa J, Vazquez MJ, Vigo E, Casanueva FF, Aguilar E, 21. Thompson EL, Patterson M, Murphy KG, Smith KL, Dhillo WS, Pinilla L, Dieguez C, Tena-Sempere M 2005 Changes in hypotha- Todd JF, Ghatei MA, Bloom SR 2004 Central and peripheral ad- lamic KiSS-1 system and restoration of pubertal activation of the ministration of kisspeptin-10 stimulates the hypothalamic-pitu- reproductive axis by kisspeptin in undernutrition. Endocrinology itary-gonadal axis. J Neuroendocrinol 16:850–858 146:3917–3925 22. Gottsch ML, Cunningham MJ, Smith JT, Popa SM, Acohido BV, 33. Tovar S, Va´zquez MJ, Navarro VM, Fernańdez-Fernańdez R, Crowley WF, Seminara S, Clifton DK, Steiner RA 2004 A role for Castellano JM, Vigo E, Roa J, Casanueva FF, Aguilar E, Pinilla L, kisspeptins in the regulation of gonadotropin secretion in the mouse. Dieguez C, Tena-Sempere M 2006 Effects of single or repeated in- Endocrinology 145:4073–4077 travenous administration of kisspeptin upon dynamic LH secretion 23. Messager S, Chatzidaki EE, Ma D, Hendrick AG, Zahn D, Dixon J, in conscious male rats. Endocrinology 147:2696–2704 Thresher RR, Malinge I, Lomet D, Carlton MB, Colledge WH, 34. Adams JM, Taylor AE, Schoenfeld DA, Crowley Jr WF, Hall JE Caraty A, Aparicio SA 2005 Kisspeptin directly stimulates gonado- 1994 The midcycle gonadotropin surge in normal women occurs in tropin-releasing hormone release via G protein-coupled receptor 54. the face of an unchanging gonadotropin-releasing hormone pulse Proc Natl Acad Sci USA 102:1761–1766 frequency. J Clin Endocrinol Metab 79:858–864 24. Shahab M, Mastronardi C, Seminara SB, Crowley WF, Ojeda SR, 35. Hayes FJ, McNicholl DJ, Schoenfeld D, Marsh EE, Hall JE 1999 Plant TM 2005 Increased hypothalamic GPR54 signaling: a poten- Free ␣-subunit is superior to luteinizing hormone as a marker of tial mechanism for initiation of puberty in primates. Proc Natl Acad Sci USA 102:2129–2134 gonadotropin-releasing hormone despite desensitization at fast 25. Caraty A, Smith JT, Lomet D, Ben Saïd S, Morrissey A, Cognie J, pulse frequencies. J Clin Endocrinol Metab 84:1028–1036 Doughton B, Baril G, Briant C, Clarke IJ 2007 Kisspeptin synchro- 36. Keen KL, Wegner FH, Bloom SR, Ghatei MA, Terasawa E 2008 An nizes preovulatory surges in cyclical ewes and causes ovulation in increase in kisspeptin-54 release occurs with the pubertal increase in seasonally acyclic ewes. Endocrinology 148:5258–5267 luteinizing hormone-releasing hormone-1 release in the stalk-me- 26. Dhillo WS, Chaudhri OB, Patterson M, Thompson EL, Murphy dian eminence of female rhesus monkeys in vivo. Endocrinology KG, Badman MK, McGowan BM, Amber V, Patel S, Ghatei MA, 149:4151–4157 Bloom SR 2005 Kisspeptin-54 stimulates the hypothalamic-pitu- 37. Ramaswamy S, Seminara SB, Pohl CR, DiPietro MJ, Crowley Jr WF, itary gonadal axis in human males. J Clin Endocrinol Metab 90: Plant TM 2007 Effect of continuous iv administration of human 6609–6615 metastin 45–54 on the neuroendocrine activity of the hypothalamic- 27. Dhillo WS, Chaudhri OB, Thompson EL, Murphy KG, Patterson pituitary-testicular axis in the adult male rhesus monkey (Macaca M, Ramachandran R, Nijher GK, Amber V, Kokkinos A, Donaldson mulatta). Endocrinology 148:3364–3370 M, Ghatei MA, Bloom SR 2007 Kisspeptin-54 stimulates gonadotro- 38. Carvalho-Bianco SD, Teles M, Kuohung W, Xu S, Latronico AC, pin release most potently during the preovulatory phase of the men- Kaiser UB, Role of GPR54 desensitization in kisspeptin action. Proc strual cycle in women. J Clin Endocrinol Metab 92:3958–3966 of the American Endocrine Society Meeting, Boston, MA, 2006 28. Navarro VM, Fernańdez-Fernańdez R, Castellano JM, Roa J, (Abstract P3-251) Mayen A, Barreiro ML, Gaytan F, Aguilar E, Pinilla L, Dieguez C, 39. Roseweir AK, Kauffman AS, Smith JT, Guerriero KA, Morgan K, Tena-Sempere M 2004 Advanced vaginal opening and precocious Pielecka-Fortuna J, Pineda R, Gottsch ML, Tena-Sempere M, activation of the reproductive axis by KiSS-1 peptide, the endoge- Moenter SM, Terasawa E, Clarke IJ, Steiner RA, Millar RP 2009 nous ligand of GPR54. J Physiol 561:379–386 Discovery of potent kisspeptin antagonists delineate physiological 29. Plant TM, Ramaswamy S, Dipietro MJ 2006 Repetitive activation mechanisms of gonadotropin regulation. J Neurosci 29:3920–3929 articles nature publishing group

Twice-Weekly Administration of Kisspeptin-54 for 8 Weeks Stimulates Release of Reproductive Hormones in Women With Hypothalamic Amenorrhea

CN Jayasena1, GMK Nijher1, A Abbara1, KG Murphy1, A Lim2, D Patel2, A Mehta2, C Todd2, M Donaldson3, GH Trew4, MA Ghatei1, SR Bloom1 and WS Dhillo1

Kisspeptin is a novel therapeutic target for infertility. A single kisspeptin-54 (KP-54) injection acutely stimulates the release of reproductive hormones in women with hypothalamic amenorrhea (HA), a commonly occurring condition characterized by absence of menstruation; however, twice-daily administration of KP-54 results in tachyphylaxis. We determined the time course of desensitization to twice-daily KP-54 injections, compared the effects of twice-daily and twice-weekly administration regimens of KP-54, and studied the effects of long-term twice-weekly administration of KP-54 on the release of reproductive hormones in women with HA. When KP-54 was administered twice daily, responsiveness to luteinizing hormone (LH) diminished gradually, whereas responsiveness to follicle-stimulating hormone (FSH) was nearly abolished by day 2. Twice-weekly KP-54 administration resulted in only partial desensitization, in contrast to the complete tolerance achieved with twice-daily administration. Women with HA who were treated with twice-weekly KP-54 injections had significantly elevated levels of reproductive hormones after 8 weeks as compared with treatment with saline. No adverse effects were observed. This study provides novel pharmacological data on the effects of KP-54 on the release of reproductive hormones in women with HA.

Infertility affects 9% of couples worldwide.1 Gonadotrophin or peripheral administration of kisspeptin induces the release of injections containing luteinizing hormone (LH) or follicle- gonadotrophin.8–16 This effect is abolished by preadministration stimulating hormone (FSH) are routinely used to treat such of an antagonist to the hypothalamic hormone gonadotrophin- patients. Although this treatment is efficacious, it is associated releasing hormone.13 Kisspeptin is therefore thought to indirectly with the risk of the potentially life-threatening condition known stimulate the release of gonadotrophin from the pituitary gland as ovarian hyperstimulation syndrome.2 It has recently emerged by stimulating the release of gonadotrophin-releasing hormone that kisspeptin plays a critical role in the mammalian reproduc- from the hypothalamus. We have previously demonstrated that tive system and may therefore offer a novel therapeutic target in single injections of kisspeptin-54 (KP-54) stimulate the secre- the treatment of patients with reproductive disorders. tion of gonadotrophin in healthy human male and female sub- Kisspeptin is a group of arginine–phenylalanine amide peptides jects.15,16 It is therefore of interest to investigate whether KP-54 encoded by the KISS1 gene, which is expressed in the hypotha- can be used to stimulate the release of gonadotrophin in patients lamus, pituitary gland, and gonads.3,4 In humans, inactivating with infertility conditions that are associated with deficient secre- mutations of the kisspeptin receptor (KISS1R) cause pubertal tion of gonadotrophin (hypogonadotrophic hypogonadism). failure,5,6 and activating mutations lead to precocious puberty.7 Hypothalamic amenorrhea (HA) is a form of hypogonado- In all the mammalian species that have been investigated, central trophic hypogonadism17 that accounts for ~30% of amenorrhea

The first two authors contributed equally to this work. 1Section of Investigative Medicine, Imperial College London, Hammersmith Hospital, London, UK; 2Imaging Department, Imperial College Healthcare NHS Trust, Charing Cross Hospital, London, UK; 3Department of Clinical Chemistry, Imperial College Healthcare NHS Trust, Charing Cross Hospital, London, UK; 4Department of Reproductive Medicine and Surgery, Assisted Conception Unit, Hammersmith Hospital, Imperial College School of Medicine, London, UK. Correspondence: SR Bloom ([email protected]) Received 26 April 2010; accepted 28 July 2010; advance online publication 27 October 2010. doi:10.1038/clpt.2010.204

840 VOLUME 88 NUMBER 6 | december 2010 | www.nature.com/cpt articles

(absence of menstruation) in women of reproductive age.18 LH responses after injection of KP-54 in women with HA Functional HA is defined as occurring in the absence of astructural (Figure 1a). On day 1, the mean maximal LH response during hypothalamo–pituitary lesion and often results from a low body the first 4 h after KP-54 injection was 23.3± 12.1 IU/l; however, weight or significant weight loss.19–24 We previously demonstrated the mean maximal LH response during this period dropped to that a single injection of KP-54 stimulates the release of gonado- 11.2 ± 5.7, 4.2 ± 1.1, 3.4 ± 0.5, and 1.1 ± 0.5 h·IU/l on days 2, 3, trophin in women with HA.25 However, women with HA who were 4, and 14 of injections, respectively (Figure 1a). On day 1, the treated with twice-daily KP-54 injections for 2 weeks were mark- mean maximal FSH response during the first 4 h after the KP-54 edly less responsive to KP-54 at the end of the study as compared injection was 7.2 ± 2.2 h·IU/l. However, the acute FSH responses with the start of the study. Interestingly, these women remained after the injection were <2 U/l on days 2, 3, 4, and 14 (Figure 1b). responsive to gonadotrophin-releasing hormone injection at the Mean estradiol levels were not significantly different at any point end of the study, thereby suggesting that the desensitization associ- during the study (Figure 1c). ated with repeated administration of KP-54 was occurring at the KISS1R. Consequently, acute administration of KP-54 to women Study 2: two-week study of release of reproductive with HA results in a potent stimulation of LH release, but twice- hormones in serum in women with HA receiving twice- daily KP-54 administration for 2 weeks leads to tachyphylaxis.25 daily KP-54 injections as compared with those receiving Determining whether KP-54 can stimulate gonadotrophin twice‑weekly injections release in a sustained manner has important implications for A. Effect of twice-daily subcutaneous injection of KP-54 (6.4 nmol/kg) the treatment of patients with infertility. We hypothesized that on the release of reproductive hormone. Twice-daily subcuta- an alternative protocol of repeated KP-54 administration would neous injection of 6.4 nmol/kg KP-54 in women with HA sustainably stimulate the release of gonadotrophin in women potently stimulated gonadotrophin release on day 1, with with HA. The aims of this randomized, double-blinded, placebo- the maximal increase being observed 240 min after the injec- controlled parallel design study were to determine (i) the time tion (mean maximal increase following KP-54 injection on course over which desensitization to the effects of KP-54 on the day 1: LH: 23.2 ± 11.3 IU/l; FSH: 7.2 ± 2.1 IU/l). However, on release of reproductive hormone occurs in women with HA, day 14, reproductive hormone responses were significantly (ii) the effects of prolongation of the KP-54 dose interval on reduced as compared with those on day 1 (mean maximal desensitization to KP-54 treatment, and (iii) whether the admin- increase after KP-54 injection on day 14: LH: 1.0 ± 0.5 IU/l, istration of KP-54 twice weekly can result in chronic stimula- P < 0.05 relative to day 1; FSH: 0.2 ± 0.3 IU/l, P < 0.001 rela- tion of gonadotrophin release and restore menstrual cyclicity in tive to day 1) (Figure 2a,b). The estradiol response was women with HA over an 8-week period. nonsignificantly lower on day 14 as compared with that at day 1 (mean maximal estradiol increase after the injection: Results day 1, 48.2 ± 29.7 pmol/l; day 14, −12.7 ± 7.0 pmol/l, P = 0.06) Characteristics of subjects recruited to the study (Figure 2c). Baseline age, weight, and body mass index were similar for all groups of subjects in this study (Table 1). B. Effect of twice-weekly subcutaneous injection of KP-54 (6.4 nmol/kg) on release of reproductive hormone. We had observed that subcu- Study 1: time course of desensitization to the taneous administration of KP-54 twice daily led to significant gonadotrophin‑inducing effects of KP-54 in women with HA desensitization of gonadotrophin responses within 2 weeks of Twice-daily subcutaneous administration of 6.4 nmol/kg treatment. We therefore investigated whether the same sub- KP-54 was associated with a progressive reduction in acute cutaneous dose of KP-54 (6.4 nmol/kg) would be associated

Table 1 Comparison of baseline characteristics of women with hypothalamic amenorrhea participating in the 2-week pilot studies of the effects of administering kisspeptin and the 8-week study of the effects of administering saline vs. those of administering kisspeptin 2-Week pilot studies 8-Week study Protocol treatment Kisspeptin-54 (n = 10) Saline (n = 5) Kisspeptin-54 (n = 5) P-value Age (years) 28.1 ± 1.1 27.0 ± 2.6 27.2 ± 2.4 NS Weight (kg) 52.6 ± 0.7 54.5 ± 3.9 53.4 ± 3.0 NS Body mass index (kg/m2) 19.1 ± 0.3 19.7 ± 1.1 19.6 ± 0.7 NS Duration of amenorrhea (months) 22.4 ± 9.9 20.4 ± 5.1 28.8 ± 12.0 NS Serum LH (IU/l) 2.5 ± 0.6 1.7 ± 0.7 1.4 ± 0.5 NS Serum FSH (IU/l) 4.8 ± 0.3 3.8 ± 0.9 3.6 ± 0.6 NS Serum estradiol (pmol/l) 133 ± 10 87 ± 17 151 ± 28 NS Mean values ± SEM data are shown. FSH, follicle-stimulating hormone; LH, luteinizing hormone; NS, nonsignificant difference between the three groups of women with hypothalamic amenorrhea.

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a 40 d 100 *

30 80 *

60 20 (IU/l) 40 10 LH increase (IU/l) AUC LH increase 20

0 0

0 30 60 90 120 150 180 210 240 Day 1 Day 2 Day 3 Day 4 Day 14 Minutes

b ** *** 10 e 25 *** 8 Day 1 20 Day 2 6 15 Day 3

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Figure 1 Time course of desensitization to the effects of kisspeptin-54 (6.4 nmol/kg) on reproductive hormone release when administered twice daily for 2 weeks in women with HA. (a–f) Changes in serum levels of LH, FSH, and estradiol (E2) during the first 4 h after kisspeptin-54 (KP-54) injections were measured on days 1, 2, 3, 4, and 14 of twice-daily subcutaneous administration of 6.4 nmol/kg of KP-54 (n = 5). (d–f) Summary of the effects of subcutaneous KP-54 injection as measured on days 1, 2, 3, 4, and 14 of twice-daily subcutaneous administration of 6.4 nmol/kg of KP-54, shown as mean AUC for (d) LH, (e) FSH, and (f) estradiol. The data are shown as mean values ± SEM. *P < 0.05 vs. day 1; **P < 0.01 vs. day 1; ***P < 0.001 vs. day 1. AUC, area under the curve; FSH, follicle- stimulating hormone; HA, hypothalamic amenorrhea; LH, luteinizing hormone. with less desensitization if administered twice weekly (rather Study 3: effects of twice-weekly KP-54 administration for than twice daily). On day 14, mean gonadotrophin responses 8 weeks on reproductive hormone levels in women with HA after the KP-54 injection were nonsignificantly lower relative The results from study 2 suggested that twice-weekly administra- to day 1 (mean maximal increase in IU/l after the injection: tion of KP-54 6.4 nmol/kg significantly reduces desensitization LH: day 1, 18.8 ± 6.6 vs. day 14, 11.5 ± 4.0, P = 0.08; FSH: day to the effects of KP-54 on the release of reproductive hormone. 1, 5.8 ± 2.0 vs. day 14, 4.1 ± 1.1, P = 0.14) (Figures 2d,e). The A randomized, double-blinded, placebo-controlled, parallel mean maximal increases in serum estradiol on day 1 after the design study was therefore performed in order to determine KP-54 injection was not significantly different from that at day whether twice-weekly administration of KP-54 at 6.4 nmol/kg 14 (day 1 44.8 ± 15.7 pmol/l vs. day 14 27.5 ± 15.5 pmol/l; P = would stimulate the release of reproductive hormone over an 0.47) (Figure 2f). 8-week period (protocol summary shown in Figure 3). Baseline A summary of the effects of subcutaneous administration of age, weight, and body mass index were not significantly different KP-54 injections on days 1 and 14 of each of the different 2-week between the group receiving KP-54 and the control group receiv- dosing regimens of KP-54 is shown in Figures 2g–i as mean ing saline (Table 1). The subjects reported no adverse effects areas under the curve for LH, FSH, and estradiol responses. after injections of either KP-54 or saline. No significant acute

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a Kisspeptin-54 Kisspeptin-54 g Summary: twice-daily 6.4 nmol/kg twice-daily d 6.4 nmol/kg twice-weekly vs. twice-weekly KP-54 * 40 40 * 100 e 30 30 80 60 LH 20 20 40 (h·IU/l) 10 10 20 LH increase (IU/l) AUC LH increas LH increase (IU/l) 0 0 0 6.4 nmol/kg 6.4 nmol/kg 0 60 120 180 240 0 60 120 180 240 twice-daily twice-weekly Minutes Minutes b * *** e h 10 10 **

e 20 8 8 15 6 6 FSH 10 4

4 (h·IU/l) 5 2 2 FSH increase (IU/l) AUC FSH increas FSH increase (IU/l) 0 0 0 6.4 nmol/kg 6.4 nmol/kg 060120 180240 060 120 180 240 twice-daily twice-weekly Minutes Minutes c f i 100 80 80 200 60

60 ) 100 40 40 E2 20 20 0 0 (h·pmol/l

0 AUC E2 increase −20 E2 increase (pmol/l) E2 increase (pmol/l) –100 −40 −20 6.4 nmol/kg 6.4 nmol/kg 0 60 120 180 240 0 60 120 180 240 twice-daily twice-weekly Minutes Minutes

First KP-54 injection First KP-54 injection Last KP-54 injection Last KP-54 injection

Figure 2 Serum reproductive hormone levels in women with HA receiving twice-daily or twice-weekly KP-54 injections for 2 weeks. (a–f) Changes in serum levels of LH, FSH, and estradiol (E2) during 4 h after subcutaneous administration of KP-54 on days 1 and 14 of the of the 2-week dosing regimens (n = 5 per dosing regimen); (a–c) after twice-daily subcutaneous injections of 6.4 nmol/kg of KP-54; (d–f) after twice-weekly subcutaneous injection of 6.4 nmol/kg of KP-54. (g–i) A summary of the effects of subcutaneous injections of KP-54 on days 1 and 14 of each of the different 2-week dosing regimens of KP-54 is shown as the mean AUC for (g) LH, (h) FSH, and (i) estradiol. The data are shown as mean values ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001 vs. day 1. AUC, area under the curve; FSH, follicle-stimulating hormone; HA, hypothalamic amenorrhea; KP-54, kisspeptin-54; LH, luteinizing hormone. changes were observed in heart rate or in systolic or diastolic P < 0.001 vs. baseline response; 4 weeks, 2.6 ± 0.7, P > 0.05 vs. blood pressure after the administration of KP-54 as compared response at 2 weeks; 6 weeks, 2.4 ± 0.8, P > 0.05 vs. response at with saline (data not shown). 2 weeks; 8 weeks, 2.7 ± 0.8, P > 0.05 vs. response at 2 weeks) Saline had no significant effects on the release of reproduc- (Figure 4e). Estradiol responses after injection of KP-54 were tive hormones at any time during the 8-week protocol of twice- similar throughout the 8-week protocol of twice-weekly injec- weekly injections (Figure 4a–c). As expected, KP-54 stimulated tions (mean maximal increase in serum estradiol after KP-54 the release of reproductive hormones after the injection on day 1. injection (pmol/l): baseline, 44.4 ± 19.9; 2 weeks, 39.2 ± 14.8; After 2 weeks of twice-weekly injections, LH responses after 4 weeks, 46.2 ± 25.8; 6 weeks, 60.8 ± 17.2; 8 weeks, 20.0 ± 7.5) the KP-54 injection were significantly lower relative to those (Figure 4f). on day 1 (mean maximal LH increase (IU/l): baseline, 21.5 ± No significant differences were observed at any stage during 10.7; 2 weeks, 10.0 ± 4.3; P < 0.001) (Figure 4d). However, no the 8-week protocol with respect to the number of follicles, further significant reductions in LH KP-54 were observed after the maximum size of the follicles, the volume of the ovary, and injection of KP-54 at 4 weeks (mean maximal LH increase: 9.0 ± endometrial thickness between subjects who received KP-54 4.1 IU/l; P > 0.05 vs. response at 2 weeks), 6 weeks (mean maxi- and those who received saline injections (Table 2). More domi- mal LH increase: 8.9 ± 3.5 IU/l; P > 0.05 vs. response at 2 weeks), nant follicles were observed in subjects treated with KP-54 than and 8 weeks (mean maximal LH increase: 7.9 ± 4.5 IU/l; P > in subjects treated with saline (six vs. three dominant follicles, 0.05 vs. response at 2 weeks) (Figure 4d). FSH responses after respectively); however, no preovulatory follicles were observed KP-54 injection showed a pattern similar to those of LH over in any subject during the study (Table 2). No significant dif- the 8-week protocol (mean maximal increase in serum FSH after ferences were observed in the values of the twice-weekly basal KP-54 injection (IU/l): baseline, 6.4 ± 3.2; 2 weeks, 2.7 ± 0.7, reproductive hormone measurements at any stage during the

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Day of protocol: 1 14 28 42 56

Twice-weekly saline injections n = 5

Randomization

n = 5 Twice-weekly KP-54 injections

4-h Blood sampling postinjection (days 1, 14, 28, 42, 56)

Twice-weekly blood sampling for LH, FSH, E2

Once-weekly pelvic ultrasound scans

Figure 3 Protocol summary for study 3: the effects of twice-weekly administration of KP-54 for 8 weeks in women with HA. Subjects with HA were randomized to receive twice-weekly injections of saline or of KP-54 at 6.4 nmol/kg for 8 weeks. Blood sampling was carried out at various time points during a 4-h period immediately after the saline or KP-54 injections on days 1, 14, 28, 42, and 56. Twice-weekly blood sampling was performed for measurement of LH, FSH, and estradiol (E2) levels. Ultrasound scans were also performed once a week. FSH, follicle-stimulating hormone; HA, hypothalamic amenorrhea; KP-54, kisspeptin-54; LH, luteinizing hormone.

8-week protocol, between subjects receiving saline and those kisspeptin-based therapies. Recent evidence suggests that receiving KP-54 injections (Table 3). One subject receiving pharmacological blockade of kisspeptin signaling may suppress saline treatment developed a preovulatory LH surge after 8 LH pulsatility rather than basal LH levels.29 Further studies weeks of saline treatment; however, none of the subjects ovu- are required in order to investigate the use of kisspeptin for lated during the study. downregulating the reproductive axis as a treatment for sex hormone–responsive tumors. Discussion In view of the tachyphylaxis associated with twice-daily Kisspeptin signaling offers an entirely novel therapeutic target administration of KP-54 (6.4 nmol/kg), we investigated whether for treating reproductive disorders; however, our understanding lengthening the dose interval to twice-weekly would reduce of the effects of kisspeptin in patients with infertility is limited. desensitization. We performed a 2-week pilot study, and the Short-term clinical studies suggest that a single injection of KP-54 results suggest that gonadotrophin responses are only partially safely stimulates the release of reproductive hormones15,16,25 but diminished after twice-weekly administration of 6.4 nmol/kg of that its repeated administration causes profound tachyphylaxis KP-54. This suggests that subjects with HA remained partially in humans.25 We present novel data suggesting that twice-weekly sensitive to KP-54 injection during this twice-weekly regimen. subcutaneous administration of KP-54 stimulates the release of To further assess the efficacy of twice-weekly subcutaneous reproductive hormones but does not restore menstrual cyclicity injection of 6.4 nmol/kg KP-54, we performed a longer, 8-week in women with HA over a 2-month period. study. As observed during the pilot study, partial desensitiza- We present detailed data of the time course of desensitization in the gonadotrophin responses to KP-54 injection was tion to the effects of KP-54 on reproductive hormone release in observed during the first 2 weeks of administration. However, women with HA. Our data suggest that desensitization of LH these responses did not significantly diminish further beyond response to twice-daily subcutaneous administration of KP-54 this initial 2-week period. Our results therefore suggest that sub- in women with HA occurs gradually over the 14-day injec- jects with HA remained partially responsive to KP-54 injection tion period. LH responses on day 4 were significantly higher during the 8-week study period. than those on day 14. By contrast, FSH responses dropped It is interesting to consider why no significant changes in to <3 h·IU/l after just 24 h of twice-daily administration of follicle growth were observed during the 8-week study of KP-54. It has also been suggested that chronic administration twice-weekly KP-54 vs. saline administration. Previous studies of kisspeptin could be useful as a therapy for sex hormone– of KP-54 administration in humans demonstrated no signifi- responsive cancers because of its inhibitory effects on the cant alterations in plasma inhibin in healthy male volunteers.15 release of sex hormones26 and on tumor metastasis.27,28 Our However, in this study, we did observe that FSH responses were data, which suggest that twice-daily subcutaneous administra- lower than LH responses after the administration of kisspep- tion of KP-54 leads to downregulation of the hypothalamo– tin. Consistent with this observation, kisspeptin has previously pituitary–gonadal axis, therefore has implications for such been shown to stimulate the release of LH more potently than it

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a Saline twice-weekly d KP-54 6.4 nmol/kg g Summary: twice-weekly twice-weekly saline vs. KP-54 6.4 nmol/kg 40 40 80 *

30 30 60

LH 20 20 40 (IU/l) (IU/l)

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Mean FSH increase 0 Mean FSH increase 0 AUC FSH increase −5 0 30 60 90 120 150 180 210 240 0 30 60 90 120 150 180 210 240 e eks eks eks Minutes Minutes lin e e e eeks ase W W W W B 2 4 6 8 c f 100 100 i 120 80 50 50 E2 40 (pmol/l) 0 (pmol/l) 0

(h·IU/l) 0 Mean E2 increase Mean E2 increase −40 −50 −50 AUC E2 increase 0 30 60 90 120 150 180 210 240 0306090 120 150 180 210 240 −80 e Minutes Minutes lin eeks eeks eeks eeks se a W W W W B 2 4 6 8

Baseline 2 Weeks 4 Weeks 6 Weeks 8 Weeks Saline KP-54

Figure 4 Reproductive hormone levels in serum after twice-weekly administration of saline or KP-54 for 8 weeks in women with HA. (a–c) Changes in levels of (a) LH, (b) FSH, and (c) estradiol (E2) in serum after bolus subcutaneous injection of saline (n = 5) on day 1 and after 2, 4, 6, and 8 weeks of administration. (d–f) Changes in levels of (d) LH, (e) FSH, and (f) estradiol after bolus s.c. injection of 6.4 nmol/kg of KP-54 (n = 5) on day 1, and at after 2, 4, 6, and 8 weeks of administration. The time point of injections is 0 min. The data are shown as mean values ± SEM. **P < 0.01; ***P < 0.001 vs. baseline. (g–i) A summary of the effects of twice-weekly administration of saline vs. KP-54 during the 8-week study period is shown as mean areas under the curve for (g) LH, (h) FSH, and (i) estradiol. The data are shown as mean values ± SEM. *P < 0.05; **P < 0.01; ***P < 0.01 vs. saline. FSH, follicle-stimulating hormone; HA, hypothalamic amenorrhea; KP-54, kisspeptin-54; LH, luteinizing hormone; s.c., subcutaneous. stimulates the release of FSH when administered to rodents,10 twice-daily administration of KP-54 in women with HA. to healthy male volunteers,15 or to healthy female volunteers.16 Twice-daily administration of KP-54 is associated with desen- Furthermore, intravenous bolus injection of gonadotrophin- sitization, a characteristic that may be utilized in the treatment releasing hormone also stimulates the release of LH release of hormone-sensitive tumors. We have also conducted the first more potently as compared with the release of FSH.30 We also long-term clinical study of KP-54 administration to women observed that FSH responses were desensitized more rapidly with HA. Twice-weekly administration of KP-54 resulted in after KP-54 injection than LH responses were. It is therefore only partial desensitization, in contrast to complete tolerance possible that inadequacy in the magnitude and duration of FSH achieved with twice-daily administration. However, this pro- stimulation might have accounted for the lack of detectable tocol of administration of KP-54 did not restore menstrual ovarian follicular growth observed in this study. cyclicity in women with HA. Further work is required to inves- The dose of KP-54 used during this study (6.4 nmol/kg) was tigate the therapeutic potential of kisspeptin in the treatment selected because it was previously shown to stimulate a mean of patients with disorders of the reproductive system. maximal rise in serum FSH of 9.1 IU, in women with HA.25 It would be interesting to investigate whether higher doses of Methods twice-weekly KP-54 administration would lead to more potent Subjects. Ethical approval for this study was granted by the research FSH stimulation and restoration of menstrual cyclicity. ethics committee of Hammersmith and Queen Charlotte’s and Chelsea In summary, we present important pharmacologic data Hospitals (registration number 05/Q0406/142). Written informed consent was obtained from all the subjects. The study was performed regarding the effects of KP-54 on the release of gonadotrophin in accordance with the Declaration of Helsinki. in a human model of infertility. We have determined the time Subjects were recruited through advertisements placed in local news- course of desensitization of LH and FSH responses during papers.25 Women were diagnosed with functional HA if they fulfilled the

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Table 2 summary of ultrasound parameters at baseline Table 3 Comparison of basal levels of reproductive hormones and during and after 8 weeks of treatment with either saline or in serum at baseline and during and after 8 weeks of treatment kisspeptin in women with hypothalamic amenorrhea with either saline or kisspeptin injections Study group Study group Characteristic Saline Kisspeptin-54 P-value Hormone Saline Kisspeptin-54 P-value Ovulation 0 0 — LH (IU/l) Baseline 1.5 ± 0.6 1.8 ± 0.7 NS (no. of subjects) Week 2 1.9 ± 0.5 1.7 ± 0.6 NS Preovulatory follicle 0 0 — Weeks 3–4 1.9 ± 0.5 1.7 ± 0.4 NS ≥18 mm (no. of subjects) Weeks 5–8 3.7 2.7 1.9 0.5 NS Dominant follicle 3 6 — ± ± ≥11 mm (no. of subjects) FSH (IU/l) Baseline 3.7 ± 0.6 3.4 ± 0.5 NS Endometrial Weeks 1–2 2.8 ± 0.1 3.2 ± 0.5 NS Week 2 2.8 ± 0.8 4.5 ± 0.9 NS thickness (mm) Weeks 3–4 3.2 ± 0.4 3.5 ± 0.4 NS Weeks 3–4 3.1 ± 1.0 3.1 ± 0.9 NS Weeks 5–6 2.9 ± 0.3 4.0 ± 0.5 NS Weeks 5–8 3.2 ± 0.8 4.3 ± 0.7 NS Weeks 7–8 3.2 ± 0.7 4.0 ± 0.5 NS Estradiol (pmol/l) Baseline 97 ± 13 78 ± 9 NS Ovarian volume (cm3) Weeks 1–2 5.7 ± 1.8 6.7 ± 2.1 NS Week 2 91 ± 10 102 ± 11 NS Weeks 3–4 5.2 ± 1.5 5.3 ± 1.0 NS Weeks 3–4 113 ± 33 90 ± 8 NS Weeks 5–6 5.4 ± 1.3 5.2 ± 0.6 NS Weeks 5–8 123 ± 37 102 ± 11 NS Weeks 7–8 6.1 ± 1.9 5.5 ± 0.7 NS Mean ± SEM values of the basal hormone levels are given for subject groups randomized to receive twice-weekly injections of saline (n = 5) or of 6.4 nmol/kg KP-54 Follicle number Weeks 1–2 12 ± 4 13 ± 3 NS (n = 5). For each subject, levels of LH, FSH, and estradiol in serum were measured at per ovary Weeks 3–4 11 ± 3 15 ± 3 NS baseline, and the mean levels were calculated from separate blood tests performed during week 2, weeks 3–4, and weeks 5–8 of the study protocol. Weeks 5–6 11 ± 3 16 ± 3 NS FSH, follicle-stimulating hormone; KP-54, kisspeptin-54; LH, luteinizing hormone; NS, Weeks 7–8 9 ± 4 13 ± 4 NS nonsignificant difference between KP-54 group vs. saline group during each 2-week time period. Maximum follicular Weeks 1–2 5.0 ± 1.0 7.0 ± 0.9 NS diameter (mm) Weeks 3–4 6.5 ± 1.5 7.8 ± 1.6 NS Study 2: two-week study of release of reproductive hormone in serum in women with HA receiving twice-daily KP-54 injections as compared Weeks 5–6 6.0 ± 1.1 7.3 ± 0.9 NS with those receiving twice-weekly injections. Subjects with HA (n = 5 Weeks 7–8 6.3 ± 0.8 7.3 ± 1.2 NS per group) were randomized to receive, in a single-blinded manner, The values given are for subjects randomized to receive twice-weekly injections of either twice-daily or twice-weekly injections of 6.4 nmol/kg KP-54 over saline (n = 5) or of 6.4 nmol/kg of KP-54 (n = 5). The change in value of each ultrasound a 2-week period (see Table 1 for baseline characteristics of the subjects). parameter during each 2-week period of the study was based on the results of The subjects attended our clinical investigation unit twice a week, and two separate scans performed during the respective 2-week period. Endometrial measurements of reproductive hormone levels were carried out at each thickness, ovarian volume, the number of follicles, and the maximum follicular of these visits. On day 1 (first day of injection) and day 14 (last day of diameter are shown as mean values ± SEM. injection), blood samples were taken from each subject at various time KP-54, kisspeptin-54; NS, nonsignificant difference between KP-54 group vs. saline points for 4 h after the injection of KP-54, as described above. group during each 2-week time period.

Study 3: randomized, double-blinded, 8-week study comparing the following criteria: body mass index <25 kg/m2; stable body weight for effects of twice-weekly KP-54 (6.4 nmol/kg) vs. saline in women with HA. 6 months; age 18–40 years; secondary amenorrhea >6 months; no oral A randomized, double-blinded, placebo-controlled, parallel-design contraceptive pill therapy within the last year; no clinical or biochemical study was performed. Ten subjects with HA (see Table 1 for baseline evidence of polycystic ovarian syndrome, thyroid dysfunction, or hyper- characteristics) were randomized to either saline or 6.4 nmol/kg KP-54 prolactinemia; a structurally normal hypothalamo–pituitary region (on injections (n = 5 per group) twice weekly for 8 weeks. Each subject magnetic resonance imaging) and female reproductive tract (on ultra- attended our clinical investigation unit twice a week. During each visit, sound); no therapeutic or recreational drug use; and no systemic disease a subcutaneous injection of either saline or KP-54 was administered to comorbidity. Twenty subjects with HA were recruited. each subject, in a double-blinded manner. After the first injection of saline or KP-54, blood samples were collected at various time points from each subject for 4 h as described above. This 4-h sampling pro- Study 1: determination of the time course of desensitization to the tocol was repeated at 2, 4, and 6 weeks and on the final day of the gonadotrophin-inducing effects of KP-54 in women with HA. Five sub- study protocol at 8 weeks. Measurements of reproductive hormones jects with HA received twice-daily injections of 6.4 nmol/kg KP-54. The were carried out twice a week, and transabdominal ultrasound scans dose of KP-54 chosen for this study has previously been shown to result were performed once a week.25 A summary of the protocol for study in robust stimulation of gonadotrophin release in healthy female volun- 3 is shown in Figure 3. During each study visit, urine was tested in teers16 and in women with HA.25 On day 1 (the first injection day) and order to exclude pregnancy (Clearview Easy HCG; Inverness Medical on days 2, 3, 4, and 14 (the last injection day) of the study protocol, blood Innovations, Waltham, MA). The subjects were interviewed by a physi- samples were taken from each subject at specific time points for 4 h after cian during each visit to check for the presence of any adverse effects. the injection of KP-54. KP-54 was administered subcutaneously at our During the 4-h blood sampling period, diastolic and systolic blood clinical investigation unit, with the subjects in the supine position. Blood pressure and heart rate were recorded; after the injection (KP-54 or samples were drawn at −30, 0, 15, 30, 45, 60, 75, 90, 120, 150, 180, 210, saline), the subjects were repeatedly asked at regular intervals about and 240 min after the injection. the presence of any adverse symptoms.

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KP-54 peptide. KP-54 was synthesized by the Advanced Biotechnology 9. Navarro, V.M. et al. Developmental and hormonally regulated messenger Centre, Imperial College London, and purified using reverse-phase high- ribonucleic acid expression of KiSS-1 and its putative receptor, GPR54, in rat performance liquid chromatography. Electrospray mass spectroscopy hypothalamus and potent luteinizing hormone-releasing activity of KiSS-1 and amino acid analysis confirmed the identity of the peptide.15,16 The peptide. Endocrinology 145, 4565–4574 (2004). 15 10. Thompson, E.L. et al. Central and peripheral administration of kisspeptin-10 peptide was tested for bioactivity and toxicity. The Limulus amebo- stimulates the hypothalamic-pituitary-gonadal axis. J. Neuroendocrinol. 16, cyte lysate assay (Associates of Cape Cod, Liverpool, UK) was nega- 850–858 (2004). tive for endotoxin, and the peptide was sterile on culture (Department 11. Gottsch, M.L. et al. A role for kisspeptins in the regulation of gonadotropin of Microbiology, Hammersmith Hospital, London, UK). Although secretion in the mouse. Endocrinology 145, 4073–4077 (2004). kisspeptin-10, -13, -14, and -54 display similar potency in vitro, we used 12. Messager, S. et al. Kisspeptin directly stimulates gonadotropin-releasing KP-54 because of its higher in vivo potency as compared with those of hormone release via G protein-coupled receptor 54. Proc. Natl. Acad. Sci. USA the other kisspeptin fragments.31,32 102, 1761–1766 (2005). 13. Shahab, M., Mastronardi, C., Seminara, S.B., Crowley, W.F., Ojeda, S.R. & Plant, T.M. Injections. Vials of freeze-dried saline or KP-54 were reconstituted in Increased hypothalamic GPR54 signaling: a potential mechanism for initiation 0.5 ml of 0.9% saline. In the studies involving twice-daily injections, the of puberty in primates. Proc. Natl. Acad. Sci. USA 102, 2129–2134 (2005). subjects were trained to self-administer these at home.25 In the studies 14. caraty, A. et al. Kisspeptin synchronizes preovulatory surges in cyclical involving twice-weekly injections, these were administered to the subjects ewes and causes ovulation in seasonally acyclic ewes. Endocrinology 148, by study investigators during scheduled visits. 5258–5267 (2007). 15. dhillo, W.S. et al. Kisspeptin-54 stimulates the hypothalamic-pituitary gonadal Collection, processing, and analysis of blood samples. Blood samples for axis in human males. J. Clin. Endocrinol. Metab. 90, 6609–6615 (2005). serum analysis were collected in plain serum Vacutainer tubes (Becton 16. dhillo, W.S. et al. Kisspeptin-54 stimulates gonadotropin release most potently Dickinson, Franklin Lakes, NJ). Clotted samples were spun for 10 min during the preovulatory phase of the menstrual cycle in women. J. Clin. Endocrinol. Metab. 92, 3958–3966 (2007). at 3,000 r.p.m. in a Hettich EBA 20 centrifuge (Hettich International, 17. Yen, S.S. Female hypogonadotropic hypogonadism. Hypothalamic Tuttlingen, Germany). Sera were separated and stored at −20 °C until amenorrhea syndrome. Endocrinol. Metab. Clin. North Am. 22, 29–58 (1993). analysis. Levels of LH, FSH, estradiol, and progesterone in the sera 18. Reindollar, R.H., Novak, M., Tho, S.P. & McDonough, P.G. Adult-onset were measured using automated chemiluminescent immunoassays amenorrhea: a study of 262 patients. Am. J. Obstet. Gynecol. 155, 531–543 (Abbott Diagnostics, Maidenhead, UK). Sex hormone–binding globu- (1986). lin was measured using a solid-phase automated enzyme immunoassay 19. Frisch, R.E. & Revelle, R. Height and weight at menarche and a hypothesis of (Immulite; Siemens, Llanberis, UK).25 critical body weights and adolescent events. Science 169, 397–399 (1970). 20. Laughlin, G.A., Dominguez, C.E. & Yen, S.S. Nutritional and endocrine- Data analysis. Data are presented as mean values ± SEM. Hormone pro- metabolic aberrations in women with functional hypothalamic amenorrhea. files during the 4-h blood sample investigations were analyzed using J. Clin. Endocrinol. Metab. 83, 25–32 (1998). repeated measures two-way analysis of variance with Bonferroni post hoc 21. Loucks, A.B., Verdun, M. & Heath, E.M. Low energy availability, not stress of correction. Pairs of means were analyzed using the unpaired two-tailed exercise, alters LH pulsatility in exercising women. J. Appl. Physiol. 84, 37–46 t-test. Multiple means were compared using one-way analysis of variance (1998). 22. Marcus, M.D., Loucks, T.L. & Berga, S.L. Psychological correlates of functional with Bonferroni’s multiple-comparison test. In all cases, P < 0.05 was hypothalamic amenorrhea. Fertil. Steril. 76, 310–316 (2001). considered to be statistically significant. 23. Reame, N.E., Sauder, S.E., Case, G.D., Kelch, R.P. & Marshall, J.C. Pulsatile gonadotropin secretion in women with hypothalamic amenorrhea: evidence Acknowledgments that reduced frequency of gonadotropin-releasing hormone secretion is the C.N.J. is supported by an NIHR Clinical Lectureship. C.N.J. and G.M.K.N. mechanism of persistent anovulation. J. Clin. Endocrinol. Metab. 61, 851–858 are supported by Wellcome Trust Research Training Fellowships. W.S.D. is (1985). supported by a HEFCE Clinical Senior Lecturer Award. This work was funded 24. castellano, J.M. et al. Changes in hypothalamic KiSS-1 system and restoration by an MRC Experimental Medicine project grant. The department is funded of pubertal activation of the reproductive axis by kisspeptin in undernutrition. by an Integrative Mammalian Biology (IMB) Capacity Building Award and Endocrinology 146, 3917–3925 (2005). the NIHR Biomedical Research Centre Funding Scheme. We are grateful 25. Jayasena, C.N. et al. Subcutaneous injection of kisspeptin-54 acutely to the Wellcome Trust and Sir John McMichael Centre for providing the stimulates gonadotropin secretion in women with hypothalamic infrastructure for this study. amenorrhea, but chronic administration causes tachyphylaxis. J. Clin. Endocrinol. Metab. 94, 4315–4323 (2009). Conflict of Interest 26. Seminara, S.B., Dipietro, M.J., Ramaswamy, S., Crowley, W.F. Jr & Plant, T.M. The authors declared no conflict of interest. Continuous human metastin 45-54 infusion desensitizes G protein-coupled receptor 54-induced gonadotropin-releasing hormone release monitored © 2010 American Society for Clinical Pharmacology and Therapeutics indirectly in the juvenile male Rhesus monkey (Macaca mulatta): a finding with therapeutic implications. Endocrinology 147, 2122–2126 (2006). 1. Boivin, J., Bunting, L., Collins, J.A. & Nygren, K.G. International estimates of 27. cho, S.G. et al. Kisspeptin-10, a KISS1-derived decapeptide, inhibits tumor infertility prevalence and treatment-seeking: potential need and demand for angiogenesis by suppressing Sp1-mediated VEGF expression and FAK/Rho infertility medical care. Hum. Reprod. 22, 1506–1512 (2007). GTPase activation. Cancer Res. 69, 7062–7070 (2009). 2. Elchalal, U. & Schenker, J.G. The pathophysiology of ovarian hyperstimulation 28. cho, S.G. et al. KiSS1 suppresses TNFα-induced breast cancer cell invasion via syndrome–views and ideas. Hum. Reprod. 12, 1129–1137 (1997). an inhibition of RhoA-mediated NF-kappaB activation. J. Cell. Biochem. 107, 3. Ohtaki, T. et al. Metastasis suppressor gene KiSS-1 encodes peptide ligand of a 1139–1149 (2009). G-protein-coupled receptor. Nature 411, 613–617 (2001). 29. Roseweir, A.K. et al. Discovery of potent kisspeptin antagonists delineate 4. Muir, A.I. et al. AXOR12, a novel human G protein-coupled receptor, activated physiological mechanisms of gonadotropin regulation. J. Neurosci. 29, by the peptide KiSS-1. J. Biol. Chem. 276, 28969–28975 (2001). 3920–3929 (2009). 5. de Roux, N., Genin, E., Carel, J.C., Matsuda, F., Chaussain, J.L. & Milgrom, E. 30. Yen, S.S., Rebar, R., Vandenberg, G., Ehara, Y. & Siler, T. Pituitary gonadotrophin Hypogonadotropic hypogonadism due to loss of function of the KiSS1-derived responsiveness to synthetic LRF in subjects with normal and abnormal peptide receptor GPR54. Proc. Natl. Acad. Sci. USA 100, 10972–10976 (2003). hypothalamic-pituitary-gonadal axis. J. Reprod. Fertil. Suppl. 20, 137–161 6. Seminara, S.B. et al. The GPR54 gene as a regulator of puberty. N. Engl. J. Med. (1973). 349, 1614–1627 (2003). 31. Thompson, E.L. et al. Chronic subcutaneous administration of kisspeptin-54 7. Teles, M.G. et al. A GPR54-activating mutation in a patient with central causes testicular degeneration in adult male rats. Am. J. Physiol. Endocrinol. precocious puberty. N. Engl. J. Med. 358, 709–715 (2008). Metab. 291, E1074–E1082 (2006). 8. Irwig, M.S. et al. Kisspeptin activation of gonadotropin releasing hormone 32. Tovar, S. et al. Effects of single or repeated intravenous administration of neurons and regulation of KiSS-1 mRNA in the male rat. Neuroendocrinology kisspeptin upon dynamic LH secretion in conscious male rats. Endocrinology 80, 264–272 (2004). 147, 2696–2704 (2006).

Clinical pharmacology & Therapeutics | VOLUME 88 NUMBER 6 | december 2010 847 JCEM ONLINE

Hot Topics in Translational Endocrinology—Endocrine Research

The Effects of Kisspeptin-10 on Reproductive Hormone Release Show Sexual Dimorphism in Humans

Channa N. Jayasena, Gurjinder M. K. Nijher, Alexander N. Comninos, Ali Abbara, Adam Januszewki, Meriel L. Vaal, Labosshy Sriskandarajah, Kevin G. Murphy, Zohreh Farzad, Mohammad A. Ghatei, Stephen R. Bloom, and Waljit S. Dhillo

Section of Investigative Medicine, Imperial College London, Hammersmith Hospital, London W12 ONN, United Kingdom

Background: Kisspeptin peptides are critical in human reproductive physiology and are potential therapies for infertility. Kisspeptin-10 stimulates gonadotropin release in both male and female rodents. However, few studies have investigated the effects of kisspeptin-10 on gonadotropin release in humans, and none have investigated the effect in women. If kisspeptin is to be useful for treating reproductive disease, its effects in both men and women must be established.

Aim: To compare the effects of kisspeptin-10 administration on reproductive hormone release in healthy men and women.

Methods: Intravenous bolus kisspeptin-10 was administered to men and women (n ϭ 4–5 per group). Subcutaneous bolus and iv infusion of kisspeptin-10 was also administered to female women (n ϭ 4–5 per group). Circulating reproductive hormones were measured.

Results: In healthy men, serum LH and FSH were elevated after iv bolus kisspeptin-10, at doses as low as 0.3 and 1.0 nmol/kg, respectively. In healthy women during the follicular phase of the menstrual cycle, no alterations in serum gonadotropins were observed after iv bolus, sc bolus, or iv infusion of kisspeptin-10 at maximal doses of 10 nmol/kg, 32 nmol/kg, and 720pmol/kg/min, respectively. In women during the preovulatory phase, serum LH and FSH were elevated after iv bolus kisspeptin-10 (10 nmol/kg).

Conclusion: Kisspeptin-10 stimulates gonadotropin release in men as well as women during the preovulatory phase of menstrual cycle but fails to stimulate gonadotropin release in women during the follicular phase. The sexual dimorphism of the responsiveness of healthy men and women to kisspeptin-10 administration has important clinical implications for the potential of kisspeptin-10 to treat disorders of reproduction. (J Clin Endocrinol Metab 96: E1963–E1972, 2011)

he kisspeptins are critical regulators of mammalian re- Central or peripheral administration of the shorter kiss- T productive physiology (1, 2). In humans, inactivating peptin peptide, kisspeptin-10, has been demonstrated to mutations of the kisspeptin receptor (KISS1R) cause puber- stimulate gonadotropin release in several mammalian spe- tal failure (3, 4), and activating mutations can lead to pre- cies (8–18). This effect is abolished by preadministration cocious puberty (5). The human kisspeptin peptides, kiss- of an antagonist to the hypothalamic hormone GnRH (10, peptin-10, -13, -14, and -54, are named according to their 15). Kisspeptin-10 is therefore thought to stimulate the number of constituent amino acids (6). All kisspeptin pep- release of GnRH from the hypothalamus, which then stim- tides share the C-terminal decapeptide sequence, kisspeptin- ulates gonadotropin release from the pituitary gland. The 10, which is required for biological activity in vitro (7). longer kisspeptin peptide, kisspeptin-54, has also been

ISSN Print 0021-972X ISSN Online 1945-7197 Abbreviations: AUC, Area under the curve; IR, immunoreactivity. Printed in U.S.A. Copyright © 2011 by The Endocrine Society doi: 10.1210/jc.2011-1408 Received May 3, 2011. Accepted September 13, 2011. First Published Online October 5, 2011

J Clin Endocrinol Metab, December 2011, 96(12):E1963–E1972 jcem.endojournals.org E1963 E1964 Jayasena et al. Kisspeptin-10 in Men and Women J Clin Endocrinol Metab, December 2011, 96(12):E1963–E1972 shown to stimulate gonadotropin release in rodents (19– Study 2: effects of iv bolus injection of kisspeptin- 21). In addition, administration of kisspeptin-54 stimu- 10 or kisspeptin-54 in healthy female volunteers lates gonadotropin secretion in humans (22–25). Kisspep- Follicular phase of the menstrual cycle tin therefore has the potential to become a novel therapy Women between d 2–10 of their menstrual cycle were ad- for treatment of reproductive disorders in humans. ministered an iv bolus injection of 0.9% saline, kisspeptin-54 The shorter amino acid sequence of kisspeptin-10 (1.0 nmol/kg), or kisspeptin-10 (at doses of 1.0, 3.0, or 10 nmol/ Ϫ makes it simpler and cheaper to synthesize than kisspep- kg) at time 0 min, and blood samples were taken at 30, 0, 10, 20, 30, 40, 50, 60, 75, 90, 120, 150, and 180 min (n ϭ 4–5 per tin-54. Future kisspeptin therapies may therefore be based group). upon kisspeptin-10 rather than kisspeptin-54. It is therefore therapeutically important to determine whether kiss- Preovulatory phase of the menstrual cycle peptin-10 can stimulate reproductive hormone release Women 15–16 d before their next predicted period received in healthy men and women. Kisspeptin-10 stimulates iv bolus injection of 10 nmol/kg kisspeptin-10 as described for the follicular phase (n ϭ 5). gonadotropin release in male rhesus monkeys (13–15). Furthermore, two recent reports have suggested that Study 3: effects of sc bolus injection of saline or kisspeptin-10 administration to healthy men stimulates kisspeptin-10 in healthy female volunteers in the gonadotropin release (26, 27). Although kisspeptin-10 follicular phase of the menstrual cycle is known to stimulate gonadotropin release in animals Kisspeptin-10 (at doses 2, 4, 8, 16, or 32 nmol/kg) or 0.9% (8, 9, 12, 17), there are no published data examining the saline was administered sc at time 0 min, and blood samples were Ϫ effects of administering kisspeptin-10 to female pri- taken at 30, 0, 15, 30, 45, 60, 75, 90, 120, 150, 180, 210, and 240 min (n ϭ 4–5 per group). mates or humans. This study aimed to determine the effects of kisspep- Study 4: effects of iv infusion of saline or tin-10 administration on reproductive hormone release in kisspeptin-10 in healthy female volunteers in the healthy men and, for the first time, in healthy women. follicular phase of the menstrual cycle Kisspeptin-10 was dissolved in saline containing gelofusine (5% vol/vol) (B. Braun Medical, Sheffield, UK) and was infused Subjects and Methods iv over 90 min. During the first 30 min of infusion, the volunteers were administered 20, 50, 90, 180, 360, or 720 pmol/kg/min. The infusion rate for each volunteer was then halved for the Subjects remaining 60 min of each infusion. Blood samples were taken at The study was conducted with Ethics Committee approval Ϫ30, 0, 15, 30, 45, 60, 75, 90, 120, 150, 180, 210, and 240 min. (reference 08/H0707/95) in accordance with The Declaration of Helsinki. Written informed consent was obtained from all sub- Study 5: determining the half-life of kisspeptin-10 jects. Thirty-five healthy female subjects and 11 healthy male in healthy female and male volunteers subjects were recruited, using criteria summarized in Supple- To determine the plasma half-life of kisspeptin-10 in men, mental Table 1 (published on The Endocrine Society’s Journals and in women during the follicular and preovulatory phases of Online web site at http://jcem.endojournals.org). the menstrual cycle, frequent blood sampling was performed during iv infusion of 360 pmol/kg/min kisspeptin-10. The pro- Study days tocol was identical to that used in study 4; except that detailed Subjects were admitted to our Clinical Investigation Unit and blood sampling was performed at 1-min (from 91–100 min) and asked to lay supine for the duration of each study. Urine was 2-min (from 102–120 min) intervals immediately after stopping tested to exclude pregnancy in women (Clearview Easy-HCG; kisspeptin-10 infusion. Blood samples were assayed for plasma Inverness Medical Innovations Inc., Waltham, MA). All blood kisspeptin IR. The decay curve of kisspeptin IR was used to samples were analyzed for measurement of serum LH, FSH, es- calculate the half-time of disappearance (t1/2) for infused kiss- tradiol (in women) or testosterone (in men), and plasma kisspeptin-10 as described previously (22). peptin immunoreactivity (IR). Heart rate, blood pressure, and the presence of adverse symptoms were recorded at regular Data analysis intervals. Data are presented as mean Ϯ SEM. Time profiles of hormone levels were compared using two-way ANOVA with Bonferroni’s Study 1: effects of iv bolus injection of saline or multiple-comparison test. Pairs of means were compared with unpaired t tests (or Mann-Whitney U test if nonparametric), and kisspeptin-10 in healthy male volunteers multiple means of area under curve (AUC) reproductive hor- Intravenous bolus injection of 0.9% saline or kisspeptin-10 mone release were compared using one-way ANOVA with Bon- (at doses of 0.3, 1.0, 3.0, or 10 nmol/kg) was administered at time feronni’s multiple-comparison test (or Kruskall-Wallis with 0 min. Blood samples were taken at Ϫ30, 0, 10, 20, 30, 40, 50, Dunn’s multiple-comparison tests if nonparametric). Slopes of 60, 75, 90, 120, 150, and 180 min (n ϭ 4–5 per group). linear regression curves were compared using an F test. In all J Clin Endocrinol Metab, December 2011, 96(12):E1963–E1972 jcem.endojournals.org E1965 cases, P Ͻ 0.05 was considered statistically significant. All data nificantly increased compared with saline injection after iv of serum reproductive hormones during treatment are presented bolus injection of 1.0 or 3.0 nmol/kg kisspeptin-10 (Fig. 1, as increases in serum levels after injection when compared with C and G). Serum testosterone was significantly in- preinjection levels. creased compared with saline injection after iv bolus injection of 0.3 or 1.0 nmol/kg kisspeptin-10 (Fig. 1, D Results and H). Serum levels of testosterone at these doses steadily increased to peak levels 150–180 min after Baseline characteristics of the subjects recruited to the injection (Fig. 1D). study are summarized in Table 1. Study 2: effects of iv bolus injection of saline or Study 1: effects of iv bolus injection of saline or kisspeptin-10 or kisspeptin-54 in healthy female kisspeptin-10 in healthy male volunteers volunteers Plasma kisspeptin IR was elevated after iv bolus injec- Follicular phase of the menstrual cycle tion of kisspeptin-10 at all doses in healthy male volunteers (Fig. 1, A and E). The highest plasma kisspeptin IR Plasma kisspeptin IR was elevated after iv bolus injec- was observed after 10 nmol/kg kisspeptin-10 (mean AUC tion of kisspeptin-10 at all doses in healthy female volun- kisspeptin IR was 700 Ϯ 160 h⅐pmol/liter, P Ͻ 0.001 vs. teers during the follicular phase of menstrual cycle (Fig. 2, saline). At this dose, mean peak kisspeptin IR (3350 Ϯ 725 A and E). The highest plasma kisspeptin IR was observed pmol/liter) was observed 10 min after injection, and after iv bolus injection of 10 nmol/kg kisspeptin-10 (mean plasma kisspeptin IR returned to undetectable levels 50 AUC kisspeptin IR in women during follicular phase was min after injection. Serum LH was elevated significantly 527 Ϯ 108 h⅐pmol/liter, P Ͻ 0.001 vs. saline); this was after administration of each tested dose of iv bolus kiss- lower when compared with kisspeptin IR after injection of peptin-10 injection compared with saline (Fig. 1, B and F). the same dose of kisspeptin-10 to men, but this difference Peak stimulation of serum LH was observed 30–40 min was not significant (P ϭ 0.42 vs. men). At this dose, mean after injection, and levels of serum LH gradually returned peak kisspeptin IR (2638 Ϯ 302 pmol/liter) was observed to baseline 180 min after injection (Fig. 1B). Maximal 10 min after injection, and plasma kisspeptin IR returned stimulation of LH was observed after iv bolus 10 nmol/kg to undetectable levels 50 min after injection. Gonadotro- kisspeptin-10 (mean AUC LH increase was 6.1 Ϯ 1.3 h⅐IU/ pin release was elevated significantly after iv bolus injec- liter, P Ͻ 0.001 vs. saline) (Fig. 1F). Serum FSH was sig- tion of 1 nmol/kg kisspeptin-54 [mean AUC increase (in h⅐IU/liter): 27.0 Ϯ 11.8 (LH), P Ͻ 0.05 vs. saline, and 7.9 Ϯ 4.1 (FSH), P Ͻ 0.05 vs. saline). Unexpectedly, no TABLE 1. Baseline characteristics of healthy male and significant changes in serum levels of reproductive hor- female volunteers administered kisspeptin-10 mones were observed after iv bolus injection of kisspep- Healthy male Healthy female tin-10 at doses up to 10 nmol/kg (Fig. 2, B–D and F–H). Baseline volunteers volunteers Preovulatory phase of the menstrual cycle (35 ؍ n) (11 ؍ characteristic (n Ϯ Ϯ Age (yr) 28.8 2.1 30.4 0.19 Kisspeptin IR was elevated significantly in women dur- BMI (kg/m2) 24.5 Ϯ 0.5 22.9 Ϯ 0.1 Length of menstrual 27 Ϯ 0.1 ing the preovulatory phase after kisspeptin-10 injection cycle (d) compared with saline, and this elevation was nonsignifi- LH (IU/liter) 2.9 Ϯ 0.2 cantly different when compared with kisspeptin IR after Follicular 3.8 Ϯ 0.6 Preovulatory 6.8 Ϯ 1.6a the same dose of kisspeptin-10 in follicular-phase women FSH (IU/liter) 2.6 Ϯ 0.2 or men (mean AUC kisspeptin IR in preovulatory phase Follicular 3.7 Ϯ 0.4 was 320 Ϯ 56 h⅐pmol/liter, P ϭ 0.13 vs. follicular phase, Ϯ b Preovulatory 6.8 1.6 ϭ Testosterone 21.6 Ϯ 1.5 and P 0.06 vs. men) (Fig. 2E). Serum LH and FSH were (nmol/liter) elevated significantly after iv bolus injection of 10 nmol/kg Estradiol (pmol/liter) kisspeptin-10 in women during the preovulatory phase of Follicular 256 Ϯ 43 Ϯ Preovulatory 562 Ϯ 195a the menstrual cycle [mean AUC increase was 30.3 7.7 h⅐IU/liter (LH), P Ͻ 0.05 vs. saline, and 6.9 Ϯ 0.9 h⅐IU/liter Female endocrine profiles are presented during the follicular and Ͻ preovulatory phases of the menstrual cycle. Data are shown as (FSH), P 0.01 vs. saline] (Fig. 2, B, C, F, and G); how- mean Ϯ SEM. ever, serum estradiol was not altered significantly (mean a P Ͻ 0.05 vs. follicular phase of the menstrual cycle. AUC estradiol increase was 111 Ϯ 96 h⅐pmol/liter, P ϭ b P Ͻ 0.01 vs. follicular phase of the menstrual cycle. 0.14 vs. saline) (Fig. 2, D and H). E1966 Jayasena et al. Kisspeptin-10 in Men and Women J Clin Endocrinol Metab, December 2011, 96(12):E1963–E1972

Study 4: effects of iv infusion of kisspeptin-10 in healthy female volunteers in the follicular phase of the menstrual cycle Intravenous infusion of peptides delivers the peptide directly into the circulation, avoiding possible degradation in sc tissue, and results in a sustained increase in circulating levels of the administered peptide. Plasma kisspeptin IR increased during iv infusion of kisspeptin-10 at all doses (Fig. 4A). The highest plasma kisspeptin IR was observed during iv infusion of 720 pmol/kg/min kisspeptin-10 (mean AUC kisspeptin IR was 2518 Ϯ 100 h⅐pmol/liter). All iv infusion doses of kisspeptin-10 were associated with a higher mean plasma kisspeptin IR than the highest studied sc dose of kisspeptin-10 (32 nmol/kg, mean AUC kisspeptin IR was 201 Ϯ 16 h⅐pmol/liter). No significant changes in serum reproductive hormone levels were observed after iv infusion of any dose of kisspeptin-10 in healthy female volunteers in the follicular phase of the menstrual cycle (Fig. 4, B–H).

Study 5: pharmacokinetic profile of kisspeptin IR during iv infusion FIG. 1. Plasma kisspeptin IR and serum reproductive hormone levels after iv bolus injection of of kisspeptin-10 in healthy male kisspeptin-10 to healthy male volunteers. A–D, Time profiles for plasma kisspeptin IR (A) and and female volunteers changes in serum LH (B), FSH (C), and testosterone (D) during 4 h after iv bolus injection of To determine the plasma half-life of saline or kisspeptin-10 to healthy male volunteers (n ϭ 4–5 per group). For 10 nmol/kg vs. saline: ␺, P Ͻ 0.05; ␺␺, P Ͻ 0.01; ␺␺␺, P Ͻ 0.001. For 3 nmol/kg vs. saline: ␭, P Ͻ 0.05; ␭␭␭, kisspeptin-10, frequent blood sampling P Ͻ 0.001. For 1 nmol/kg vs. saline: ⌬, P Ͻ 0.05; ⌬⌬⌬, P Ͻ 0.001. E–H, AUC for plasma was performed in women during the kisspeptin IR (E) and changes in serum LH (F), FSH (G), and testosterone (H) during 4 h after iv follicular and preovulatory phases of bolus injection of saline or kisspeptin-10 to healthy male volunteers (n ϭ 4–5 per group). *, P Ͻ 0.05; **, P Ͻ 0.01; ***, P Ͻ 0.001. T, Testosterone. Data are shown as mean Ϯ SEM. menstrual cycle, and in men, after cessation of an iv infusion of 360 pmol/ kg/min kisspeptin-10 (Fig. 5). When Study 3: effects of sc bolus injection of saline or plotted on a natural log scale, linear regression slopes were kisspeptin-10 in healthy female volunteers in the not significantly different among the three groups (F ϭ follicular phase of the menstrual cycle 0.099; degrees of Freedom numerator ϭ 2; degrees of Free- Plasma kisspeptin IR was significantly elevated during dom denominator ϭ 111; P ϭ 0.91), and plasma half-lives the 4 h after sc injection of kisspeptin-10 at doses of 4 of kisspeptin-10 were calculated as 3.8 Ϯ 0.3 min (men), nmol/kg and higher, when compared with saline (Fig. 3A). 4.1 Ϯ 0.4 min (follicular-phase women), and 4.1 Ϯ 0.4 The highest plasma kisspeptin IR after sc injection was min (preovulatory-phase women) (Fig. 5E). observed after injection of 32 nmol/kg kisspeptin-10 (mean AUC kisspeptin IR was 201 Ϯ 16 h⅐pmol/liter, P Ͻ 0.001 vs. saline). Discussion No significant changes in serum reproductive hormone levels were observed after sc bolus injection of kisspep- These studies reveal a previously unknown sexual dimor- tin-10 at any dose (Fig. 3, B–H). phism in responsiveness to kisspeptin-10 administration J Clin Endocrinol Metab, December 2011, 96(12):E1963–E1972 jcem.endojournals.org E1967

tested. These results are consistent with our previous observation that administration of the longer form of kisspeptin, kisspeptin-54, stimulates reproductive hormone release in men (22). Further- more, two recent reports have shown that iv injection of kisspeptin-10 potently stimulates LH release in healthy male volunteers at doses similar to those used in this study (26, 27) and that prolonged infusion of kisspeptin-10 significantly stimulates LH pulsatility in healthy male volunteers (27). Studies in rodents suggest that kisspeptin-10 stimulates gonadotropin release in both males and females (8, 9). Furthermore, we observed that the kisspeptin-10 peptide used in this study stimulated LH release in female adult mice (Supplemental Fig. 1), although with a much lower potency when compared with kisspeptin-54. We were therefore surprised to observe that doses of kisspeptin-10 identical to those used in men failed to stimulate reproductive hormone release in healthy female volunteers during the follicular phase of the menstrual cycle when administered as an iv bolus injection. It is possible that the lack of effect of iv administration of kisspeptin-10 to stimulate reproductive hor- FIG. 2. Plasma kisspeptin IR and serum reproductive hormone levels after iv bolus injection of kisspeptin-10 to healthy female volunteers. A–D, Time profiles for plasma kisspeptin IR (A) mone release in women in the follicular and changes in serum LH (B), FSH (C), and estradiol (D) during 4 h after iv bolus injection of phase of their menstrual cycle was due saline, kisspeptin-10 (KP10), or kisspeptin-54 (KP54) to healthy female volunteers. For 10 to the short period of time that circu- nmol/kg KP10 vs. saline: ␺, P Ͻ 0.05; ␺␺, P Ͻ 0.01; ␺␺␺, P Ͻ 0.001. For 3 nmol/kg KP10 vs. saline: ␭␭␭, P Ͻ 0.001. For 1 nmol/kg KP54 vs. saline: ␬, P Ͻ 0.05; ␬␬, P Ͻ 0.01; ␬␬␬, P Ͻ lating kisspeptin-10 levels were ele- 0.001. For 10 nmol/kg KP10 (Preov) vs. saline: *, P Ͻ 0.05; **, P Ͻ 0.01; ***, P Ͻ 0.001. vated after iv bolus administration. E–H, AUC for plasma kisspeptin IR (E) and changes in serum LH (F), FSH (G), and estradiol (H) Subcutaneous bolus administration of during 4 h after iv bolus injection of saline or kisspeptin-10 to healthy female volunteers. kisspeptin-10 is likely to result in a lon- *, P Ͻ 0.05; **, P Ͻ 0.01; ***, P Ͻ 0.001. Data are shown as mean Ϯ SEM. E2, Estradiol; Preov, preovulatory phase of the menstrual cycle. ger period of elevated circulating levels of kisspeptin-10 than iv bolus administration (22, 23). Furthermore, we in healthy men and women. Numerous studies in animals have previously determined that sc bolus injection of kiss- have demonstrated that kisspeptin-10 robustly stimulates peptin-54 at doses as low as 0.4 nmol/kg potently stimu- gonadotropin release, thus implicating kisspeptin signaling lates serum LH in healthy women during the follicular as a potential therapeutic target for treating female patients with infertility (8, 9, 12, 17). Kisspepin-10 stimulates gonad- phase of the menstrual cycle (23). However, serum go- otropin release in male rhesus monkeys (13–15), and recent nadotropin levels were not elevated by sc bolus injection reports have suggested that kisspepeptin-10 administration of kisspeptin-10 in healthy women in the follicular phase stimulates gonadotropin release in healthy men (26, 27). of the menstrual cycle, despite elevations in plasma kiss- However, the effects of administration of kisspeptin-10 in peptin IR for up to 90 min after injection. female primates or women have not previously been studied. The lack of effect of sc bolus administration of kiss- We observed that iv bolus injection of kisspeptin-10 peptin-10 to stimulate reproductive hormone release in robustly stimulated LH release in healthy men at all doses women in the follicular phase of their menstrual cycle may E1968 Jayasena et al. Kisspeptin-10 in Men and Women J Clin Endocrinol Metab, December 2011, 96(12):E1963–E1972

the follicular phase of the menstrual cycle, despite markedly raised plasma levels of kisspeptin IR up to concentrations of 2000 pmol/liter. Our data therefore suggest that women in the follicular phase of the menstrual cycle are markedly less responsive to kisspeptin-10 administration than men. The underlying mechanism for this observation is unclear. However, sexual dimorphism of hypothalamic kisspeptin signaling pathways has been demonstrated in rodents (28–30). It is important to consider whether the contrasting effects of kisspeptin-10 in men and women may have been attributable to factors other than differential sensitivity to the peptide. We observed that levels of plasma kisspeptin IR appeared slightly lower in women when compared with men when identical weight-adjusted doses of kisspeptin-10 were administered. It is possible that levels of kisspeptin-10 may have been modified by factors known to differ between the sexes, such as body fat content or clearance of peptide from the circulation (19). However, it is note- worthy that only a marginal elevation of plasma kisspeptin IR (approximately 10 h⅐pmol/liter) was necessary to stimulate significant LH secretion in men after injection of kisspeptin-10 (0.3 nmol/kg iv bolus); by contrast, a 200- fold greater elevation in plasma kisspeptin IR failed to stimulate LH release FIG. 3. Plasma kisspeptin IR and serum reproductive hormone levels after sc bolus injection of kisspeptin-10 to healthy female volunteers in the follicular phase of the menstrual cycle. in women during the follicular phase of A–D, Time profiles for plasma kisspeptin IR (A) and changes in serum LH (B), FSH (C), and the menstrual cycle. Furthermore, we estradiol (D) during 4 h after sc bolus injection of kisspeptin-10 to healthy female volunteers observed no significant differences in in the follicular phase of the menstrual cycle (n ϭ 4–5 per group). For 32 vs. 2 nmol/kg: *, P Ͻ 0.05; ***, P Ͻ 0.001. For 16 vs. 2 nmol/kg: ƒƒƒ, P Ͻ 0.001. For 8 vs. 2 nmol/kg: ␺␺␺, the plasma half-lives of kisspeptin-10 P Ͻ 0.001. For 4 vs. 2 nmol/kg: ␭, P Ͻ 0.05. E–H, AUC for plasma kisspeptin IR (E) and between men and women. It is therefore changes in serum LH (F), FSH (G), and testosterone (H) during 4 h after sc bolus injection of unlikely that the striking contrast be- ϭ kisspeptin-10 to healthy female volunteers in the follicular phase of the menstrual cycle (n tween male and female responsiveness 4–5 per group). *, P Ͻ 0.05; **, P Ͻ 0.01; ***, P Ͻ 0.001. E2, Estradiol. Data are shown as mean Ϯ SEM. to kisspeptin-10 administration reflects differences in metabolism of kisspeptin-10. We observed nonsignificant re- have been due to breakdown of kisspeptin-10 in the sc ductions in serum LH (P ϭ 0.19) and FSH (P ϭ 0.91) after tissue. Intravenous infusion of peptides delivers the pep- saline injection in men when compared with saline injec- tide directly into the circulation, avoiding possible degradation in sc tissue, and results in a sustained increase in tion in women. Although both men and women under- circulating levels of the administered peptide. However, went identical study protocols, it is possible that minor serum gonadotropin levels were not elevated during iv differences in factors inhibiting reproductive function infusion of kisspeptin-10 to healthy female volunteers in such as stress may be greater in the male group. However, J Clin Endocrinol Metab, December 2011, 96(12):E1963–E1972 jcem.endojournals.org E1969

kisspeptin-54 when compared with kisspeptin-10 may explain why exogenous kisspeptin-54 injection (but not exogenous kisspeptin-10) stimulates reproductive hormone release in women during the follicular phase of the menstrual cycle. The differences between kisspeptin-10 and kisspeptin-54 may be a consequence of the rapid breakdown of kisspeptin-10 in the circulation. The in vivo plasma half-life of iv kisspeptin-10 in this study was calculated to approximately 4 min, which is 7-fold shorter than the calculated in vivo plasma half-life of kisspeptin-54 (22). It is interesting to consider whether men and women have differential responses to kisspeptin-54 (as they appear to be to kisspeptin-10) based on previous studies. Intravenous infusion of kisspeptin-54 at doses as low as 1.2 nmol (0.25 pmol/kg/min) stimulate LH secretion in healthy men (22). By comparison, sc bolus injection of kisspeptin-54 at doses of 28 nmol (0.4 nmol/ kg) or more stimulate LH secretion in healthy women during the follicular phase of the menstrual cycle (23); furthermore, this study suggests that iv bo- FIG. 4. Plasma kisspeptin IR and serum reproductive hormone levels during iv infusion of kisspeptin-10 to healthy female volunteers in the follicular phase of the menstrual cycle. A–D, lus injection of approximately 60 nmol Time profiles for plasma kisspeptin IR (A) and changes in serum LH (B), FSH (C), and estrogen (1 nmol/kg) kisspeptin-54 stimulates sig- (D) during 4 h after commencement of a 90-min iv infusion of kisspeptin-10 to healthy female nificant LH secretion in these women. volunteers in the follicular phase of the menstrual cycle (n ϭ 4–5 per group). For 720 vs. 20 pmol/kg ⅐ min: ***, P Ͻ 0.001. For 360 vs. 20 pmol/kg ⅐ min: ƒƒƒ, P Ͻ 0.001. For 180 vs. 20 These data demonstrate that, unlike pmol/kg ⅐ min: ␺, P Ͻ 0.05; ␺␺␺, P Ͻ 0.001. For 50 vs. 20 pmol/kg ⅐ min: ⌬, P Ͻ 0.05. E–H, kisspeptin-10, kisspeptin-54 can stim- AUC for plasma kisspeptin IR (E) and changes in serum LH (F), FSH (G), and testosterone (H) ulate reproductive hormone release in during 4 h after commencement of a 90-min iv infusion of kisspeptin-10 to healthy female volunteers in the follicular phase of the menstrual cycle (n ϭ 4–5 per group). **, P Ͻ 0.01; women in the follicular phase of their ***, P Ͻ 0.001. E2, Estradiol. Data are shown as mean Ϯ SEM. menstrual cycle. In the current study, gonadotropin responses during kisspeptin-10 infu- such a difference would not explain why men are more sion appeared more variable than after iv or sc bolus in- responsive to kisspeptin-10 than women in the follicular jections. These alterations in gonadotropin secretion did phase of the menstrual cycle. not appear to be related to either the dose or to the onset In the current study, we observed that exogenous kiss- of kisspeptin infusion; however, it is possible that subtle peptin-54 stimulated significant gonadotropin secretion in women during the follicular phase of the menstrual effects of kisspeptin-10 infusion on gonadotropin release cycle, despite a failure of exogenous kisspeptin-10 to stim- were not detected in the current study protocol. ulate gonadotropin secretion at a 10-fold higher molar We have previously demonstrated that women in the dose. Furthermore, kisspeptin-54 injection stimulated LH preovulatory phase of the menstrual cycle are significantly in female mice more potently when compared with kiss- more sensitive to the effects of kisspeptin-54 on gonado- peptin-10, and it has been previously observed that kiss- tropin release when compared with women in the follic- peptin-52 (31) and kisspeptin-54 (21) stimulate LH more ular phase of the menstrual cycle (23). In keeping with potently when compared with kisspeptin-10 in male rats. these observations, iv bolus injection of kisspeptin-10 sig- Collectively, these data suggest that the greater potency of nificantly stimulated LH and FSH release in women dur- E1970 Jayasena et al. Kisspeptin-10 in Men and Women J Clin Endocrinol Metab, December 2011, 96(12):E1963–E1972

secretion (30 min after injection); this phenomenon was also observed after sc bolus injection of kisspeptin-54 in healthy women (23). Our results therefore suggest that both kisspeptin-10 and -54 stimulate LH secretion more potently and more rapidly than FSH. It is interesting to consider why we did not observe any consistent stimulation of testosterone secretion after iv bolus kisspeptin-10 injection in healthy men when compared with the robust increases in serum LH and FSH observed at all tested doses. Significant increases in serum testosterone were observed only at 0.3 and 1.0 nmol/kg iv bolus kisspeptin-10, but these rises were marginal (no more than 10 h⅐nmol/liter above baseline). Furthermore, iv bolus injection of kisspeptin-10 stimulated go- FIG. 5. Detailed time profiles of plasma kisspeptin IR after iv infusion of kisspeptin-10 to nadotropin release in women during healthy male and female volunteers. A–D, Blood sampling was performed for measurement the follicular phase of the menstrual cy- of plasma kisspeptin IR during 4 h after commencement of a 90-min iv infusion of kisspeptin- cle but did not increase serum estradiol 10 (360 pmol/kg ⅐ min) to healthy male volunteers (A and D) and female volunteers during the follicular (B and D) and preovulatory (C and D) phases of the menstrual cycle (n ϭ 4–5 per during 3 h after injection. Our previous group). For men vs. women during follicular phase: ␾␾␾, P Ͻ 0.001; ␾, P Ͻ 0.05. For men vs. data suggest that at least 4 h are re- women during preovulatory phase: ***, P Ͻ 0.001; *, P Ͻ 0.05. E, Blood sampling was quired for serum levels of sex steroids performed at 1-min intervals immediately after stopping infusion of kisspeptin-10. Linear curve fits of natural log plasma kisspeptin IR (dotted lines) were similar for all three groups to peak after a sc bolus injection of (P ϭ 0.91). Data are shown as mean Ϯ SEM. kisspeptin-54 (23–25). A longer period of blood sampling after injection may have revealed more pronounced ing the preovulatory phase of the menstrual cycle in the alterations in sex steroid secretion in subjects after injec- current study. Because we observed that kisspeptin-10 has tion of kisspeptin-10. We observed that serum gonado- a similar pharmacokinetic profile in both follicular and tropins almost returned to baseline levels within 3 h after preovulatory phases of the menstrual cycle, our results iv bolus injection of kisspeptin-10; it is possible that iv suggest that women have heightened sensitivity to kiss- bolus injection of kisspeptin-10 has a duration of action peptin-10 during the preovulatory phase of the menstrual inadequate to stimulate significant gonadal sex steroid cycle. In female rodents, c-fos expression within kisspeptin release. neurons and levels of kiss1 expression are increased within the anteroventral periventricular nucleus of the hypothal- In the current study, all tested iv bolus doses of kiss- amus immediately before ovulation (35). Furthermore, peptin-10 (0.3–10 nmol/kg) were associated with similar levels of kiss1r expression are increased in rat hypotha- degrees of gonadotropin secretion in healthy male sub- lamic fragments at diestrus when compared with proestrus jects. A recent study by George et al. (27) suggests that (36). It is therefore possible that women in the preovula- kisspeptin-10 stimulates serum LH secretion at doses as ␮ tory phase of the menstrual cycle have heightened respon- low as 0.01 g/kg (equivalent to 0.008 nmol/kg), with a ␮ siveness to kisspeptin-10 administration when compared maximal response seen at 1 g/kg (0.8 nmol/kg) in men. with women during the follicular phase of the menstrual Taking these data into consideration, all doses of kisspep- cycle. tin-10 selected during our study may have stimulated near- In the current study, we observed that kisspeptin-10 maximal levels of gonadotropin secretion in healthy male stimulated LH secretion more potently than FSH. This is volunteers. Interestingly, George et al. (27) observed that consistent with our previous studies of kisspeptin-54 ad- the increase in serum LH at 3 ␮g/kg (2.4 nmol/kg) was ministration in healthy men and women. After iv bolus lower than the increase in serum LH at 1 ␮g/kg, which they kisspeptin-10 injection, peak FSH secretion was observed speculated was attributable to tachyphylaxis, a phenom- 45–150 min after injection, which was later than peak LH enon that we have previously observed after chronic kiss- J Clin Endocrinol Metab, December 2011, 96(12):E1963–E1972 jcem.endojournals.org E1971 peptin-54 injections (24, 25, 27). We therefore cannot P, Steplewski K, Shabon U, Miller JE, Middleton SE, Darker JG, exclude that the similarity between LH responses at doses Larminie CG, Wilson S, Bergsma DJ, Emson P, Faull R, Philpott KL, Harrison DC 2001 AXOR12, a novel human G protein-coupled between 0.3 and 10 nmol/kg kisspeptin-10 in men might receptor, activated by the peptide KiSS-1. J Biol Chem 276:28969– be explained in part by tachyphylaxis to kisspeptin-10 at 28975 the higher tested doses. Additional studies are required to 3. de Roux N, Genin E, Carel JC, Matsuda F, Chaussain JL, Milgrom E 2003 Hypogonadotropic hypogonadism due to loss of function of investigate these observations. the KiSS1-derived peptide receptor GPR54. Proc Natl Acad Sci USA In summary, this is the first clinical study to report the 100:10972–10976 effects of kisspeptin-10 administration on gonadotropin 4. Seminara SB, Messager S, Chatzidaki EE, Thresher RR, Acierno Jr JS, Shagoury JK, Bo-Abbas Y, Kuohung W, Schwinof KM, Hendrick release in women and to compare the effects of kisspep- AG, Zahn D, Dixon J, Kaiser UB, Slaugenhaupt SA, Gusella JF, tin-10 between men and women. Kisspeptin-10 robustly O’Rahilly S, Carlton MB, Crowley Jr WF, Aparicio SA, Colledge stimulates gonadotropin release in men but fails to stim- WH 2003 The GPR54 gene as a regulator of puberty. N Engl J Med ulate gonadotropin release in healthy female volunteers in 349:1614–1627 5. Teles MG, Bianco SD, Brito VN, Trarbach EB, Kuohung W, Xu S, the follicular phase of the menstrual cycle when adminis- Seminara SB, Mendonca BB, Kaiser UB, Latronico AC 2008 A tered by iv bolus injection, sc bolus injection, or iv infu- GPR54-activating mutation in a patient with central precocious pu- sion. These experiments reveal sexual dimorphism in the berty. N Engl J Med 358:709–715 6. Bilban M, Ghaffari-Tabrizi N, Hintermann E, Bauer S, Molzer S, responsiveness of healthy human volunteers to kisspep- Zoratti C, Malli R, Sharabi A, Hiden U, Graier W, Kno¨ fler M, tin-10 administration. These findings have important clin- Andreae F, Wagner O, Quaranta V, Desoye G 2004 Kisspeptin-10, ical implications for the potential therapeutic use of kiss- a KiSS-1/metastin-derived decapeptide, is a physiological invasion inhibitor of primary human trophoblasts. J Cell Sci 117:1319–1328 peptin-10 to treat disorders of reproduction. 7. Kotani M, Detheux M, Vandenbogaerde A, Communi D, Vander- winden JM, Le Poul E, Bre´zillon S, Tyldesley R, Suarez-Huerta N, Vandeput F, Blanpain C, Schiffmann SN, Vassart G, Parmentier M 2001 The metastasis suppressor gene KiSS-1 encodes kisspeptins, Acknowledgments the natural ligands of the orphan G protein-coupled receptor GPR54. J Biol Chem 276:34631–34636 We are grateful to the Wellcome Trust and Sir John McMichael 8. Irwig MS, Fraley GS, Smith JT, Acohido BV, Popa SM, Cunningham Centre for providing infrastructure for this study. MJ, Gottsch ML, Clifton DK, Steiner RA 2004 Kisspeptin activation of gonadotropin releasing hormone neurons and regulation of Address all correspondence and requests for reprints to: Prof. KiSS-1 mRNA in the male rat. Neuroendocrinology 80:264–272 Stephen R. Bloom, Section of Investigative Medicine, Imperial 9. Navarro VM, Castellano JM, Ferna´ndez-Ferna´ndez R, Barreiro College London, 6th Floor, Commonwealth Building, Hammer- ML, Roa J, Sanchez-Criado JE, Aguilar E, Dieguez C, Pinilla L, smith Hospital, Du Cane Road, London W12 ONN, United Tena-Sempere M 2004 Developmental and hormonally regulated messenger ribonucleic acid expression of KiSS-1 and its putative Kingdom. E-mail: [email protected]. receptor, GPR54, in rat hypothalamus and potent luteinizing hor- The Section of Investigative Medicine is funded by grants mone-releasing activity of KiSS-1 peptide. Endocrinology 145: from the Medical Research Council (MRC), Biotechnology and 4565–4574 Biological Sciences Research Council, National Institute of 10. Gottsch ML, Cunningham MJ, Smith JT, Popa SM, Acohido BV, Health Research (NIHR), an Integrative Mammalian Biology Crowley WF, Seminara S, Clifton DK, Steiner RA 2004 A role for Capacity Building Award, and an FP7-HEALTH-2009-241592 kisspeptins in the regulation of gonadotropin secretion in the mouse. EurOCHIP Grant and is supported by the NIHR Imperial Bio- Endocrinology 145:4073–4077 medical Research Centre Funding Scheme. This work was also 11. Messager S, Chatzidaki EE, Ma D, Hendrick AG, Zahn D, Dixon J, funded by an MRC project grant. C.N.J. is supported by an Thresher RR, Malinge I, Lomet D, Carlton MB, Colledge WH, NIHR Clinical Lectureship, Society for Endocrinology Early Ca- Caraty A, Aparicio SA 2005 Kisspeptin directly stimulates gonadotropin-releasing hormone release via G protein-coupled receptor reer Grant, and Academy of Medical Sciences/Wellcome Starter 54. Proc Natl Acad Sci USA 102:1761–1766 Grant for Clinical Lecturers. G.M.K.N. is supported by a Well- 12. Caraty A, Smith JT, Lomet D, Ben Saïd S, Morrissey A, Cognie J, come Clinical Training Fellowship. A.N.C. is supported by an Doughton B, Baril G, Briant C, Clarke IJ 2007 Kisspeptin synchro- Imperial College Healthcare NHS Trust Charity Award. W.S.D. nizes preovulatory surges in cyclical ewes and causes ovulation in is supported by an NIHR Career Development Fellowship. seasonally acyclic ewes. Endocrinology 148:5258–5267, Disclosure Summary: The authors have nothing to disclose. 13. Plant TM, Ramaswamy S, Dipietro MJ 2006 Repetitive activation of hypothalamic G protein-coupled receptor 54 with intravenous pulses of kisspeptin in the juvenile monkey (Macaca mulatta) elicits a sustained train of gonadotropin-releasing hormone discharges. References Endocrinology 147:1007–1013 14. Ramaswamy S, Seminara SB, Pohl CR, DiPietro MJ, Crowley Jr WF, 1. Ohtaki T, Shintani Y, Honda S, Matsumoto H, Hori A, Kanehashi Plant TM 2007 Effect of continuous iv administration of human K, Terao Y, Kumano S, Takatsu Y, Masuda Y, Ishibashi Y, Wa- metastin 45–54 on the neuroendocrine activity of the hypothalamic- tanabe T, Asada M, Yamada T, Suenaga M, Kitada C, Usuki S, pituitary-testicular axis in the adult male rhesus monkey (Macaca Kurokawa T, Onda H, Nishimura O, Fujino M 2001 Metastasis mulatta). Endocrinology 148:3364–3370 suppressor gene KiSS-1 encodes peptide ligand of a G-protein-cou- 15. Shahab M, Mastronardi C, Seminara SB, Crowley WF, Ojeda SR, pled receptor. Nature 411:613–617 Plant TM 2005 Increased hypothalamic GPR54 signaling: a poten- 2. Muir AI, Chamberlain L, Elshourbagy NA, Michalovich D, Moore tial mechanism for initiation of puberty in primates. Proc Natl Acad DJ, Calamari A, Szekeres PG, Sarau HM, Chambers JK, Murdock Sci USA 102:2129–2134 E1972 Jayasena et al. Kisspeptin-10 in Men and Women J Clin Endocrinol Metab, December 2011, 96(12):E1963–E1972

16. Greives TJ, Mason AO, Scotti MA, Levine J, Ketterson ED, Kriegs- Subcutaneous injection of kisspeptin-54 acutely stimulates gonad- feld LJ, Demas GE 2007 Environmental control of kisspeptin: im- otropin secretion in women with hypothalamic amenorrhea, but plications for seasonal reproduction. Endocrinology 148:1158– chronic administration causes tachyphylaxis. J Clin Endocrinol 1166 Metab 94:4315–4323 17. Lents CA, Heidorn NL, Barb CR, Ford JJ 2008 Central and periph- 25. Jayasena CN, Nijher GM, Abbara A, Murphy KG, Lim A, Patel D, eral administration of kisspeptin activates gonadotropin but not Mehta A, Todd C, Donaldson M, Trew GH, Ghatei MA, Bloom SR, somatotropin secretion in prepubertal gilts. Reproduction 135: Dhillo WS 2010 Twice-weekly administration of kisspeptin-54 for 879–887 8 weeks stimulates release of reproductive hormones in women with 18. Kadokawa H, Matsui M, Hayashi K, Matsunaga N, Kawashima C, hypothalamic amenorrhea. Clin Pharmacol Ther 88:840–847 Shimizu T, Kida K, Miyamoto A 2008 Peripheral administration of 26. Chan YM, Butler JP, Pinnell NE, Pralong FP, Crowley Jr WF, Ren kisspeptin-10 increases plasma concentrations of GH as well as LH C, Chan KK, Seminara SB 2011 Kisspeptin resets the hypothalamic in prepubertal Holstein heifers. J Endocrinol 196:331–334 GnRH clock in men. J Clin Endocrinol Metab 96:E908–E915 19. Tovar S, Va´zquez MJ, Navarro VM, Fernańdez-Fernańdez R, Cas- 27. George JT, Veldhuis JD, Roseweir AK, Newton CL, Faccenda E, tellano JM, Vigo E, Roa J, Casanueva FF, Aguilar E, Pinilla L, Di- Millar RP, Anderson RA 2010 Kisspeptin 10 rapidly and potently eguez C, Tena-Sempere M 2006 Effects of single or repeated intra- stimulates LH secretion in men. J Clin Endocrinol Metab 96:E1228– venous administration of kisspeptin upon dynamic LH secretion in E1236 conscious male rats. Endocrinology 147:2696–2704 28. Clarkson J, Herbison AE 2006 Postnatal development of kisspeptin 20. Patterson M, Murphy KG, Thompson EL, Patel S, Ghatei MA, neurons in mouse hypothalamus; sexual dimorphism and projec- Bloom SR 2006 Administration of kisspeptin-54 into discrete re- tions to gonadotropin-releasing hormone neurons. Endocrinology gions of the hypothalamus potently increases plasma luteinising hor- 147:5817–5825 mone and testosterone in male adult rats. J Neuroendocrinol 18: 29. Adachi S, Yamada S, Takatsu Y, Matsui H, Kinoshita M, Takase K, 349–354 Sugiura H, Ohtaki T, Matsumoto H, Uenoyama Y, Tsukamura H, 21. Thompson EL, Murphy KG, Patterson M, Bewick GA, Stamp GW, Inoue K, Maeda K 2007 Involvement of anteroventral periventricu- Curtis AE, Cooke JH, Jethwa PH, Todd JF, Ghatei MA, Bloom SR lar metastin/kisspeptin neurons in estrogen positive feedback action 2006 Chronic subcutaneous administration of kisspeptin-54 causes on luteinizing hormone release in female rats. J Reprod Dev 53: testicular degeneration in adult male rats. Am J Physiol Endocrinol 367–378 Metab 291:E1074–E1082 30. Kauffman AS, Gottsch ML, Roa J, Byquist AC, Crown A, Clifton 22. Dhillo WS, Chaudhri OB, Patterson M, Thompson EL, Murphy DK, Hoffman GE, Steiner RA, Tena-Sempere M 2007 Sexual dif- KG, Badman MK, McGowan BM, Amber V, Patel S, Ghatei MA, ferentiation of Kiss1 gene expression in the brain of the rat. Endo- Bloom SR 2005 Kisspeptin-54 stimulates the hypothalamic-pitu- crinology 148:1774–1783 itary gonadal axis in human males. J Clin Endocrinol Metab 90: 31. Soldin OP, Chung SH, Mattison DR 2011 Sex differences in drug 6609–6615 disposition. J Biomed Biotechnol 2011:187103 23. Dhillo WS, Chaudhri OB, Thompson EL, Murphy KG, Patterson 32. Smith JT, Popa SM, Clifton DK, Hoffman GE, Steiner RA 2006 M, Ramachandran R, Nijher GK, Amber V, Kokkinos A, Donald- Kiss1 neurons in the forebrain as central processors for generating son M, Ghatei MA, Bloom SR 2007 Kisspeptin-54 stimulates go- the preovulatory luteinizing hormone surge. J Neurosci 26:6687– nadotropin release most potently during the preovulatory phase of 6694 the menstrual cycle in women. J Clin Endocrinol Metab 92:3958– 33. Roa J, Vigo E, Castellano JM, Navarro VM, Fernańdez-Fernańdez 3966 R, Casanueva FF, Dieguez C, Aguilar E, Pinilla L, Tena-Sempere M 24. Jayasena CN, Nijher GM, Chaudhri OB, Murphy KG, Ranger A, 2006 Hypothalamic expression of KiSS-1 system and gonadotropin- Lim A, Patel D, Mehta A, Todd C, Ramachandran R, Salem V, releasing effects of kisspeptin in different reproductive states of the Stamp GW, Donaldson M, Ghatei MA, Bloom SR, Dhillo WS 2009 female Rat. Endocrinology 147:2864–2878