Kisspeptin treatment induces gonadotropic responses and rescues ovulation in a subset of preclinical models and women with polycystic ovary syndrome

Abstract

Study question: Can kisspeptin treatment induce gonadotrophin responses and ovulation in preclinical models and anovulatory women with polycystic ovary syndrome (PCOS)?

Summary answer: Kisspeptin administration in some anovulatory preclinical models and women with PCOS can stimulate reproductive hormone secretion and ovulation, albeit with incomplete efficacy.

What is known already: PCOS is a prevalent, heterogeneous endocrine disorder, characterized by ovulatory dysfunction, hyperandrogenism and deregulated gonadotrophin secretion, in need of improved therapeutic options. Kisspeptins (encoded by Kiss1) are master regulators of the reproductive axis, acting mainly at GnRH neurons, with kisspeptins being an essential drive for gonadotrophin-driven ovarian follicular maturation and ovulation. Altered Kiss1 expression has been found in rodent models of PCOS, although the eventual pathophysiological role of kisspeptins in PCOS remains unknown.

Study design, size, duration: Gonadotrophin and ovarian/ovulatory responses to kisspeptin-54 (KP-54) were evaluated in three preclinical models of PCOS, generated by androgen exposures at different developmental windows, and a pilot exploratory cohort of anovulatory women with PCOS.

Participants/materials, setting, methods: Three models of PCOS were generated by exposure of female rats to androgens at different periods of development: PNA (prenatal androgenization; N = 20), NeNA (neonatal androgenization; N = 20) and PWA (post-weaning androgenization; N = 20). At adulthood (postnatal day 100), rats were subjected to daily treatments with a bolus of KP-54 (100 μg/kg, s.c.) or vehicle for 11 days (N = 10 per model and treatment). On Days 1, 4, 7 and 11, LH and FSH responses were assessed at different time-points within 4 h after KP-54 injection, while ovarian responses, in terms of follicular maturation and ovulation, were measured at the end of the treatment. In addition, hormonal (gonadotrophin, estrogen and inhibin B) and ovulatory responses to repeated KP-54 administration, at doses of 6.4-12.8 nmol/kg, s.c. bd for 21 days, were evaluated in a pilot cohort of anovulatory women (N = 12) diagnosed with PCOS, according to the Rotterdam criteria.

Main results and the role of chance: Deregulated reproductive indices were detected in all PCOS models: PNA, NeNA and PWA. Yet, anovulation was observed only in NeNA and PWA rats. However, while anovulatory NeNA rats displayed significant LH and FSH responses to KP-54 (P < 0.05), which rescued ovulation, PWA rats showed blunted LH secretion after repeated KP-54 injection and failed to ovulate. In women with PCOS, KP-54 resulted in a small rise in LH (P < 0.05), with an equivalent elevation in serum estradiol levels (P < 0.05). Two women showed growth of a dominant follicle with subsequent ovulation, one woman displayed follicle growth but not ovulation and desensitization was observed in another patient. No follicular response was detected in the other women.

Limitations, reasons for caution: While three different preclinical PCOS models were used in order to capture the heterogeneity of clinical presentations of the syndrome, it must be noted that rat models recapitulate many but not all the features of this condition. Additionally, our pilot study was intended as proof of principle, and the number of participants is low, but the convergent findings in preclinical and clinical studies reinforce the validity of our conclusions.

Wider implications of the findings: Our first-in-rodent and -human studies demonstrate that KP-54 administration in anovulatory preclinical models and women with PCOS can stimulate reproductive hormone secretion and ovulation, albeit with incomplete efficacy. As our rat models likely reflect the diversity of PCOS phenotypes, our results argue for the need of personalized management of anovulatory dysfunction in women with PCOS, some of whom may benefit from kisspeptin-based treatments.

Study funding/competing interest(s): This work was supported by research agreements between Ferring Research Institute and the Universities of Cordoba and Edinburgh. K.S. was supported by the Wellcome Trust Scottish Translational Medicine and Therapeutics Initiative (STMTI). Some of this work was undertaken in the MRC Centre for Reproductive Health which is funded by the MRC Centre grant MR/N022556/1. M.T.-S. is a member of CIBER Fisiopatología de la Obesidad y Nutrición, which is an initiative of Instituto de Salud Carlos III. Dr Mannaerts is an employee of Ferring International PharmaScience Center (Copenhagen, Denmark), and Drs Qi, van Duin and Kohout are employees of the Ferring Research Institute (San Diego, USA). Dr Anderson and Dr Tena-Sempere were recipients of a grant support from the Ferring Research Institute, and Dr Anderson has undertaken consultancy work and received speaker fees outside this study from Merck, IBSA, Roche Diagnostics, NeRRe Therapeutics and Sojournix Inc. Dr Skorupskaite was supported by the Wellcome Trust through the Scottish Translational Medicine and Therapeutics Initiative 102419/Z/13/A. The other authors have no competing interest.

Keywords: gonadotrophins; kisspeptin; ovarian stimulation; ovulation; polycystic ovary syndrome; preclinical models.

Figure 1

Baseline reproductive features of the three preclinical rat models of PCOS. Major reproductive features are presented from the three preclinical models of polycystic ovary syndrome (PCOS) (PNA: prenatal androgenization, NeNA: neonatal androgenization and PWA: post-weaning androgenization) at adulthood, prior to pharmacological intervention. Bar graphs represent data on serum LH and FSH levels, uterus weight (UW), ovarian weight (OW) and number of fresh corpora lutea (i.e. of the current cycle) in a representative subset of randomly selected animals (N = 10/group) of the three models. Data from the corresponding non-androgenized control animals are also shown. In addition, representative histological images of ovarian sections from the three PCOS models are presented. Note that, like control cycling females, PNA rats showed several generations of corpora lutea, as sign of ovulation. In contrast, corpora lutea were absent in NeNA and PWA rats, which presented atretic follicles that at advanced stages appeared as follicular cysts. Each histogram represents the mean ± SEM. Data were analyzed by Student t tests (^*P < 0.05, ^**P < 0.01, ^***P < 0.001 versus corresponding control groups). CL: corpus luteum; Cy: cyst-like structures; AtF: atretic follicle. Scale bar: 600 μm.

Figure 2

Baseline metabolic features of the three preclinical rat models of PCOS. Major metabolic indices are presented in a representative subset of randomly selected animals (N = 10/group) of the three preclinical models of PCOS (PNA, NeNA and PWA) at adulthood, prior to pharmacological intervention. Graphs correspond to data of body weight (BW) evolution, body fat composition (% Fat), basal glucose levels, glucose tolerance tests (GTT) and insulin tolerance tests (ITT). Data from the corresponding non-androgenized control animals are also show. Data are presented as the mean ± SEM. Data were analyzed by Student t tests or ANOVA followed by Student–Newman–Keuls tests (^*P < 0.05, ^**P < 0.01 versus corresponding control groups).

Figure 3

LH responses to KP-54 in the three preclinical rat models of PCOS. Profiles of serum LH responses to kisspeptin-54 (KP-54) administration are presented from the three preclinical models of PCOS (PNA, NeNA and PWA) at adulthood. The animals were injected for 11 days with a daily bolus of KP-54 (100 μg/kg; N = 10 per PCOS model) or 0.9% saline (N = 10 per model), and serial blood sampling was conducted on Days 1, 4, 7 and 11 of treatment, just before (0) or at the indicated time intervals after s.c. KP-54 injection. Androgenized animals from each PCOS model, injected with vehicle, were run in parallel for comparative purposes. In addition, the profiles of LH responses to a single bolus of KP-54 or vehicle in control (non-androgenized) female rats are also shown in the left panels. Data are presented as the mean ± SEM. PNA 1D: postnatal day 1.

Figure 4

FSH responses to KP-54 in the three preclinical rat models of PCOS. Profiles of serum FSH responses to KP-54 administration are presented from the three preclinical models of PCOS (PNA, NeNA and PWA) at adulthood. The animals were injected for 11 days with a daily bolus of KP-54 (100 μg/kg; N = 10 per PCOS model) or 0.9% saline (N = 10 per model), and serial blood sampling was conducted on Days 1, 4, 7 and 11 of treatment, just before (0) or at the indicated time intervals after s.c. KP-54 injection. Androgenized animals from each PCOS model, injected with vehicle, were run in parallel for comparative purposes. In addition, the profiles of LH responses to a single bolus of KP-54 or vehicle in control (non-androgenized) female rats are also shown in the left panels. Data are presented as the mean ± SEM.

Figure 5

Integral gonadotrophin responses to KP-54 in the three preclinical rat models of PCOS. Integral LH (upper panel) and FSH (lower panel) responses to KP-54 administration are presented from the three preclinical models of PCOS (PNA, NeNA and PWA; N = 10 per model) at adulthood. Integral responses were calculated as AUC over the 240-min period following KP-54 administration, for each indicated day of treatment. Data in these graphs are the AUC values of the hormonal profiles presented in Figs 3 and 4. Data are presented as the mean ± SEM. Data were analyzed by ANOVA followed by Student–Newman–Keuls tests (^**P < 0.01 versus corresponding PNA values; aP < 0.01 versus corresponding NeNA values).

Figure 6

Ovarian responses to KP-54 in the three preclinical rat models of PCOS. In panel I, UW and OW, as well as the number of fresh CL per ovary, after 11-day treatment with KP-54 or vehicle are shown from the three preclinical models of PCOS (PNA, NeNA and PWA); dotted lines indicate reference OW, UW and CL in control (non-androgenized) adult female rats. Data are presented as mean ± SEM and were analyzed by ANOVA followed by Student–Newman–Keuls multiple range tests (^*P < 0.05, ^**P < 0.01, ^***P < 0.001 versus control rats; aP < 0.01 versus PNA rats treated with Vehicle). In panel II, representative images of ovarian histology of adult NeNA rats treated as adults with vehicle (A, B) or KP-54 (C, D). The presence of CL, Cy, atrophic follicles (AF) and cumulus oocyte complexes (COCs) is indicated. Note that while NeNA rats treated with vehicle failed to show CL (as index of anovulation), KP-54 administration to NeNA rats rescued ovulation, as denoted by the appearance of CL and COC. Scale bars are as follows: A (300 μm); B (75 μm); C (200 μm); and D (300 μm). In panel III, scoring of follicular maturation and ovulatory status in PNA, NeNA and PWA rats, following treatment as adults with either vehicle (blue dots) or KP-54 (red dots), is presented. Note that each dot represents the score of individual ovaries. Depending on initial morphometric data and optimally fixed tissue availability, ovaries from 5–10 independent animals were studied per model. Control, non-androgenized rats are included (black dots) for reference purposes. Analyses were conducted in ovarian samples obtained after completion of the 11-day period of treatment with KP-54 or vehicle. Scoring of follicular maturation was based on the identification of the most advanced stage of large growing follicles, divided into these classes: F1 (275–350 μm), F2 (351–400 μm), F3 (401–450 μm), F4 (451–575 μm) and F5 (>575 μm). The presence of one or two generations (GEN) of corpora lutea denoted the occurrence of one or two ovulatory cycles.

Figure 7

Schematic of protocol, and gonadotrophin and ovarian hormone responses to KP-54 in 12 individual women with PCOS. In the left panel, a schematic showing the timing of KP-54 administration in the (A) dose-exploration arm and (B) constant dose arm of the study. In both cases, KP-54 was started after a progestogen-induced withdrawal menses and administered s.c. bd for 21 days. Group sizes were N = 5 in (A; split into N = 3 and 2, in two escalation protocols, as depicted) and N = 7 in (B). In (A), multiple blood samples were taken over periods of 7.5 h, as indicated for pharmacokinetic (PK) analysis, and in both, blood samples and transvaginal ultrasound were performed twice weekly as indicated. In the right panels, serum hormone concentrations before and after repeated administration of KP-54 at 3.2, 6.4, 9.6 and 12.8 nmol/kg, and 7 days following the last dose. KP-54 was administered s.c. twice daily for 21 days; Day 1 samples were taken immediately before first dose. Pre- (day-1) and post-KP (7-day after completion of treatments) hormonal levels are shown. Significant overall rises in LH and estradiol (E2) levels during the period of KP-54 administration were detected (Student t tests), with no changes in FSH or inhibin B levels. Blue symbols indicate those patients treated with increasing doses of KP-54, and red symbols denote those treated with a fixed dose of 9.6 nmol/kg twice daily throughout. MPA: medroxyprogesterone acetate, 10 mg bd for 7 days to induce a withdrawal bleed. USS: ultrasound scan.

Figure 8

Individual gonadotrophin and ovarian hormone responses to KP-54 in women with PCOS. Serum hormones and ultrasound-measured diameter of the leading follicle and endometrial thickness (ET) in individual women (panels A/B, C/D and E display data from each of the three women) administered 9.6 nmol/kg s.c. bd for 21 days to illustrate the variation in response. Blue line: LH; black line, FSH; orange line, E2; grey line, inhibin B. Blue columns, follicle diameter; orange columns, ET. In the woman whose results are shown in panels A/B, there was a small but consistent increase in the size of the dominant follicle during 3 weeks of KP-54 treatment, with a more marked rise immediately thereafter, with a coincident increase in serum E₂ and ET. This culminated in an LH surge, disappearance of the dominant follicle and a subsequent serum progesterone level of 25.4 nmol/L detected thereafter, indicating ovulation. In panels C/D, a rise in LH with a limited ovarian response is illustrated, with a rise in E₂ during the third week of treatment and a transient increase in diameter of the leading follicle to >10 mm. Panel E shows evidence of desensitization, with declines in LH and E2, accompanied by some light vaginal bleeding.