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Investigation of the Novel Hormone Kisspeptin in Disorders Of

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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 1 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. 2 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 3 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 ..........................................................................
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