A second dose of kisspeptin-54 improves oocyte maturation in women at high risk of ovarian hyperstimulation syndrome: a Phase 2 randomized controlled trial

Abstract STUDY QUESTION Can increasing the duration of LH-exposure with a second dose of kisspeptin-54 improve oocyte maturation in women at high risk of ovarian hyperstimulation syndrome (OHSS)? SUMMARY ANSWER A second dose of kisspeptin-54 at 10 h following the first improves oocyte yield in women at high risk of OHSS. WHAT IS KNOWN ALREADY Kisspeptin acts at the hypothalamus to stimulate the release of an endogenous pool of GnRH from the hypothalamus. We have previously reported that a single dose of kisspeptin-54 results in an LH-surge of ~12–14 h duration, which safely triggers oocyte maturation in women at high risk of OHSS. STUDY DESIGN, SIZE, DURATION Phase-2 randomized placebo-controlled trial of 62 women at high risk of OHSS recruited between August 2015 and May 2016. Following controlled ovarian stimulation, all patients (n = 62) received a subcutaneous injection of kisspeptin-54 (9.6 nmol/kg) 36 h prior to oocyte retrieval. Patients were randomized 1:1 to receive either a second dose of kisspeptin-54 (D; Double, n = 31), or saline (S; Single, n = 31) 10 h thereafter. Patients, embryologists, and IVF clinicians remained blinded to the dosing allocation. PARTICIPANTS/MATERIALS, SETTING, METHODS Study participants: Sixty-two women aged 18–34 years at high risk of OHSS (antral follicle count ≥23 or anti-Mullerian hormone level ≥40 pmol/L). Setting: Single centre study carried out at Hammersmith Hospital IVF unit, London, UK. Primary outcome: Proportion of patients achieving an oocyte yield (percentage of mature oocytes retrieved from follicles ≥14 mm on morning of first kisspeptin-54 trigger administration) of at least 60%. Secondary outcomes: Reproductive hormone levels, implantation rate and OHSS occurrence. MAIN RESULTS AND THE ROLE OF CHANCE A second dose of kisspeptin-54 at 10 h following the first induced further LH-secretion at 4 h after administration. A higher proportion of patients achieved an oocyte yield ≥60% following a second dose of kisspeptin-54 (Single: 14/31, 45%, Double: 21/31, 71%; absolute difference +26%, CI 2–50%, P = 0.042). Patients receiving two doses of kisspeptin-54 had a variable LH-response following the second kisspeptin dose, which appeared to be dependent on the LH-response following the first kisspeptin injection. Patients who had a lower LH-rise following the first dose of kisspeptin had a more substantial ‘rescue’ LH-response following the second dose of kisspeptin. The variable LH-response following the second dose of kisspeptin resulted in a greater proportion of patients achieving an oocyte yield ≥60%, but without also increasing the frequency of ovarian over-response and moderate OHSS (Single: 1/31, 3.2%, Double: 0/31, 0%). LIMITATIONS, REASONS FOR CAUTION Further studies are warranted to directly compare kisspeptin-54 to more established triggers of oocyte maturation. WIDER IMPLICATIONS OF THE FINDINGS Triggering final oocyte maturation with kisspeptin is a novel therapeutic option to enable the use of fresh embryo transfer even in the woman at high risk of OHSS. STUDY FUNDING/COMPETING INTEREST(S) The study was designed, conducted, analysed and reported entirely by the authors. The Medical Research Council (MRC), Wellcome Trust & National Institute of Health Research (NIHR) provided research funding to carry out the studies. There are no competing interests to declare. TRIAL REGISTRATION NUMBER Clinicaltrial.gov identifier NCT01667406 TRIAL REGISTRATION DATE 8 August 2012. DATE OF FIRST PATIENT'S ENROLMENT 10 August 2015.


SUPPLEMENTARY DATA
Supplemental methods Further detail of study protocol From Day 2 or 3 of the menstrual cycle, patients received daily SC recombinant FSH (recFSH) injections (Gonal F 112.5 iU, Merck Serono, Geneva, Switzerland, administered daily at midday); pelvic ultrasound scans were performed 5 days after commencing recFSH, to determine ovarian follicular development and appropriately titrate the dose of recFSH. Daily subcutaneous GnRH antagonist injections (Cetrotide 0.25 mg, Merck Serono, injected at 9 p.m. daily) were commenced from Day 5 of recFSH injections until 24 h prior to kisspeptin trigger injection. If serum LH was undetectable (<0.5 iU/L) on Day 7 of recFSH injections, then the dose of cetrotide was halved to 0.125 mg daily. When at least three ovarian follicles ≥18 mm diameter were visible on ultrasound, all patients received a subcutaneous bolus injection of kisspeptin administered by a study investigator to trigger oocyte maturation at 36 h prior to oocyte retrieval (between 20:30 and 23:00 h). The dose of kisspeptin was weight-adjusted (9.6 nmol/kg), and injection volumes ranged from a minimum of 300 -580 μl. Patients were then randomized to receive either a second dose of kisspeptin (9.6 nmol/kg), or saline placebo injection at 10 h following the first kisspeptin trigger injection. Patients, IVF physicians and embryologists were blinded to the randomization.
Transvaginal ultrasound-directed oocyte retrieval (TVOR) was carried out 36 h following kisspeptin administration under intravenous anaesthesia (propofol). ICSI was performed in all study cycles using sperm from the male partner to allow for assessment of oocyte maturation.
Definitions of other clinical outcomes reported include fertilization rate (percentage of mature oocytes which fertilized to form two pronuclear (2PN) zygotes following ICSI), embryo formation and quality, biochemical pregnancy rate (serum βhCG > 10iU/L 11 days after embryo transfer) and clinical pregnancy rate (intrauterine gestational sac with heartbeat on ultrasound at 6 weeks gestation).

Assessment of ovarian hyperstimulation syndrome (OHSS)
Women were 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 was graded according to the criteria of Golan et al. 1989with updated categorization by Navot et al. (1992) (Golan et al., (1989Navot et al., 1992;Abbara et al., 2015). OHSS was independently graded by two experienced IVF clinicians external to the study team (S.L. and R.S.), who were 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 was used. Women who did not have fresh embryo transfer due to lack of oocyte maturation/ fertilization were screened by symptoms alone due to low risk of OHSS. In order to safeguard participant well-being throughout the study, a clinical decision was requested from the senior IVF study clinician (G.T.) prior to kisspeptin trigger administration in all patients with ≥18 follicles ≥11 mm, or serum estradiol level ≥18 000 pmol/L, as to whether to proceed with fresh embryo transfer, or to cryopreserve all embryos and conduct embryo transfer in a subsequent cycle (segmentation). Women who had segmentation were screened for early OHSS at 5 days following oocyte retrieval, but were screened for late OHSS by symptoms alone unless the results of early screening revealed any abnormality on ultrasound or blood parameters.

Embryo grading
All embryos were graded at Day 3 by an embryologist, 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. If at Day 3 at least two embryos had six or more cells, <20% difference in blastomere diameter and <20% fragmentation, they were incubated until Day 5 post oocyte retrieval in order that the strongest embryos could be identified for transfer. On Day 5, embryos were graded for blastocyst expansion (1-6), inner cell mass (A-E) and trophectoderm (A-C). At blastocyst stage, embryos which scored at least 3 for blastocyst expansion and A or B for inner cell mass and trophectoderm were classified as 'high quality embryos'.

Peptide synthesis
Kisspeptin-54 was synthesized and purified by Bachem (Bachem Holding AG, Bubendorf. Switzerland). Sterile vials of Kisspeptin-54 were produced by the Clinalfa brand of Bachem (Bachem Distribution Services GmbH, Weil am Rhein, Germany). Both productions were prepared according to Good Manufacturing Practice (GMP). Vials of freeze-dried kisspeptin-54 were stored at -20°C and reconstituted in 0.9% saline as previously described (Abbara et al., 2015).