Title: Kisspeptin expression in the human infundibular nucleus in relation to sex, gender identity and sexual orientation

word count: 249 / max 250) Context. Since the discovery of its central role in reproduction, our functional neuroanatomical knowledge of the hypothalamic kisspeptin system is predominantly based on animal studies. Although sex differences in kisspeptin expression have been shown in humans in adulthood, the developmental origin of this sex difference o is unknown. Objectives. Our objectives were to determine 1) when during development the sex difference in kisspeptin expression in the infundibular nucleus (INF) would emerge and 2) whether this sex difference is related to sexual orientation or transsexuality. Design and setting. Post-mortem hypothalamic tissues were collected by the Netherlands Brain Bank and sections were stained for kisspeptin by immunohistochemistry. Patients. Hypothalami of 44 control subjects were categorized into 3 periods: infant/prepubertal (6 girls/7 boys), adult (11 women/7 men) and elderly (6 aged women/7 aged men). Eight male-to-female (MTF) transsexuals, 3 HIV+ heterosexual men and 5 HIV+ homosexual men were also analyzed. Main outcome measure. We estimated the total number of kisspeptin-immunoreactive neurons within the INF. Results. Quantitative analysis confirmed that the human infundibular kisspeptin system exhibits a female-dominant sex difference. The number of kisspeptin neurons is significantly greater in the infant/prepubertal and elderly periods compared to the adult period. Finally, in MTF transsexuals – but not homosexual men -, a female-typical kisspeptin expression was observed. Conclusions. These findings suggest 1) that infundibular kisspeptin neurons are sensitive to both early organizing and activating actions of sex steroids and 2) that the sex-reversal observed in MTF transsexuals probably reflects, at least partially, an atypical brain sexual differentiation. Introduction The human brain is thought to be sexually differentiated under the influence of testosterone acting in the male fetus [1]. A large number of morphological and neurochemical sex differences induced by sex steroid hormones during the perinatal period are found predominantly in limbic-hypothalamic regions that participate in the neural control of reproduction, including the secretion of gonadotropin-releasing hormone (GnRH; [2]). In the human hypothalamus, post-mortem and neuroimaging studies have identified several structural [3-8] and functional [9-10] sex differences, some of which seemed to be related to gender identity and sexual orientation [1, 11-13]. Human genetic studies demonstrated that kisspeptin [14] and neurokinin B (NKB) signaling [15] are both potent regulators of GnRH secretion and are therefore critically involved in the onset of puberty and the maintenance of adult reproductive function. We recently reported that the NKB system exhibits a female-dominant sex difference in the human infundibular nucleus (INF) that reached only significance in adulthood as well as that male-to-female (MTF) transsexuals had a female-typical infundibular NKB system [16]. At present, only a fragmented overview of kisspeptin expression throughout life is available in humans. Kisspeptin expression in the human brain has been reported in fetuses [17] and adult subjects [18-20]. Sex differences were observed in adulthood with women having a greater number of kisspeptin neurons in the INF than men [19-20]. In addition, an increase and hypertrophy of neurons expressing kisspeptin mRNA was found after menopause [18]. However, in humans, it is unknown when sex differences in kisspeptin expression arise during development under the influence of sex hormones. Therefore, in the present study, we analyzed infundibular kisspeptin expression from birth to old age in both sexes. By including brain material from MTF transsexuals and homosexual men, we also investigated whether this sex difference was related to gender identity and sexual orientation or rather would reflect circulating steroid levels. Materials and methods