Conditions: Infertility
Keywords: kisspeptin infertility in vitro fertilisation
Allocation: RANDOMIZED | Intervention model: PARALLEL
Enrollment: 175
Healthy volunteers: no

Brief summary:
We want to find out if kisspeptin is successful in stimulating oocyte maturation when it is used as a 'trigger' injection during IVF therapy for infertility.

Detailed description:
Participants in the study will have a standard Gonadotrophin Releasing Hormone (GnRH) antagonist IVF cycle, but instead of having a trigger injection of hCG (human chorionic gonadotrophin) they will be randomised to receive one of 4 doses of kisspeptin injection.

Oocytes will be retrieved and graded by an embryologist to see whether or not they have matured.

The reproductive hormones Luteinising hormone, follicle stimulating hormone, oestradiol and progesterone will also be measured

Arms:
arm 1: Participant undergoing in vitro fertilisation (IVF) treatment will receive a single trigger injection of Kisspeptin, dose of 1.6 nmol/kg arm 2: Participant undergoing in vitro fertilisation (IVF) treatment will receive a single trigger injection of Kisspeptin, dose of 3.2 nmol/kg arm 3: Participant undergoing in vitro fertilisation (IVF) treatment will receive a single trigger injection of Kisspeptin, dose of 6.4 nmol/kg arm 4: Participant undergoing in vitro fertilisation (IVF) treatment will receive a single trigger injection of Kisspeptin, dose of 12.8 nmol/kg arm 5: Participant undergoing in vitro fertilisation (IVF) treatment and who is at high risk of ovarian hyper stimulation syndrome (OHSS) will receive a single trigger injection of Kisspeptin, dose of 3.2 nmol/kg arm 6: Participant undergoing in vitro fertilisation (IVF) treatment and who is at high risk of ovarian hyper stimulation syndrome (OHSS) will receive a single trigger injection of Kisspeptin, dose of 6.4 nmol/kg arm 7: Participant undergoing in vitro fertilisation (IVF) treatment and who is at high risk of ovarian hyper stimulation syndrome (OHSS) will receive a single trigger injection of Kisspeptin, dose of 9.6 nmol/kg arm 8: Participant undergoing in vitro fertilisation (IVF) treatment and who is at high risk of ovarian hyper stimulation syndrome (OHSS) will receive a single trigger injection of Kisspeptin, dose of 12.8 nmol/kg arm 9: Participant undergoing in vitro fertilisation (IVF) treatment and who is at high risk of ovarian hyper stimulation syndrome (OHSS) will receive a trigger injection of Kisspeptin, dose of 9.6 nmol/kg and a further injection of Kisspeptin, dose of 9.6 nmol/kg 10 hours later arm 10: Participant undergoing in vitro fertilisation (IVF) treatment and who is at high risk of ovarian hyper stimulation syndrome (OHSS) will receive a trigger injection of Kisspeptin, dose of 9.6 nmol/kg and a further injection of saline10 hours later

Interventions: intervention 1: Kisspeptin 1.6nmol/kg intervention 2: Kisspeptin 3.2nmol/kg intervention 3: Kisspeptin 6.4nmol/kg intervention 4: Kisspeptin 9.6nmol/kg intervention 5: Kisspeptin 12.8nmol/kg intervention 6: Kisspeptin 9.6 nmol/kg double intervention 7: Kisspeptin 9.6 nmol/kg + saline
Intervention descriptions: intervention 1: single kisspeptin dose 1.6 nmol/kg subcutaneously intervention 2: single kisspeptin dose 3.2 nmol/kg subcutaneously intervention 3: single kisspeptin dose 6.4 nmol/kg subcutaneously intervention 4: single kisspeptin dose 9.6 nmol/kg subcutaneously intervention 5: single kisspeptin dose 12.8 nmol/kg subcutaneously intervention 6: kisspeptin dose 9.6 nmol/kg given twice 10hrs apart subcutaneously intervention 7: kisspeptin dose 9.6 nmol/kg subcutaneously and saline subcutaneously, 10hrs apart

Protocol document:
Prot_000.pdf: 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
The use of the hormone kisspeptin in in vitro fertilisation 
(IVF) treatment 
 
 
 
 
 
 
 
Version 9                     1st February 2015 
 
 
MAIN SPONSOR: Imperial College London  
FUNDERS: Medical Research Council (MRC) 
STUDY COORDINATION CENTRE: Imperial College London 
Protocol Code Number ICL/KISS/12/01 
EudraCT number 2012-000154-61 
Research Ethics Committee (REC) reference: 10/H0707/2 
   
 
NCT No.  NCT01667406                        
 
 
Protocol authorised by: 
 
Name & Role 
Date 
Signature 
 
 
 
Prof. Waljit Dhillo 
Professor in Endocrinology 
and Metabolism 
Imperial College London 
 
7th 1st February 2015 
 
 
 


The use of kisspeptin in IVF treatment 
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The use of the hormone kisspeptin in in vitro fertilisation 
(IVF) treatment 
 
 
 
 
 
 
 
Version 9                     1st February 2015 
 
 
 
 
 
MAIN SPONSOR: Imperial College London  
FUNDERS: Medical Research Council (MRC) 
STUDY COORDINATION CENTRE: Imperial College London 
Protocol Code Number ICL/KISS/12/01 
EudraCT number 2012-000154-61 
Research Ethics Committee (REC) reference: 10/H0707/2 
                          
 
 
Protocol authorised by: 
 
Name & Role 
Date 
Signature 
 
 
 
Prof. Waljit Dhillo 
Professor in Endocrinology 
and Metabolism 
Imperial College London 
 
7th 1st February 2015 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 


The use of kisspeptin in IVF treatment 
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Study Management Group 
 
Chief Investigator: Professor Stephen R. Bloom (Professor of Medicine and Head of Department of 
Investigative Medicine, Imperial College London) 
 
Co-investigators: Professor Waljit Dhillo (Reader in Endocrinology and Metabolism, Department of 
Investigative Medicine, Imperial College London). Dr Geoff Trew (Director of the IVF Unit at Hammersmith 
Hospital). 
 
Statistician: Professor Ashby (Professor of Medical Statistics and Clinical Trials, Clinical Trials Unit, Imperial 
College London) 
 
Trial Management: Clinical Trials Unit, Imperial College London 
 
 
Study Coordination Centre 
 
For general queries, supply of trial documentation, and collection of data, please contact: 
 
Study Coordinator: Professor. Waljit Dhillo 
Address: Department of Investigative Medicine, Hammersmith Hospital, Du Cane Road, London W12 
ONN  
 
 
 
 
 
Tel: 020 8383 2820   
Fax:  0208 383 8320 
 
 
 
 
 
 
 
E-mail:  w.dhillo@imperial.ac.uk 
Web address: http://www1.imperial.ac.uk/medicine/people/w.dhillo/  
Randomisations: no randomisations in this study 
 
 
Clinical Queries 
 
Clinical queries should be directed to Professor. Waljit Dhillo who will direct the query to the appropriate 
person 
 
Sponsor 
 
Imperial College is the main research sponsor for this study.  For further information regarding the 
sponsorship conditions, please contact the Research Governance Manager: 
 
 
 
Becky Ward 
Research Governance Manager 
Joint Research Compliance Office 
Imperial College London and Imperial College Healthcare NHS Trust 
Room 5L10A 
5th Floor, Lab Block 
Charing Cross Hospital 
Fulham Palace Road 
London, W6 8RF 
0203 311 0205 
E-mail: becky.ward@imperial.ac.uk 
www.imperial.ac.uk/clinicalgoveranceoffice 
 


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Funder 
 
MRC / National Institute of Health Research ( NIHR) 
This protocol describes a study to investigate the use of the hormone kisspeptin in IVF treatment and provides information 
about procedures for entering participants. The protocol should not be used as a guide for the treatment of other participants; 
every care was taken in its drafting, but corrections or amendments may be necessary. These will be circulated to investigators 
in the study.  
Problems relating to this trial should be referred, in the first instance, to the study coordinator Dr. Waljit Dhillo.  
This trial will adhere to the principles outlined in the Medicines for Human Use (Clinical Trials) Regulations 2004 Statutory 
Instrument 2004/1031 (SI 2004/1031) and the International Conference on Harmonisation Good Clinical Practice (ICH GCP) 
guidelines. It will be conducted in compliance with the protocol, the Data Protection Act and other regulatory requirements as 
appropriate.  
 
Table of Contents 
STUDY SUMMARY 
6 
1. 
INTRODUCTION 
8 
1.1 
BACKGROUND 
8 
1.2 
RATIONALE FOR CURRENT STUDY 
9 
2. 
STUDY OBJECTIVES 
10 
3. 
STUDY DESIGN 
10 
3.1 
STUDY OUTCOME MEASURES 
10 
4. 
PARTICIPANT ENTRY 
11 
4.1 
PRE-RANDOMISATION EVALUATIONS 
11 
4.2 
INCLUSION CRITERIA 
11 
4.3 
EXCLUSION CRITERIA 
11 
4.4 
WITHDRAWAL CRITERIA 
11 
5. 
RANDOMISATION AND ENROLMENT PROCEDURE 
11 
5.1 
RANDOMISATION OR REGISTRATION PRACTICALITIES 
11 
5.2 
UNBLINDING 
11 
6. 
TREATMENTS 
11 
6.1 
TREATMENT ARMS 
12 
6.2 
DOSE MODIFICATIONS FOR TOXICITY 
12 
6.3 
PREMEDICATION 
12 
6.4 
INTERACTION WITH OTHER DRUGS 
12 
6.5 
DISPENSING AND ACCOUNTABILITY 
12 
7. 
PHARMACOVIGILANCE 
12 
7.1 
DEFINITIONS 
12 
7.2 
CAUSALITY 
13 
7.3 
REPORTING PROCEDURES 
14 
7.3.1 NON SERIOUS AR/AES 
14 
7.3.2 SERIOUS AR/AES 
14 
8. 
ASSESSMENT AND FOLLOW-UP 
15 


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8.1 
LOSS TO FOLLOW-UP 
15 
8.2 
TRIAL CLOSURE 
15 
9. 
STATISTICS AND DATA ANALYSIS 
15 
10. 
MONITORING 
16 
10.1 
RISK ASSESSMENT 
16 
10.2 
MONITORING AT LOCAL SITE 
16 
11. 
REGULATORY ISSUES 
16 
11.1 
CLINICAL TRIALS AUTHORISATION (CTA) 
16 
11.2 
ETHICS APPROVAL 
16 
11.3 
CONSENT 
16 
11.4 
CONFIDENTIALITY 
17 
11.5 
INDEMNITY 
17 
11.6 
SPONSOR 
17 
11.7 
FUNDING 
17 
11.8 
AUDITS AND INSPECTIONS 
17 
12. 
TRIAL MANAGEMENT 
17 
13. 
PUBLICATION POLICY 
17 
14. 
REFERENCES 
17 
APPENDIX 1. SUMMARY OF INVESTIGATIONS, TREATMENT AND ASSESSMENTS 19 
APPENDIX 2.  STANDARD FSH/GNRH ANTAGONIST IVF PROTOCOL WITH LUTEAL 
PHASE SUPPLEMENTATION 
19 
APPENDIX 3.  DETAILS OF THE IMP 
20 
  
GLOSSARY OF ABBREVIATIONS 
AE 
Adverse event 
AMH 
Anti-Mullerian hormone 
AR 
Adverse reaction 
ARDS 
Acute respiratory distress syndrome 
CRF 
Case Report Form 
CTA 
Clinical Trials Authorisation 
DMEC 
Data Monitoring & Ethics Committee 
FSH  
Follicle stimulating hormone 
GCP 
Good Clinical Practice 
GnRH 
 
Gonadotrophin releasing hormone 
GPR54 
G-Protein Coupled Receptor 54 
h 
Hour 
hCG  
Human chorionic gonadotrophin 
HPG 
Hypothalamic-pituitary gonadal 
ICH GCP 
International Conference on Harmonisation Good Clinical 
Practice  


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ICSI 
Intracytoplasmic sperm injection  
ICV 
Intracerebroventricular 
IMP 
Investigational Medicinal Product 
IU/L 
International Unit/Litre 
IVF  
 
In vitro fertilisation 
LH 
 
Luteinising hormone 
MHRA 
Medicines and Healthcare Regulatory Agency 
MRC 
Medical Research Council 
nmol/kg 
Nanomole/kilogram 
NICE 
National Institute for Health and Clinical Excellence 
NIHR 
National Institute of Health Research 
OHSS 
Ovarian hyperstimulation syndrome 
RCOG 
Royal College of Obstetricians and Gynaecologists 
REC 
Research Ethics Committee 
SAE 
Serious Adverse Event 
SAR 
Serious Adverse Reaction 
SD 
Standard deviation 
SI 2004/1031 Statutory Instrument 2004/1031 
SmPC 
Summary of product characteristics 
SUSAR 
Suspected Unexpected Serious Adverse Reaction 
TMG 
Trial Management Group 
KEYWORDS 
Infertility, hormone, kisspeptin 
 
STUDY SUMMARY 
 
TITLE The use of the hormone kisspeptin in IVF treatment 
DESIGN Open label, dose escalation, phase 2 clinical trial investigating the 
administration of Kisspeptin to induce oocyte maturation in  102 women 
undergoing IVF treatment 
AIMS To investigate whether kisspeptin can stimulate oocyte maturation in IVF 
therapy for infertility 
OUTCOME MEASURES Primary outcome measure: Oocyte maturation 
POPULATION Study participants will be women needing IVF treatment attending the IVF unit 
at Imperial College Healthcare National Health Service Trust. 
ELIGIBILITY Eligibility for the study will be determined at screening which will comprise a 
medical history, routine physical examination and basic investigations 
(including full blood count, urea and electrolytes, liver function tests, thyroid 
function tests, plasma glucose, lipid profile and electrocardiogram). 
TREATMENT All of the women in this study will undergo a standard follicle stimulating 
hormone  (FSH) / gonadotrophin releasing hormone  (GnRH) antagonist IVF 
protocol which is used for women undergoing IVF treatment at Hammersmith 
Hospital under the supervision of Dr Geoff Trew (Director of the IVF Unit). The 
only difference will be that women taking part in the study will have a single 


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subcutaneous injection of kisspeptin in place of human chorionic gonadotrophin 
(hCG) in order to trigger oocyte maturation. 
DURATION We will aim to complete the study in 3 years 


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1. 
INTRODUCTION  
 
1.1 
BACKGROUND 
Infertility is commonly defined as the inability to conceive after two years of regular unprotected sexual 
intercourse (1). 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 (1). The inability to have children can be 
devastating, and has important implications for mental, social, and reproductive health.  
 
In healthy women, the reproductive cycle is controlled by the hypothalamic-pituitary gonadal (HPG) axis. 
GnRH released from the hypothalamus stimulates the release of the gonadotrophins, luteinising 
hormone (LH) and FSH from the anterior pituitary gland. These gonadotrophins in turn regulate ovarian 
function and oocyte maturation and ovulation which are driven by an LH surge. Recently kisspeptin has 
been shown to be responsible for the endogenous LH surge, via the stimulation of hypothalamic GnRH 
release (6). After ovulation, progesterone produced by the corpus luteum prepares the endometrium for 
embryo implantation. Normal function of the corpus luteum is dependent on the pituitary secretion of 
LH. IVF treatment uses the exogenous administration of hormones to stimulate ovarian follicular 
development and oocyte maturation. FSH injections are used to stimulate ovarian follicular development 
and endogenous LH production is blocked in order to prevent premature ovulation. Once 3 dominant 
follicles have developed, hCG is administered to stimulate oocyte maturation. The oocytes are collected 
and fertilised with sperm in vitro. The embryo is then replaced into the endometrial cavity to allow 
implantation and pregnancy to occur.  
 
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 (NICE). However, the most common serious 
complication of IVF is ovarian hyperstimulation syndrome (OHSS), a potentially life threatening condition 
which has significant morbidity and mortality (2). OHSS is a systemic disease resulting from the release 
of vasoactive products from hyper stimulated ovaries. This potentially life threatening condition can 
cause massive ovarian enlargement, ovarian torsion, ascites, hydrothorax, liver dysfunction, 
thromboembolism, electrolyte imbalance, renal failure and acute respiratory distress syndrome (ARDS) 
and death (2). Mild forms of OHSS are common, occurring in 1 in 3 of all IVF cycles, whilst almost 1 in 
10 IVF cycles are complicated by moderate or severe OHSS (3).  
 
The major cause of OHSS is the use of hCG in current IVF protocols for oocyte maturation (4). 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 underlying cause of OHSS (12). Human chorionic gonadotrophin circulates 
for up to a week after injection (13), whereas with the physiological stimulus, the LH surge only lasts for 
48 hours (14). This overstimulation of the corpus luteum also results in an environment more hostile to 
embryo implantation (5). A more physiological method for oocyte maturation in IVF treatment should 
prevent overstimulation of the corpus luteum and thus the subsequent development of OHSS (5).  
 
Kisspeptin is a neuropeptide which stimulates the HPG axis. Kisspeptin offers a novel approach in IVF 
therapy to improve pregnancy rates and avoid the complication of OHSS. Kisspeptin is the endogenous 
agonist of the G protein-coupled receptor, GPR54 (19-22). The kisspeptin / GPR54 system plays a 
critical role in the hypothalamic regulation of the HPG axis. Inactivating GPR54 mutations in humans 
have been shown to cause hypogonadotrophic hypogonadism (absence of sexual maturation with low 
sex hormones and gonadotrophins) and failure of pubertal development (23-24). GPR54 or kisspeptin 
deficient mice have a similar phenotype with abnormal sexual development, low circulating 
gonadotrophin concentrations, and infertility (23, 25, 26). Furthermore, administration of kisspeptin 
potently stimulates activity of the HPG axis. Acute intracerebroventricular (ICV) or peripheral 
administration of kisspeptin to rodents or primates potently stimulates gonadotrophin release (10, 27). 
Kisspeptin stimulates the release of a limited pool of endogenous GnRH, which results in release of 
endogenous gonadotrophins. (9-11). We have recently demonstrated for the first time that acute 
administration of kisspeptin to healthy male and female volunteers with normal reproductive function, 
and in women with infertility due to hypothalamic amenorrhea potently stimulates the HPG axis without 


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side effects (7, 8, 33). The evidence to date demonstrates that the kisspeptin / GPR54 system is critical 
for normal pubertal development and reproductive function.  
 
Kisspeptin is a critical upstream regulator of GnRH neurones necessary for normal reproductive 
function. Kisspeptin is found in hypothalamic regions important in the regulation of the HPG axis (9). In 
rodents, hypothalamic kisspeptin neurons are activated during proestrus (ie the time of the cycle just 
prior to ovulation), and blockade of the actions of hypothalamic kisspeptin with a specific monoclonal 
antibody abolishes the proestrus LH surge, suggesting that kisspeptin signalling is required for a normal 
menstrual cycle (28). Recent evidence demonstrates that kisspeptin is the physiological regulator of the 
LH surge (6) and exogenous administration of kisspeptin has been shown to induce ovulation in rats 
and sheep (31, 32). In keeping with this, our previous human studies demonstrate that kisspeptin 
stimulates LH release most potently in the pre-ovulatory phase of the menstrual cycle in females with 
regular normal menstrual cycles (8). 
 
The major cause of OHSS in current IVF protocols is the pharmacological use of hCG which is used for 
oocyte maturation (4). Since kisspeptin stimulates the release of physiological levels of GnRH and the 
consequent release of endogenous gonadotrophins (9-11) it should lead to a physiological LH surge 
which would be dependent on the sensitivity of the HPG axis of each woman. A more physiological 
approach for oocyte maturation in IVF treatment would prevent overstimulation of the corpus luteum and 
OHSS in IVF treatment (5). The significant advantage of kisspeptin over current treatments is that its 
effects would depend on the sensitivity of an individual’s 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 (5).  
 
GnRH antagonists are widely used in IVF protocols to suppress the increase in endogenous LH which 
would otherwise cause premature ovulation (29). These agents bind to GnRH receptors in the pituitary 
gland and competitively inhibit the actions of endogenous GnRH. The onset of the suppression of 
pituitary gonadotrophin release is rapid, as is the recovery of pituitary function after cessation. This 
makes GnRH antagonists ideal agents for short term pituitary suppression (29). In addition since 
pituitary function recovers almost immediately when GnRH antagonist is withdrawn, they allow the 
potential use of physiological stimulators of endogenous gonadotrophins (e.g. kisspeptin) in IVF 
protocols to generate a more physiological LH surge and oocyte maturation in IVF protocols (29).  
 
We hypothesise that 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 the current study is to provide proof of concept that administration of kisspeptin can induce 
oocyte maturation in infertile women undergoing IVF treatment. 
 
1.2 
RATIONALE FOR CURRENT STUDY 
 
Research question: Can kisspeptin stimulate oocyte maturation in IVF therapy for infertility ? 
 
Hypothesis: kisspeptin will stimulate oocyte maturation in IVF therapy for infertility  
 
Infertility affects one in six couples in the UK (1). IVF treatment is now widely and successfully used to 
enable infertile couples to conceive. Approximately 45,000 cycles of IVF are performed in Britain each 
year. However, IVF treatment can result in the potentially life threatening condition, OHSS (2). Mild 
forms of OHSS occur in approximately 1 in 3 of all IVF cycles, whilst approximately 1 in 10 IVF cycles 
result in moderate or severe OHSS (3). The major cause of OHSS is the pharmacological use of hCG 
to stimulate oocyte maturation in current IVF protocols (4). The development of a more physiological 
stimulus for oocyte maturation should avoid this dangerous side effect and improve the safety and 
efficacy of IVF treatment (5). Kisspeptin is a hormone which stimulates reproductive hormone secretion 
in animals and is responsible for the endogenous LH surge which results in ovulation (6). In keeping 


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with this, exogenous administration of kisspeptin has been shown to induce ovulation in rats and sheep 
(31, 32). We have conducted the first studies of kisspeptin administration to humans; in which we have 
shown that acute administration of kisspeptin potently stimulates reproductive hormone secretion in 
healthy men and women (7, 8), as well as women with infertility due to hypothalamic amenorrhea (33). 
Importantly, we have demonstrated that healthy women with regular menstrual cycles are most sensitive 
to the effects of kisspeptin immediately prior to ovulation (8). Recent evidence demonstrates that 
kisspeptin stimulates the release of a limited pool of endogenous GnRH which results in the release of 
endogenous gonadotrophins (9-11). Therefore, the significant advantage of kisspeptin over current 
treatments is that its effects would depend on the sensitivity of an individual’s 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 (5). 
 
 
 
2. 
STUDY OBJECTIVES 
 
To investigate whether kisspeptin can stimulate oocyte maturation in IVF therapy for infertility. 
 
 
 
3. 
STUDY DESIGN 
 
Type of study: Open label, dose escalation, phase 2 clinical trial investigating the administration of 
Kisspeptin to induce oocyte maturation in 90 women undergoing IVF treatment  
 
Duration: the treatment that is specific to this study will only be the single injection of kisspeptin and 
blood sample following kisspeptin injection subsequently. The rest of the IVF protocol is part of 
standard care.  
 
Number and type of patients recruited: 90 women undergoing IVF treatment for infertility 
 
 
 
3.1 
STUDY OUTCOME MEASURES 
 
Primary outcome: To investigate whether kisspeptin can stimulate oocyte maturation in IVF therapy for 
infertility. 
 
Secondary outcome measures: 
Rates of OHSS 
Plasma kisspeptin levels 
Serum LH  
Serum FSH 
Serum Estradiol 
Ovarian follicular number and size 
Oocyte quality  
Embryo quality 
βhCG concentration 12 to16 days after embryo transfer 
Clinical pregnancy rate 
Fertilisation rate 
Pregnancy outcomes 
Live births 
 


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4. 
PARTICIPANT ENTRY  
 
4.1 
PRE-RANDOMISATION EVALUATIONS  
Study participants will be women needing IVF treatment for male factor infertility requiring 
intracytoplasmic sperm injection (ICSI), attending the IVF unit at Queen Charlottes and Chelsea 
Hospital, Imperial College Healthcare NHS Trust. Eligibility for the study will be determined at 
screening which will comprise a medical history, routine physical examination and basic investigations 
(including full blood count, urea and electrolytes, liver function tests, thyroid function tests, plasma 
glucose, lipid profile and electrocardiogram). Participants will be free to withdraw from the study at any 
time.   
 
4.2 
INCLUSION CRITERIA 
• 
Aged 18 – 34 years 
• 
Body mass index between 18 and 29 kg/m2 
• 
Stable body weight for at least 3 months 
• 
Normal early menstrual cycle follicular phase serum FSH concentration (≤12iu/l) 
• 
Serum anti-Mullerian hormone (AMH) > 40pmol/L or antral follicle count (AFC) on ultrasound 
scanning > 23 follicles 
or those women who have had a good follicular response in a previous IVF cycle in the previous 6 
months with a dose of FSH of 150iu 
• 
 No more than one previous IVF treatment cycle 
• 
 Both ovaries intact 
4.3 
EXCLUSION CRITERIA 
• 
History of any medical, psychological or other condition, or use of any medications, including 
over-the-counter products, which, in the opinion of the investigators, would either interfere with 
the study or potentially cause harm to the volunteer 
• 
Without access at home to a telephone, or other factor likely to interfere with ability to 
participate reliably in the study 
• 
Treatment with an investigational drug within the preceding 2 months 
• 
Donated blood during the preceding 3 months or intention to do so before the end of the study  
• 
Previous poor response to IVF treatment 
 
4.4 
WITHDRAWAL CRITERIA 
It will be made clear to participants that they will be free to withdraw from the study at any time without 
providing any reason and this will not affect their standard care in any way. 
 
5. 
RANDOMISATION AND ENROLMENT PROCEDURE 
 
5.1 
RANDOMISATION OR REGISTRATION PRACTICALITIES 
This is an open label, dose escalation, phase 2 clinical trial investigating the administration of 
Kisspeptin to induce oocyte maturation – there will therefore be no randomisation as all women will 
receive kisspeptin 
 
5.2 
UNBLINDING 
This is an open label, dose escalation, phase 2 clinical trial investigating the administration of 
Kisspeptin to induce oocyte maturation and all women will receive at least one dose of kisspeptin. 
Women will receive either one, or two doses of kisspeptin at least 8 hours apart. Women will be 
advised that the first injection will be kisspeptin for all women, but the second injection received may 
be either kisspeptin or saline. 
 
 
 
 
6. 
TREATMENTS 
 


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6.1 
TREATMENT ARMS 
All of the women in this study will undergo a standard FSH/GnRH antagonist IVF protocol with luteal 
phase supplementation which is used for women undergoing IVF treatment at Hammersmith Hospital 
under the supervision of Dr Geoff Trew (Director of the IVF Unit) (please see appendix 2). The only 
difference will be that women taking part in the study will have either one or two subcutaneous 
injections of kisspeptin in place of hCG in order to trigger oocyte maturation. This injection will be 
administered by two experienced physicians at the Hammersmith Hospital or in the participant’s home 
according to the patients preference. 
The patients will be administered with the first dose of kisspeptin 12-24 hours after the last injection of 
FSH/GnRH antagonist for final oocyte maturation. Women will be randomised to receive either a 
single dose of kisspeptin (9.6nmol/kg), or two doses of kisspeptin 8-12 hours apart. The optimal 
dosing regimen of kisspeptin will be the dose of kisspeptin which causes the highest level of oocyte 
maturation. 
Since kisspeptin causes a rapid and potent rise in LH release following sc injection we would expect 
that kisspeptin will induce oocyte maturation at a similar timing as when hCG or GnRH are used to 
trigger oocyte maturation which is 32-36 hours after injection (13, 16, 17, 18). In order to determine the 
changes in reproductive hormones which occur during oocyte maturation, participants will be given 
two options for blood testing following injection of kisspeptin. These options will be discussed at 
screening, and participants are entirely free to decline overnight blood sampling without altering their 
study participation or clinical care: 
Option 1. Overnight blood sampling: Participants opting for overnight blood sampling will be admitted 
to the Wellcome / NIHR Clinical Research Facility (CRF), Hammersmith Hospital. Blood will be 
sampled for measurement of plasma LH, FSH, progesterone, kisspeptin and oestradiol. Total blood 
loss will not exceed 100ml, and blood sampling will not be performed more frequently than every 
15min. 
Option 2. Less intensive blood testing: In participants who do not opt into overnight blood sampling, 
plasma LH, FSH, progesterone and oestradiol will be measured immediately before kisspeptin 
injection, on up to six occasions within the first 24h following kisspeptin injection (according to 
participant availability), and then at the time of oocyte retrieval. 
These hormone measurements will determine the time course following kisspeptin injection at which 
an endogenous LH surge occurs which will be used to confirm the timing of oocyte retrieval.  
This study will determine the potential of kisspeptin as a trigger for oocyte maturation in IVF treatment. 
 
6.2 
DOSE MODIFICATIONS FOR TOXICITY  
Not applicable 
 
6.3 
PREMEDICATION 
Not applicable 
 
6.4 
INTERACTION WITH OTHER DRUGS 
The only medication which participants should avoid is the oral contraceptive pill or hormone 
replacement therapy. All patients attending for IVF treatment will be asked to stop these medications 
as part of their standard care before IVF treatment whether they participate in the study or not. 
  
6.5 
DISPENSING AND ACCOUNTABILITY 
The pharmacy at Hammersmith will be responsible for storing and dispensing the study drug and full 
accountability documentation of the Investigational Medicinal Product (IMP) will be kept in pharmacy. 
 
7. 
PHARMACOVIGILANCE 
 
7.1 
DEFINITIONS   
Adverse Event (AE): any untoward medical occurrence in a patient or clinical trial subject administered 
a medicinal product and which does not necessarily have a causal relationship with this treatment.  An 
AE can therefore be any unfavourable and unintended sign (including an abnormal laboratory finding), 
symptom, or disease temporally associated with the use of an IMP, whether or not considered related 
to the IMP. 


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Adverse Reaction (AR): all untoward and unintended responses to an IMP related to any dose 
administered.  All AEs judged by either the reporting investigator or the sponsor as having reasonable 
causal relationship to a medicinal product qualify as adverse reactions.  The expression reasonable 
causal relationship means to convey in general that there is evidence or argument to suggest a causal 
relationship. 
 
Unexpected Adverse Reaction: an AR, the nature or severity of which is not consistent with the 
applicable product information (eg investigator’s brochure for an unapproved investigational product or 
summary of product characteristics (SmPC) for an authorised product).  When the outcome of the 
adverse reaction is not consistent with the applicable product information this adverse reaction should 
be considered as unexpected.  Side effects documented in the SmPC which occur in a more severe 
form than anticipated are also considered to be unexpected. 
 
Serious Adverse Event (SAE) or Serious Adverse Reaction: any untoward medical occurrence or 
effect that at any dose 
• 
Results in death 
• 
Is life-threatening – refers to an event in which the subject was at risk of death at the time 
of the event; it does not refer to an event which hypothetically might have caused death if 
it were more severe 
• 
Requires hospitalisation, or prolongation of existing inpatients’ hospitalisation 
• 
Results in persistent or significant disability or incapacity 
• 
Is a congenital anomaly or birth defect 
 
Medical judgement should be exercised in deciding whether an AE/AR is serious in other situations.  
Important AE/ARs that are not immediately life-threatening or do not result in death or hospitalisation 
but may jeopardise the subject or may require intervention to prevent one of the other outcomes listed 
in the definition above, should also be considered serious. 
 
Suspected Unexpected Serious Adverse Reaction (SUSAR): any suspected adverse reaction 
related to an IMP that is both unexpected and serious.   
 
7.2 
CAUSALITY 
Most adverse events and adverse drug reactions that occur in this study, whether they are serious or 
not, will be expected treatment-related toxicities due to the drugs used in this study.  The assignment of 
the causality should be made by the investigator responsible for the care of the participant using the 
definitions in the table below. 
 
If any doubt about the causality exists the local investigator should inform the study coordination centre 
who will notify the Chief Investigators.  The pharmaceutical companies and/or other clinicians may be 
asked to advise in some cases. 
 
In the case of discrepant views on causality between the investigator and others, all parties will discuss 
the case.  In the event that no agreement is made, the MHRA will be informed of both points of view.  
 
Relationship 
Description 
Unrelated 
There is no evidence of any causal relationship 
Unlikely 
There is little evidence to suggest there is a causal relationship (e.g. the event 
did not occur within a reasonable time after administration of the trial 
medication).  There is another reasonable explanation for the event (e.g. the 
participant’s clinical condition, other concomitant treatment). 
Possible 
There is some evidence to suggest a causal relationship (e.g. because the 
event occurs within a reasonable time after administration of the trial 
medication).  However, the influence of other factors may have contributed to 


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the event (e.g. the participant’s clinical condition, other concomitant 
treatments). 
Probable 
There is evidence to suggest a causal relationship and the influence of other 
factors is unlikely. 
Definitely 
There is clear evidence to suggest a causal relationship and other possible 
contributing factors can be ruled out. 
Not assessable 
There is insufficient or incomplete evidence to make a clinical judgement of the 
causal relationship. 
 
 
7.3 
REPORTING PROCEDURES 
All adverse events will be reported.  Depending on the nature of the event the reporting procedures 
below will be followed.  A flowchart is given below to aid in the reporting procedures. 
 
7.3.1 Non serious AR/AEs 
All such toxicities, whether expected or not, will be recorded in the toxicity section of the relevant case 
report form and sent to the study coordination centre within one month of the form being due.   
 
7.3.2 Serious AR/AEs 
Fatal or life threatening SAEs and SUSARs will be reported on the day that the investigators are aware 
of the event.  The SAE form asks for nature of event, date of onset, severity, corrective therapies given, 
outcome and causality (i.e. unrelated, unlikely, possible, probably, definitely).  The responsible 
investigator (Dr Waljit Dhillo) will sign the causality of the event.  Additional information should be sent 
within 5 days if the reaction has not resolved at the time of reporting.   
 
 
 
SAEs 
An SAE form will be completed and faxed to the Imperial College Joint Research Compliance Office for 
review.  
 
SUSARs  
In the case of serious, unexpected and related adverse events, the staff involved in the study will:  
 
 
Complete the SAE case report form & send it immediately (within 24 hours, preferably by 
fax), signed and dated to Dr Waljit Dhillo together with relevant treatment forms and 
anonymised copies of all relevant investigations. 
Or 
 
Contact the Dr Waljit Dhillo by phone and then send the completed SAE form to Dr Waljit 
Dhillo within the following 24 hours as above. 
 
Dr Waljit Dhillo will notify the MHRA, main REC and Sponsor of all SUSARs occurring during the study 
according to the following timelines; fatal and life-threatening within 7 days of notification and non-life 
threatening within 15 days.   All investigators will be informed of all SUSARs occurring throughout the 
study. 
 


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Contact details for reporting SAEs and SUSARs 
Fax: 0208 383 8320, attention Dr Waljit Dhillo 
Please send SAE forms to: Dr Waljit Dhillo 
Tel: 020 8383 2820 (Mon to Fri 09.00 – 17.00) 
 
8. 
ASSESSMENT AND FOLLOW-UP  
 
Each participant will be in the study for the period of their IVF treatment, which will be no longer than 
eight weeks. Following this if the patient becomes pregnant they will have routine antenatal follow up 
for the duration of their pregnancy and delivery at Queen Charlottes' Hospital. There will be no 
additional interventions needed during the pregnancy related to the study but we will follow up patients 
until the birth of their child.  
 
8.1 
LOSS TO FOLLOW-UP 
The patients who are lost to follow from this study will be contacted using their telephone and address 
details supplied at the time of entry into the study to ensure safety. As far as data analysis an intention 
to treat analysis will be carried out for this study and sensitivity analysis will be used in data analysis to 
take account of the small number of drop outs that may occur in the study.  
 
8.2 
TRIAL CLOSURE 
The study will stop recruiting patients when the optimal dose of kisspeptin required to trigger oocyte 
maturation has been determined. The optimal dose of kisspeptin will be the dose of kisspeptin which 
causes oocyte maturation in >90% of women (n=15). However, we will continue to follow up all women 
in the study until they have completed their pregnancy. 
 
9. 
STATISTICS AND DATA ANALYSIS 
 


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Sample size calculation: For the primary endpoint of oocyte maturation in the proposed patient 
population, there is no directly relevant data that has been published previously upon which sample 
sizes estimates could be based. However, there is evidence in the literature (13) of a relationship 
between successful oocyte maturation and rises in luteinising hormone (LH) which can be used as a 
basis for sample size calculations for this study. In that study, successful oocyte maturation occurs 
following a mean rise in LH of 106IU/L at 4hours following a GnRH agonist trigger in a FSH/GnRH 
antagonist IVF protocol. Our previous dose finding studies of kisspeptin on LH release in normal women 
in the preovulatory phase of their menstrual cycle show that kisspeptin (12.8nmol/kg) can increase LH 
by up to 148IU/L with an estimated SD of 78IU/L. Using the 2-group t-test approach, we have calculated 
that with 13 patients in for each dose of kisspeptin tested will have at least 90% power to detect a 
difference of 148IU/L amongst any 2 groups (2-sided 5% significance level without correction for 
multiplicity and common standard deviation of 78 IU/L) (nQuery Advisor Version 5.0. Statistical 
Solutions, Cork, Ireland). This n number is similar to that used in our recent studies of kisspeptin on LH 
release in normal women in the preovulatory phase of their menstrual cycle (n=8) to show a significant 
effect of kisspeptin on LH release compared to controls (8).  It is anticipated that the drop-out rate will 
be less than 10% (based on similar previous studies in these highly motivated infertile patients). 
Therefore, fifteen women for each of the 6 doses of kisspeptin (0.4, 0.8, 1.6, 3.2, 6.4 and 12.8nmol/kg) 
administered will be recruited to allow for dropouts during the study and each woman will only participate 
in the study once. As such a total of 90 women will be recruited to the study. 
 
Data analysis for the study: 
The statistical data analysis will be carried out by Imperial Clinical Trials Unit under the supervision of 
Professor Ashby (Professor of Medical Statistics and Clinical Trials, Imperial College London). An 
intention to treat analysis will be carried out for this study and sensitivity analysis will be used in data 
analysis to take account of the small number of drop outs that may occur in the study. The primary 
analysis of the primary endpoint (oocyte maturation) will employ a logistic model to characterise the 
relationship between dose of kisspeptin and oocycte maturation. Additionally Bayesian analysis 
methods will be employed to characterise the relationship between dose of kisspeptin, LH and oocyte 
maturation. 
 
10. 
MONITORING 
 
10.1 
RISK ASSESSMENT 
A risk assessment will be carried out according to the sponsor’s standard operating procedures 
(SOPs). 
 
10.2 
MONITORING AT LOCAL SITE 
Imperial College will risk assess the project and monitoring will be carried out according to the 
sponsors SOPs. 
 
11. 
REGULATORY ISSUES 
 
11.1 
CLINICAL TRIALS AUTHORISATION (CTA) 
A Clinical Trials Authorisation from the UK Competent Authority will be obtained. 
 
11.2 
ETHICS APPROVAL 
The Study Coordination Centre has submitted an ethics application to the Hammersmith and Queen 
Charlotte’s Research Ethics Committee.  The study will be conducted in accordance with the 
recommendations for physicians involved in research on human subjects adopted by the 18th World 
Medical Assembly, Helsinki 1964 and later revisions. 
 
11.3 
CONSENT  
Consent to enter the study will be sought from each participant only after a full explanation has been 
given, an information leaflet offered and time allowed for consideration.  Signed participant consent will 
be obtained.  The right of the participant to refuse to participate without giving reasons will be respected.  
After the participant has entered the trial the clinician remains free to give alternative treatment to that 


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specified in the protocol at any stage if he/she feels it is in the participant’s best interest, but the reasons 
for doing so will be recorded.  In these cases the participants remain within the study for the purposes 
of follow-up and data analysis.  All participants are free to withdraw at any time from the protocol 
treatment without giving reasons and without prejudicing further treatment. 
 
11.4 
CONFIDENTIALITY 
Participants’ identification data will be required for the registration process.  The Study Coordination 
Centre will preserve the confidentiality of participants taking part in the study and is registered under the 
Data Protection Act. 
 
11.5 
INDEMNITY 
Imperial College holds Public Liability ("negligent harm") and Clinical Trial ("non-negligent harm") 
insurance policies which apply to this trial. 
 
11.6 
SPONSOR 
Imperial College London will act as the main sponsor for this study.  Delegated responsibilities will be 
assigned to the NHS trusts taking part in this study.   
 
11.7 
FUNDING 
An MRC Developmental Clinical Studies grant application has been submitted to fund this study 
(outcome July 2010). This is an academic study and the investigators will not be paid for recruiting 
patients into this study.  
 
11.8 
AUDITS AND INSPECTIONS  
The study may be subject to inspection and audit by Imperial College London under their remit as 
sponsor, the Study Coordination Centre and other regulatory bodies to ensure adherence to GCP.  
 
 
12. 
TRIAL MANAGEMENT 
 
A Trial Management Group (TMG) will be appointed and will be responsible for overseeing the progress 
of the trial.  The day-to-day management of the trial will be co-ordinated by Professor Waljit Dhillo. In 
addition a Data Monitoring Committee will be convened (for further details please see the Data 
Monitoring & Ethics Committee (DMEC) Standard Operating Procedure enclosed in with this application.  
 
 
13. 
PUBLICATION POLICY 
 
All publications and presentations relating to the study will be authorised by the Trial Management 
Group.  The first publication of the trial results will be in the name of the Trial Management Group, if this 
does not conflict with the journal’s policy.  If there are named authors, these will include at least the 
trial’s Chief Investigator, Statistician and Trial Coordinator.  Members of the TMG and the Data 
Monitoring Committee will be listed and contributors will be cited by name if published in a journal where 
this does not conflict with the journal’s policy.   
 
14. 
REFERENCES 
 
1. HFEA facts and figures http://www.hfea.gov.uk/en/406.html.  
2. RCOG Guideline No.5. The Management of Ovarian Hyperstimulation Syndrome. Sept 2006.          
3. Delvigne A, Rozenberg S. Epidemiology and prevention of ovarian hyperstimulation syndrome (OHSS).Hum 
Reprod Update. 2002 8(6):559-77.         
4. Golan A, Ron-el R, Herman A, Soffer Y, Weinraub Z, Caspi E. Ovarian hyperstimulation syndrome: an update 
review. Obstet Gynecol Surv. 1989 44(6):430-40.  
 
 
5. 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.   


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6. Clarkson J, d'Anglemont de Tassigny X, Moreno AS, Colledge WH, Herbison AE. Kisspeptin-GPR54 signaling is 
essential for preovulatory gonadotropin-releasing hormone neuron activation and the luteinizing hormone 
surge. J Neurosci. 2008 28(35):8691-7. 
7. Dhillo WS, Chaudhri OB, Patterson M, Thompson EL, Murphy KG, Badman MK et al. Kisspeptin-54 stimulates 
the hypothalamic-pituitary gonadal axis in human males. J Clin Endocrinol Metab. 2005; 90(12):6609-15.            
8. Dhillo WS, Chaudhri OB, Thompson EL, Murphy KG, Patterson M, Ramachandran R et al. Kisspeptin-54 
stimulates gonadotropin release most potently during the preovulatory phase of the menstrual cycle in women. 
J Clin Endocrinol Metab. 2007; 92(10):3958-66.  
9. Irwig MS, Fraley GS, Smith JT, Acohido BV, Popa SM, Cunningham MJ et al. Kisspeptin activation of 
gonadotropin releasing hormone neurons and regulation of KiSS-1 mRNA in the male rat. Neuroendocrinology. 
2004; 80(4):264-72.  
 
10. Thompson EL, Patterson M, Murphy KG, Smith KL, Dhillo WS, Todd JF et al. Central and peripheral 
administration of kisspeptin-10 stimulates the hypothalamic-pituitary-gonadal axis. J Neuroendocrinol. 2004; 
16(10):850-8. 
 
11. Messager S, Chatzidaki EE, Ma D, Hendrick AG, Zahn D, Dixon J et al. Kisspeptin directly stimulates 
gonadotropin-releasing hormone release via G protein-coupled receptor 54. Proc Natl Acad Sci U S A. 2005; 
102(5):1761-6.            
12. 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.       
13. 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. 
             
14. Hoff JD, Quigley ME, Yen SS. Hormonal dynamics at midcycle: a reevaluation. J Clin Endocrinol Metab. 1983 
Oct;57(4):792-6. 
 
 
15. Kol S. Luteolysis induced by a gonadotropin-releasing hormone agonist is the key to prevention of ovarian 
hyperstimulation syndrome. Fertil Steril. 2004 Jan;81(1):1-5.                         
16. Humaidan P, Bredkjaer HE, Bungum L, Bungum M, Grøndahl ML, Westergaard L et al. GnRH agonist 
(buserelin) or hCG for ovulation induction in GnRH antagonist IVF/ICSI cycles: a prospective randomized study. 
Hum Reprod. 2005; 20(5):1213-20. 
 
17. Kolibianakis EM, Schultze-Mosgau A, Schroer A, van Steirteghem A, Devroey P, Diedrich K et al. A lower 
ongoing pregnancy rate can be expected when GnRH agonist is used for triggering final oocyte maturation 
instead of HCG in patients undergoing IVF with GnRH antagonists. Hum Reprod. 2005; 20(10):2887-92.  
18. Griesinger G, Diedrich K, Devroey P, Kolibianakis EM. GnRH agonist for triggering final oocyte maturation in 
the GnRH antagonist ovarian hyperstimulation protocol: a systematic review and meta-analysis. Hum Reprod 
Update. 2006; 12(2):159-68. 
 
 
19. Kotani M, Detheux M, Vandenbogaerde A, Communi D, Vanderwinden JM, Le Poul E et al. The metastasis 
suppressor gene KiSS-1 encodes kisspeptins, the natural ligands of the orphan G protein-coupled receptor GPR54. 
J Biol Chem 2001; 276(37):34631-34636.  
          
20. Lee JH, Miele ME, Hicks DJ, Phillips KK, Trent JM, Weissman BE et al. KiSS-1, a novel human malignant 
melanoma metastasis-suppressor gene. J Natl Cancer Inst 1996; 88(23):1731-1737.  
 
21. Muir AI, Chamberlain L, Elshourbagy NA, Michalovich D, Moore DJ, Calamari A et al. AXOR12, a novel human 
G protein-coupled receptor, activated by the peptide KiSS-1. J Biol Chem 2001; 276(31):28969-28975. 
22. Ohtaki T, Shintani Y, Honda S, Matsumoto H, Hori A, Kanehashi K et al. Metastasis suppressor gene KiSS-1 
encodes peptide ligand of a G-protein-coupled receptor. Nature 2001; 411(6837):613-617. 
     
23. Seminara SB, Messager S, Chatzidaki EE, Thresher RR, Acierno JS Jr, Shagoury JK et al. The GPR54 gene as a 
regulator of puberty. N Engl J Med 2003; 349(17):1614-1627. 24. de Roux N, Genin E, Carel JC, Matsuda F, 
Chaussain JL, Milgrom E. Hypogonadotropic hypogonadism due to loss of function of the KiSS1-derived peptide 
receptor GPR54. Proc Natl Acad Sci U S A 2003; 100(19):10972-10976. 
 
25. Lapatto R, Pallais JC, Zhang D, Chan YM, Mahan A, Cerrato F et al. Kiss1-/- mice exhibit more variable 
hypogonadism than Gpr54-/- mice. Endocrinology. 2007; 148(10):4927-36.                        
26. d'Anglemont de Tassigny X, Fagg LA, Dixon JP, Day K, Leitch HG, Hendrick AG et al. Hypogonadotropic 
hypogonadism in mice lacking a functional Kiss1 gene. Proc Natl Acad Sci U S A. 2007 Jun 19;104(25):10714-9.           


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27. Plant TM, Ramaswamy S, Dipietro MJ. 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. 2006; 147(2):1007-13 
28. Kinoshita M, Tsukamura H, Adachi S, Matsui H, Uenoyama Y, Iwata K et al. Involvement of central metastin 
in the regulation of preovulatory luteinizing hormone surge and estrous cyclicity in female rats. Endocrinology 
2005; 146(10):4431-4436.        
29. DiLuigi AJ, Nulsen JC. Effects of gonadotropin-releasing hormone agonists and antagonists on luteal function. 
Curr Opin Obstet Gynecol. 2007; 19(3):258-65.  
30. Staessen C, Camus M, Bollen N, Devroey P, Van Steirteghem AC. The relationship between embryo quality 
and the occurrence of multiple pregnancies. Fertil Steril. 1992 Mar;57(3):626-30. 
31. Matsui H, Takatsu Y, Kumano S, Matsumoto H, Ohtaki T. Peripheral administration of metastin induces 
marked gonadotropin release and ovulation in the rat. Biochem Biophys Res Commun. 2004 Jul 23;320(2):383-
8. 
32. Caraty A, Smith JT, Lomet D, Ben Saïd S, Morrissey A, Cognie J, Doughton B, Baril G, Briant C, Clarke IJ. 
Kisspeptin synchronizes preovulatory surges in cyclical ewes and causes ovulation in seasonally acyclic ewes. 
Endocrinology. 2007 Nov;148(11):5258-67. 
33. Jayasena CN, Nijher GM, Chaudhri OB, Murphy KG, Ranger A, Lim A, Patel D, Mehta A, Todd C, Ramachandran 
R, Salem V, Stamp GW, Donaldson M, Ghatei MA, Bloom SR, Dhillo WS. Subcutaneous injection of kisspeptin-54 
acutely stimulates gonadotropin secretion in women with hypothalamic amenorrhea, but chronic administration 
causes tachyphylaxis. J Clin Endocrinol Metab. 2009 Nov;94(11):4315-23. 
 
 
 
APPENDIX 1. 
SUMMARY OF INVESTIGATIONS, TREATMENT AND 
ASSESSMENTS 
 
All of the women in this study will undergo a standard FSH/GnRH antagonist IVF protocol with luteal 
phase supplementation which is used as part of routine care for women undergoing IVF treatment at 
Hammersmith Hospital (see appendix 2) under the supervision of Dr Geoff Trew (Director of the IVF 
Unit). The only difference will be that women taking part in the study will have: 
1. a single subcutaneous injection of kisspeptin in place of hCG in order to trigger oocyte 
maturation.  
2.  Plasma LH, FSH, progesterone and oestradiol will be measured immediately before kisspeptin 
injection and then after kisspeptin injectionon up to 6 occasions during the first 24h and finally 
at the time of oocyte retrieval. These hormone measurements will determine the time course 
following kisspeptin injection at which an endogenous LH surge occurs which will be used to 
confirm the timing of oocyte retrieval. 
All of the other procedures that the patients will have are part of routine care and would be the same 
for volunteers not participating in the study. 
 
 
 
 
APPENDIX 2.  
STANDARD FSH/GNRH ANTAGONIST IVF PROTOCOL 
WITH LUTEAL PHASE SUPPLEMENTATION  
All of the women in this study will undergo a standard FSH/GnRH antagonist IVF protocol with luteal 
phase supplementation which is used as part of routine care for women undergoing IVF treatment at 
Hammersmith Hospital (see appendix 2) under the supervision of Dr Geoff Trew (Director of the IVF 
Unit). The current protocol is detailed below. This will be updated according to best clinical care.  
 
Daily subcutaneous (sc) FSH injections (Gonal F) will be commenced on day 2 of the patient’s 
menstrual cycle. Daily GnRH antagonist administration (Cetrotide) will be used to inhibit a premature 


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LH surge and will be started on the 5th day of FSH injections. The patient will attend 5 days after 
commencing the FSH injections for a pelvic ultrasound scan (USS) to determine ovarian follicular 
development. The patient will be asked to attend for further scans according to follicular size and 
response (if the largest follicle is 10-12mm the next USS will be 3 days later, if the largest follicle is 13-
15mm the next USS will be 2 days later, if the largest follicle is 16mm daily USS will be performed). 
When 3 ovarian follicles of ≥ 17mm have developed, the FSH and GnRH antagonist injections will be 
stopped. Kisspeptin will be given in place of hCG at this point in the protocol as detailed in section 6.1 
of the protocol to cause oocyte maturation. 
Thirty two to thirty six hours later, transvaginal ultrasound-directed oocyte retrieval will be carried out. 
Oocyte maturation will be determined using standard criteria (calculations of the percentage of 
metaphase II (MII) oocytes and fertilisation rate (number of two pronuclear (2PN) oocytes / number of 
metaphase oocytes injected) (15). ICSI will be carried out using fresh sperm from the male partner. 
The quality of the embryos will be classified as high quality (score 1), medium quality (score 2), and 
low quality (score 3) as previously described (30). One or two embryos of highest quality will be 
transferred to the uterine cavity of the patient on day 3-5 after oocyte collection. The remaining 
embryos will be frozen for the patient to use in future if required. Progesterone  and oestradiol (at 
dosages recommended by the IVF team) will be started on the morning after oocyte collection until 12 
weeks of pregnancy for luteal phase supplementation. After oocyte retrieval doxycycline (100mg bd) 
will be given for 7 days as well as analgesia as required (diclofenac suppository or paracetamol). 
Women will be followed up according to the standard protocol of the IVF Unit at Hammersmith 
Hospital. Women in whom kisspeptin does not result in oocyte maturation will receive a further IVF 
cycle (i.e. with hCG as a trigger for oocyte maturation) as part of their standard care. Pregnancy will be 
defined biochemically by a plasma βhCG concentration >10iu/l 12 to 16 days after embryo transfer. 
Pregnancy will be defined clinically as an intrauterine gestational sac with a heartbeat 2-3 weeks after 
a positive hCG test. An additional ultrasound scan may also be performed at 7 weeks if there is clinical 
suspicion of an ectopic pregnancy.  In women with a clinical pregnancy, standard pregnancy follow up 
will occur at Queen Charlotte’s Hospital. Pregnancy outcomes and live births will be documented.   
IVF clinicians will make decisions in the best clinical interest of patients to safeguard patient safety 
where necessary as would occur in normal clinical practice. 
 
APPENDIX 3.  
DETAILS OF THE IMP 
The IMP is kisspeptin-54. Doses between 0.4 and 12.8nmol/kg will be administered during this study. 
The IMP has been manufactured and labelled in compliance with Good Manufacturing Practice Annex 
13 by Bachem Switzerland. Please see the Investigators Brochure for further details of the IMP and for 
storage conditions. 

SAP_001.pdf: 
 
 
 
 
The use of the Hormone Kisspeptin in in vitro fertilisation (IVF) treatment. 
Statistical Analysis Plan, version 3.0 
Date: 24/06/2015 
 
MAIN SPONSOR: Imperial College London  
FUNDERS: Medical Research Council (MRC) 
STUDY COORDINATION CENTRE: Imperial College London 
Protocol Code Number ICL/KISS/12/01 
EudraCT number 2012-000154-61 
Research Ethics Committee (REC) reference: 10/H0707/2 
   
 
NCT No.  NCT01667406                        
 
 
 
 


Statistical Analysis Plan, version 3.0 
Date: 24/06/2015 
Study: One versus 2 doses of kisspeptin (10hrs apart) 
Participants will undergo controlled ovarian stimulation using a recombinant FSH/GnRH antagonist 
protocol and receive either one (Single) or two doses (Double) of kisspeptin-54 (9.6nmol/kg each 
dose) to trigger oocyte maturation.  
On day 2 or 3 of the menstrual cycle, patients will receive daily SC recombinant FSH (recFSH) 
injections (Gonal F 112.5 IU, Merck Serono, Geneva, Switzerland, administered daily at midday); 
pelvic ultrasound scan will be performed five days after commencing recFSH, to determine ovarian 
follicular development. Daily subcutaneous GnRH antagonist injections (Cetrotide 0•25mg, Merck 
Serono, injected at 9pm daily) will be used to inhibit a premature LH surge, and will be commenced 
on day 5 of recombinant FSH injections. If serum LH is undetectable (<0•5 IU/L) on Day 7 of 
recombinant FSH injections, then the dose of cetrotide will be halved to 0•125mg daily. Further 
ultrasound scans will be performed according to follicular size and response to superovulation. When 
at least three ovarian follicles ≥18mm diameter are visible on ultrasound, all patients will receive a 
subcutaneous bolus injection of kisspeptin-54 administered by a study investigator to trigger oocyte 
maturation at 36hrs pior to egg collection (between 2030 and 2300h). The dose of kisspeptin-54 will 
be weight-adjusted (9.6nmol/kg). Patients will then be randomized to receive either a second dose of 
kisspeptin (9.6nmol/kg), or saline placebo injection at 10hrs following the first kisspeptin trigger 
injection. 
The final dose of GnRH antagonist will be administered 24 hours prior to kisspeptin-54. Serum LH, 
FSH, oestradiol and progesterone will be measured immediately before, 12hrs and 36hrs after 
kisspeptin-54 injection, and at the time of egg retrieval in all women. Transvaginal ultrasound-
directed oocyte retrieval (TVOR) will be carried out 36h following kisspeptin administration under 
intravenous anaesthesia (propofol). Intracytoplasmic sperm injection (ICSI) will be performed in all 
study cycles using sperm from the male partner to allow for assessment of oocyte maturation. All 
embryos will be graded at day 3 by an independent embryologist, blinded to doses of kisspeptin 
administered, using the British Fertility Society (BFS) and Association of Clinical Embryologist 
(ACE) embryo grading scheme for cleavage stage embryos, which describes embryos based on cell 
number, blastomere size and fragmentation.(30) If at day 3 at least 2 embryos have 6 or more cells, 
<20% difference in blastomere diameter and <50% fragmentation, they will be incubated until day 5 
post oocyte retrieval in order that the strongest embryos can be identified for transfer. At Day 5, 
embryos will be graded for blastocyst expansion (1 to 6), inner cell mass (A to E) and trophectoderm 
(A to C).(30) At blastocyst stage, embryos which score at least 3 for blastocyst expansion and A or B 
for inner cell mass and trophectoderm will be classified as ‘high quality embryos’. One or two 
embryos of highest quality during morphological assessment will be transferred to the uterine cavity 
3-5 days following oocyte retrieval. 
 
Assessment of Ovarian Hyperstimulation Syndrome (OHSS) 
All women recruited to the study will be regarded as being at high risk of OHSS. Therefore women 
will be routinely screened for the development of early OHSS (assessed at embryo transfer 3-5days 
following oocyte retrieval) and late OHSS (assessed 11days following embryo transfer). Women will 
be screened by symptoms (abdominal pain, abdominal bloating, diarrhoea, nausea, vomiting, 
subjective reduction in urine output), blood analysis (hemaglobin, hematocrit, white cell count, liver 
function, renal function, coagulation profile) and ultrasound parameters (ovarian size, presence of free 


fluid in pouch of douglas, adnexae, abdomen or pleural cavity). OHSS will be graded according to the 
criteria of Golan et al. 1989 with updated categorization by Navot et al. 1992.(16,17) OHSS will ne 
independently graded by two experienced IVF clinicians external to the study team (S.L. and R.S.), 
who will ne provided with blinded data concerning patient symptom, blood analysis and ultrasound 
parameters. In the event of any discrepancy in categorization of OHSS, the more severe classification 
will be used.  
 
Study outcomes 
The primary outcome is proportion of patients achieving optimal oocyte yield (percentage of mature 
(metaphase 2; M2) oocytes collected from the number of follicles ≥14mm in size on final ultrasound 
scan prior to kisspeptin-54 trigger administration of at least 60%). All women will have an ultrasound 
on the morning of kisspeptin trigger administration. Oocytes will be independently classified as M2 
by presence of the first polar body and round ooplasm, by an embryologist blinded to whether one or 
2 doses of kisspeptin-54 were administered. 
Secondary outcomes are the occurrence of OHSS, fertilization rate (percentage of M2 oocytes which 
fertilize to form two pronuclear (2PN) zygotes following intracytoplasmic injection with sperm; 
ICSI), embryo formation and quality, biochemical pregnancy rate (serum βhCG >10mIU/mL eleven 
days after embryo transfer) and clinical pregnancy rate (intrauterine gestational sac with heartbeat on 
ultrasound at six weeks gestation). 
Primary Outcome:  
Proportion of patients achieving optimal oocyte yield (defined as proportion of mature eggs collected 
from follicles of at least 14mm in size on final ultrasound prior to egg collection being at least 60%) 
(Binary Outcome). 
The main covariates to be considered are the number of follicles on final ultrasound scan prior to 
trigger (follicles ≥14mm on day of trigger), serum AMH, Antral Follicle Count, age, pretrigger 
estradiol level.  
Absolute difference between two groups with CIs will be calculated.  
 
Secondary outcomes:  
-Change in reproductive hormones LH, FSH, Estradiol and progesterone from pretrigger values to 
4hr, 10hr, 14hr and 20hr values.  
Variables to be included in the model: weight, AMH, pretrigger reproductive hormone levels. 
 
Pregnancy (binary) and Implantation rates (ordinal- can be 0, 50 or 100%)-                              
Baseline covariates which may need adjusting for include age, number of embryos transferred, 
number of high quality embryos transferred, cause of infertility and BMI.  
 
 
 
 


-Rates of OHSS : 
Covariates which may need to be adjusted for would include number of follicles >11mm on final 
ultrasound scan prior to trigger, serum AMH or antral follicle count, number of eggs collected, 
pretrigger oestradiol level. 
 
List trial data as per CONSORT guidance:  
Number of women screened, number who withdrew, number not eligible, number of women recruited 
and meeting inclusion criteria, but excluded prior to randomisation for complications requiring other 
IVF treatments prior to receiving kisspeptin eg hydrosalpinx. 
We expect to recruit 62 women eligible for randomization (31 per group). 
 
Example:  
 
 
 
 
 
 
 
 
 
 
 


Tables to be generated following study completion: 
Table 1:  
 
Sort by column A 
SINGLE 
DOIUBLE 
BOTH 
N 
31 
31 
62 
 
Age (y) C 
 
 
 
Weight (kg) I 
 
 
 
Body mass index (kg/m2) J 
 
 
 
Antral Follicle Count (AFC) F 
 
 
 
Serum AMH* (pmol/L) E 
 
 
 
% with average cycle length ≥35 
days D≥35 
 
 
 
 
Cause of infertility: G 
 
 
 
PCOS‡ 
 
 
 
Tubal defect** 
 
 
 
Male Factor† 
 
 
 
Mixed 
 
 
 
Idiopathic (unexplained) 
 
 
 
 
Number of follicles¶ BF 
 
 
 
Number of Follicles ≥ 11mm¶ 
BK 
 
 
 
Number of Follicles  ≥ 14mm¶ 
BN 
 
 
 
 
Table 1: Baseline characteristics of patients who received kisspeptin-54 trigger. Contains 
medians (lower quartile, upper quartile) for continuous variables and totals (percentages) for 
categorical variables. 
* AMH = anti-Müllerian hormone 
‡ Anovulation due to Polycystic Ovarian Syndrome. 
** Blocked or removed Fallopian tubes. 
† Infertility is due to a problem with male partner’s fertility. 
¶ On final ultrasound scan during controlled ovarian stimulation prior to kisspeptin-54 trigger 
administration. 


Table 2: 
 
SINGLE 
DOUBLE 
BOTH 
 
(n = 31) 
(n = 31) 
(n = 62) 
 
Number of Oocytes BZ 
 
 
 
Number of Mature (M2) Oocytes CA 
 
 
 
Oocyte Maturation Rate (%) * CD 
 
 
 
Oocyte Yield (%) † CG 
Proportion achieving ≥60% oocyte yield (%) 
CH 
 
 
 
Number of 2 pronuclei (2PN) zygotes CE 
 
 
 
Proportion with at Least 4 eggs collected (%) 
CB 
 
 
 
Fertilization rate (%) ‡ CF 
 
 
 
Number of patients with embryo transfer 
CN≥1 
 
 
 
Number of Cleaved embryos at day 3 post 
ICSI CI 
 
 
 
Number of Embryos at day 3 graded as ≥623 
CJ 
 
 
 
Number of Patients with day 5 transfer % with 
CM=5 
 
 
 
Number of Embryos at day 5 CK 
 
 
 
Number of High quality blastocysts (≥3A/B) 
CL 
 
 
 
Number of High quality blastocysts (>3A/B) 
transferred CO 
 
 
 
Number of Embryos suitable for freezing CV 
 
 
 
Biochemical Pregnancy rate per protocol (%) 
CP including those who did not have transfer 
as 0 
 
 
 
Biochemical Pregnancy rate per transfer (%) 
CP not including those who did not have 
transfer  
 
 
 
Clinical Pregnancy rate per protocol (%) CQ 
 
 
 


including those who did not have transfer as 0 
Clinical Pregnancy rate per transfer (%) CQ 
not including those who did not have transfer 
 
 
 
Implantation rate (%)Ψ CU 
 
 
 
 
 
 
 
Live Birth rate per protocol (%)  
 
 
 
Table 2: IVF outcome measures following kisspeptin-54 trigger. Contains means (standard 
deviation) for continuous variables and totals (percentages of N) for categorical variables 
* Oocyte Maturation Rate is the percentage of oocytes collected which were mature.  
† Oocyte Yield is the percentage of mature oocytes collected from the number of follicles  
≥14mm in diameter on the final ultrasound scan prior to kisspeptin-54 trigger administration.  
‡ Fertilization rate is the percentage of mature oocytes which fertilize following intracytoplasmic 
injection with sperm (ICSI).  
Ψ Implantation rate is defined as the percentage of embryos transferred which implant on assessment 
by ultrasound at 6 weeks of gestation. 
 
 
 
 
 
 
 
 
 


Table 3: 
Kisspeptin-54 Dose 
SINGLE 
DOUBLE 
BOTH 
N 
31 
31 
62 
At least 1 oocyte retrieved BZ ≥1 
 
 
 
At least 1 mature oocyte retrieved CA ≥1 
 
 
 
At least 1 2PN zygote formed ‡ CE ≥1 
 
 
 
Segmentation Ψ  
 
 
 
Number of patients with embryo transfer CN≥1 
 
 
 
Number of patients with day 5 transfer* % with CM=5 
 
 
 
High quality embryo transfer† CO≥1 
 
 
 
Biochemical pregnancy at 11 days CP 
 
 
 
Clinical pregnancy at 6 weeks CQ 
 
 
 
Miscarriage / Still Birth (PENDING) 
 
 
 
Live Birth (PENDING) 
 
 
 
 
Table 3: Summary of patient response following kisspeptin-54 triggering.  
The number of patients completing each criterion is shown (% in brackets).  
‡ 2PN is two pronuclear zygote assessed on day following oocyte retrieval 
Ψ Segmentation refers to cryopreservation of all embryos and embryo transfer in a subsequent frozen 
cycle due to high risk of OHSS 
* Embryo transfer on day 5  
† Transfer of at least one high quality embryo. High quality embryos were blastocyst embryos graded 
on day 5 as being at least 3A or B in quality (Gardner et al. 2000).  
 
 
 


Table 4:  
 
 
 
Early OHSS 
By single vs 
double 
 
 
Late OHSS 
By single by 
double 
 
 
OHSS Symptomatology 
Number of patients screened by OHSS symptoms 
 
 
At least 1 symptom potentially consistent with OHSS  
 
 
Number of patients requiring medical intervention or hospitalization 
for OHSS 
 
 
 
Sonographical Screening 
Number of patients screened with pelvic ultrasound 
 
 
 
 
 
 
 
Mean ovarian volume (mls) 
  
  
Increase in ovarian volume as proportion of baseline scan 
  
  
Max ovarian diameter (mm) 
  
  
Number of patients with maximum ovarian diameter >5cm  
 
 
Number of patients with maximum ovarian diameter >8cm  
 
 
Number of patients with pleural effusion 
 
 
Number of patients with free fluid in abdomen  
 
 
Number of patients with fluid in POD / adnexa  
 
 
 
Blood parameters 
Number of patients screened with Blood analysis 
 
 
Number of patients with Hematocrit >45%  
 
 
Number of patients with White Cell Count >15 x109/L  
 
 
Number of patients with ALT or AST >2xULN  
 
 
Number of patients with total protein >80 g/L  
 
 
Number of patient with creatinine >110 µmol/L  
 
 


 
 
 
Number of patients with OHSS ¥ 
 
 
Normal 
 
 
Mild 
 
 
Moderate 
 
 
Severe 
 
 
Critical 
 
 
 
 
 
Table 4: OHSS in high risk women following kisspeptin-54 trigger 
¥ Diagnosis of OHSS was performed by two experienced IVF physicians independent of the study 
team provided with blinded data according to the criteria of Golan et al. 1989 with updated 
categorization of severe and critical OHSS by Navot et al. 1992.(16,17) 
POD, Pouch of douglas; ALT, Alanine transaminase; AST, Aspartate transaminase; ULN, Upper 
Limit of Normal.