Association of Follicular Fluid Kisspeptin-54 Levels and Ovarian Response to Stimulation

INTRODUCTION

Kisspeptin-54 (Kp-54) is a neuropeptide produced by the hypothalamus and is thought to be an important regulator of several biological processes. Kp-54 is encoded by the KISS-1 gene, which was initially identified in melanoma cells as a metastasis suppressor gene.^[1] It was originally named “metastin” due to its inhibitory effect on chemotaxis, invasion, and metastasis.^[2] It has since been identified in many other tissues throughout the body, including the pancreas, intestine, ovaries, and placenta.^[2–4] Kp-54 is secreted by kisspeptin neurones, which in humans exist primarily in the infundibular nucleus of the hypothalamus.^[5] The target of Kp-54 is a G-protein coupled receptor called GPR-54, which is similarly expressed in diverse tissues. The seemingly widespread role of Kp-54 led researchers to suspect diverse functions outside of melanoma regulation; this was later confirmed when a study by de Roux et al. demonstrated that a mutation in GPR-54 led to hypogonadotropic hypogonadism.^[6] A similar study by Seminara et al. showed that GPR-54 mutations result in autosomal recessive idiopathic hypogonadotropic hypogonadism in humans and mice.^[7] Kp-54 later emerged as a potent activator of gonadotropin-releasing hormone (GnRH) neurones, further implicating the role of Kp-54 in reproductive function.^[8–10]

Kp-54 has since been identified as a key member of the hypothalamic-pituitary-gonadal (HPG) axis, though its entire regulatory role remains undetermined. Kp-54 likely influences the secretion of GnRH by integrating central and peripheral signals; GnRH secretion can then stimulate the release of luteinising hormone and follicle-stimulating hormone.^[11] These gonadotropins stimulate the release of sex steroid hormones, which ultimately exert feedback on the hypothalamus and complete the regulatory loop. Though GnRH neurones in the hypothalamus are known to respond to sex steroid hormone levels, the mechanism by which this occurs is unclear because these neurones lack oestrogen and progesterone receptors.^[12,13] Kp-54 may represent the “missing link” that connects the players of the HPG axis, as Kp-54 neurones express sex steroid receptors and project over GnRH neurones, which express Kp-54 receptors.^[14–18]

Recent studies have identified even broader functions of Kp-54, including the regulation of puberty,^[6,7,10] ovulation,^[19–21] placentation,^[22–25] and pregnancy.^[26–29] In the realm of assisted reproductive technology (ART), Kp-54 has already been used as an effective oocyte maturation trigger with very low rates of ovarian hyperstimulation syndrome.^[30,31] It is currently regarded as the most potent stimulator of GnRH secretion.^[32,33] It has also been shown to increase granulosa cell receptivity to gonadotropin stimulation during in vitro fertilisation (IVF).^[34]

In light of Kp-54’s many physiologic roles, there has become interest in exploring its potential as a fertility biomarker or even a therapeutic target in addressing reproductive disorders. Because of this, a deeper understanding of Kp-54’s role in the ovary and HPG axis is desired. Several studies have measured serum Kp-54 concentrations in association with clinical features such as pregnancy, polycystic ovarian syndrome, and precocious puberty.^[22,35–38] Very little is known about Kp-54 concentrations in follicular fluid, though this information could be useful in providing insight into the role of Kp-54 in the local environment of the ovary and maturing oocyte. Furthermore, a deeper understanding of the relationship between follicular fluid Kp-54 (ff Kp-54) levels and response to controlled ovarian hyperstimulation in IVF patients could be informative in the prediction of fertility outcomes.

We hypothesised that there is an association between ff Kp-54 concentration and response to controlled ovarian stimulation. To test this hypothesis, we compared the ff Kp-54 concentrations of IVF patients with moderate versus high responses to controlled ovarian hyperstimulation. We also evaluated whether ff Kp-54 levels vary with age, BMI, gravidity, parity, or pregnancy outcomes.

MATERIAL AND METHODS

RESULTS

Of the 45 study participants, 23 were categorised into the moderate responder group and 22 were categorised into the high responder group. The mean ff Kp-54 concentration in the moderate responder group was 0.2077 ± 0.124 ng/ml, compared to 0.1905 ± 0.0886 ng/ml in the high responder group. These concentrations were not statistically significantly different (p = 0.5971).

Baseline characteristics of age, BMI, gravidity, and parity were evaluated. The mean age of participants in the moderate responder group was 34.9130 ± 3.4066 and 33.1364 ± 4.1332 in the high responder group. The mean BMI was 26.6227 ± 5.5835 for moderate responders and 28.4300 ± 5.5035 for high responders. Similarly, there was little difference between moderate and high responder groups with respect to gravidity and parity. The mean gravidity for moderate responders was 0.9333 ± 1.0998 and for high responders was 0.6923 ± 1.1821, whereas the mean for parity was 0.2000 ± 0.4140 for moderate responders and 0.3846 ± 0.6504 for high responders. No statistically significant differences in these baseline characteristics were noted between the comparison groups [Table 1]. Lastly, associations between ff Kp-54 concentration and age, BMI, gravidity, parity, and live birth per embryo transfer were assessed [Table 2]. No statistically significant associations were identified.

DISCUSSION

In this study population, we found no statistically significant differences in the ff Kp-54 concentration between moderate and high responders to ovarian stimulation. Similarly, no statistically significant associations were noted between ff Kp-54 concentration and age, BMI, gravidity, parity, or birth outcomes. Kp-54 has a variety of functions throughout the body, including roles in puberty,^[6,7,10] ovulation,^[19–21] placentation,^[22–25] and pregnancy.^[26–29] Kp-54 is already in use as an oocyte maturation trigger and may have other applications in ART.^[30,31] This study sought to better understand how knowledge of Kp-54 concentrations at the level of the ovarian follicle might predict treatment outcomes.

This study is limited by ff Kp-54 concentrations that were lower and more similar than expected, resulting in an underpowered study design. This was unanticipated, as our initial power calculation was appropriate and indicated a need for 8 participants in each group; over 45 total patients were recruited. Of note, the Kp-54 assays used in these studies were from different manufacturers. Strengths of the study include the similarity of the study populations at baseline. A study with more participants who have a more diverse age, BMI, gravidity, and parity would likely be more informative.

Though ff Kp-54 concentrations were found to be similar in patients with moderate and high response to controlled ovarian stimulation, there remains biological plausibility for Kp-54 as a fertility marker in other applications. Given its known physiologic roles in fertility and reproduction, many questions remain: How much could Kp-54 concentrations vary using different assays? Could there be different utilities for serum versus follicular fluid Kp-54 levels? Is one better than the other for predicting early pregnancy outcomes or IVF outcomes? If Kp-54 were to become commercially available (i.e., could be ordered easily in the hospital setting), could we correlate these levels with β-hCG or another hormonal marker for better predictive value?

CONCLUSION

In this study, we aimed to identify whether a difference in ff Kp-54 concentrations exists between moderate and high responders to controlled ovarian hyperstimulation. We found that no such difference exists in this study population. We also identified no association between ff Kp-54 concentration and age, BMI, gravidity, parity, or pregnancy outcomes. Additional investigation is needed to better understand the role of Kp-54 in the ovarian follicle and how it may be targeted to improve fertility outcomes.