Minipuberty of human infancy – A window of opportunity to evaluate hypogonadism and differences of sex development?

Activation of the hypothalamic-pituitary-gonadal (HPG) axis happens in 3 phases during life. The first phase is during fetal life and is only separated from the second phase, called minipuberty, by the high concentration of placental hormones at birth. The third period of activation of the HPG axis is puberty and is well-described. Minipuberty consists of the neonatal activation of the HPG axis, mainly in the first 1–6 months, where the resulting high levels of gonadotropins and sex steroids induce the maturation of sexual organs in both sexes. With gonadal activation, testosterone levels rise in boys with peak levels after 1–3 months, which results in penile and testicular growth. In girls, gonadal activation leads to follicular maturation and a fluctuating increase in estrogen levels, with more controversy regarding the actual influence on the target tissue. The regulation of the HPG axis is complex, involving many biological and environmental factors. Only a few of these have known effects. Many details of this complex interaction of factors remain to be elucidated in order to understand the mechanisms underlying the first postnatal activation of the HPG axis as well as mechanisms shutting down the HPG axis, resulting in the hormonal quiescence observed between minipuberty and puberty. Minipuberty allows for the maturation of sexual organs and forms a platform for future fertility, but the long-term significance is still not absolutely clear. However, it provides a window of opportunity in the early detection of differences of sexual development, offering the possibility of initiating early medical treatment in some cases.


Introduction
The age of pubertal onset has changed markedly over time especially in girls. 1,2) Thus, we have recently demonstrated in systematic review and meta-analysis a worldwide decline in age at thelarche corresponding to 3 months per decade from 1977 to 2013. 3) The mechanisms behind this secular trend are quite complex and multifactorial. There is a wide interindividual variation in the timing of puberty, which can be partially explained by genetic and environmental changes such as nutrition, but most parts remain unexplained. Onset of puberty is directly linked to the activation of the hypothalamic-pituitary-gonadal (HPG) hormone axis resulting in the development of secondary sexual characteristics and achievement of reproductive capacity. The pulsatile hypothalamic gonadotropin releasing hormone (GnRH) stimulates the secretion from pituitary gonadotrophs in terms of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) into the bloodstream, and subsequent stimulation of gonadal hormone production. However, important developmental steps occur much earlier during the transient activation of the HPG axis already in fetal life during the second trimester Minipuberty of human infancy -A window of opportunity to evaluate hypogonadism and differences of sex development?
www.e-apem.org of pregnancy [4][5][6][7][8] and again after birth at 1-6 months of postnatal age during the so-called minipuberty. [8][9][10][11][12] Minipuberty has been suggested as a critical window of programming with lifelong implications. It has been proposed that minipuberty is linked to the timing of puberty and that it may be a marker of reproductive capacity in adult life, although the physiological role and clinical importance of the elevated sex steroids during minipuberty still remain to be elucidated. We suggest that minipuberty may be used as an early "window of opportunity" to detect and even treat reproductive disorders.

Sexual differentiation
Sexual differentiation is a complex genetic and hormonal event starting in early fetal life. Until week 6 of gestation, the gonads are indifferent. Genetic sex is determined by the sex chromosome constitution (46,XX or 46,XY) and the presence or absence of the sex-determining region of the Y (SRY) gene, which together with other genes determines the fate of the primitive gonad -will it differentiate into a testis or an ovary? In the presence of testes and specific testicular hormones, internal and external genitalia will follow the male pathway, whereas their absence will direct the development into the female pathway. 13) In fetal life, marked changes in the HPG-axis hormone activity occur especially in the second trimester of pregnancy, with the secretion of pituitary FSH and LH increasing already from week 10 of gestation. Peak levels of FSH and LH are seen in midgestation followed by a decrease to almost undetectable levels at term. In females, the concentration of both LH and FSH is significantly higher than in males. 7) By contrast, inhibin B secretion from gonads are significantly higher in male than in female fetuses at gestational age 26 to 28 weeks, although the levels decrease with ongoing gestation to similar levels at term. 5) In the male fetus, the masculinization of genitalia and testicular descent depends on the production of testosterone and insulin-like peptide 3 (INSL3) from the Leydig cells and anti-Müllerian hormone (AMH) produced by the Sertoli cells.  14) www.e-apem.org cells with the stimulation from human chorionic gonadotropin (hCG) in the first trimester, where hCG also controls the secretion of testosterone. 14) After the 9th week of gestation, the secretion of testosterone is increasingly controlled by LH, and a clear increase in testosterone is observed. The concentration reaches its maximum between 11 and 14 weeks of gestation, with peak levels similar to adult values. Hereafter the testosterone concentration decreases towards term (Fig. 1). 15,16) During fetal life and early infancy, Sertoli cells in the male testes are immature. The immature Sertoli cells secrete AMH with the main function of triggering the involution of the Müllerian ducts in the male fetus. 17) While testosterone and AMH are key to the masculinization of the male fetus, inducing the formation of male urogenital structures and involution of the Müllerian ducts, respectively, estrogen and the lack of AMH is central to the development of the female reproductive system. In the absence of AMH, the Müllerian duct develops into fallopian tubes, uterus, and the upper third of the vagina, 18) and the Wolffian ducts regress. The main source of fetal estrogen is the placenta 8) and ovarian follicles are created in the female ovaries when estrogen concentrations are high, which happens rapidly from 14 weeks of gestation and stabilizes at around week 34. While there is a correlation between the levels of circulating estrogen and the formation of primordial follicles in the fetus, further studies are needed to interpret the roles of FSH and LH during fetal life. 14)

Regulation of the HPG hormone axis
During early embryogenesis, GnRH neurons migrate from their origin in the olfactory placode to the hypothalamus and the median eminence, where GnRH is released into the pituitary portal vessels (for review, see 19) ). Several neurotransmitters have been identified to play a key role in regulating the GnRH release. Many of them derive from the same hypothalamic cell type, the KNDy neurons, which releases kisspeptin, neurokinin B (NKB), and dynorphin. Kisspeptin is a peptide, which stimulates the release of GnRH by activating the Kisspeptin receptor (KISS1R), while NKB and dynorphin participate in regulating the release of kisspeptin. 20) Studies have shown, that the inactivation of the gene encoding KISS1R results in pubertal failure. 21,22) In contrast to kisspeptin and neurokinin B, which have been identified to stimulate the activation of the HPG axis, Makorin RING-finger Protein 3 (MKRN3) has been found to act as an inhibitor. A study identified mutations causing loss of function in the MKRN3 gene to cause central precocious puberty (CPP). 23) Interestingly, MKRN3 mutations have later proven to be the most common genetic cause of familial CPP. Even though children with MKRN3 mutations are rare, such findings significantly contribute to the understanding of the central regulation of the HPG axis. MKRN3 is considered the brake on the HPG-axis, which is activated in minipuberty and released again in childhood resulting in pubertal onset. In accordance, circulating MKRN3 levels decline in boys and girls a few years before the HPG axis is reactivated in puberty. 24,25) The hypothalamic GnRH pulse generator releases GnRH pulses of 1-2 per hour into the hypophyseal portal circulation and stimulates the gonadotrophs cells in the anterior pituitary gland. This leads to the pulsatile release of gonadotropins, FSH and LH, into the systemic bloodstream and to the target tissue in testes or ovaries.
In the testes, FSH stimulates Sertoli cells to produce inhibin B and AMH, while LH stimulates Leydig cells to produce testosterone and INSL3. In the ovaries, LH stimulates theca cells to produce testosterone, which is converted into estradiol by FSH-driven aromatase activity in ovarian granulosa cells. Consequently, estradiol is released into circulation. In addition, inhibin A and B and AMH are produced in granulosa cells and released from the ovaries. 26)

Minipuberty in healthy infants
In postnatal life, the HPG axis is transiently activated in the first months of life, during the minipuberty. At birth, serum concentrations of LH and FSH are low due to the suppressive effects of the high concentrations of placental hormones, 10,12) which are gradually cleared with total clearance after approximately one week. This removes the restraint, leading to elevated gonadotropin levels until 6 to 9 months of age, with the exception of FSH, which remains elevated in girls for up to 3 to 4 years. 10,12) The LH/FSH ratio increases markedly in minipuberty and clearly separates male infants from female infants. 27) In boys, the postnatal rise in LH and FSH is associated with increases in testosterone and INSL-3 from the Leydig cells and inhibin B and AMH from the Sertoli cells. 28,29) Similar to gonadotropin, testosterone levels are low in cord blood, but increase shortly after birth. It then reaches its peak between 1 and 3 months of age, with levels reported just beneath adult concentrations, before declining and stabilizing after approximately 4-6 months at undetectable levels ( Fig. 1). 10,12) The number of Leydig cells increases concomitantly until 3 months of age when the fetal Leydig cells go into apoptosis. 30) Similarly, the Sertoli cells develop and the number of germ cells increases in the first months of age. 31) However, they do not express androgen receptors during infancy. Consequently, despite high intratesticular testosterone levels, spermatogenesis is not initiated and the production of AMH is not inhibited. Thus, the Sertoli cells produce AMH at very high concentrations during infancy, peaking at around 3 months of age, even in the presence of high levels of testosterone. 32) From the time of puberty, the main role of AMH remains to be determined. 28) At birth, circulating AMH is 35 times lower in female than in male cord blood. 33) The following postnatal surge in AMH in healthy female infants correlates with the rise in the number of growing ovarian follicles. 34) This correlation is supported by the undetectable AMH levels in postmenopausal women 35) and patients with Turner syndrome, 33) and by reports of very high levels of AMH in ovarian tumors. 36) Minipuberty and the associated sex steroid levels are important for genital development in boys. Thus, penile growth is www.e-apem.org positively associated with testosterone levels from birth until 3 years of age with the highest growth velocity of 1 mm per month present during the first 3 months. 37) Likewise, minipuberty is associated with testicular growth 38,39) which is due to increased length of seminiferous tubules, total germ cell number, and Sertoli cell number, as well as decreased apoptosis of Sertoli cells (for review, see 40) ). A positive correlation between FSH levels and testicular growth has been found, with testicular volume increasing from 0.27 to 0.44 cm 3 from birth until 5-6 months, and then decreasing to 0.31 cm 3 at 9 months of age. 38) Genital development during minipuberty is well described in boys whereas the association between elevated reproductive hormones and the development of the female reproductive organs in minipuberty is more unclear. Estradiol levels are high in cord blood in both sexes but decrease during the following days. However, in girls, the endogenous production increases after approximately one week, corresponding with the increase in follicular development. 41) The estradiol levels then proceed to fluctuate while decreasing gradually towards the second year of life. 42) In fact, large ovarian follicles are seen more often during the first than during the second year of life. 42,43) Estradiol levels are significantly higher in the intrauterine environment, leading to the development of the uterus and mammary glands. Thus, both boys and girls have developed breast tissue at birth and later in infancy, but breast tissue grows more in girls than in boys, which is likely an effect of the estrogen surge in girls. 44,45) FSH, AMH, and breast tissue size in female minipuberty are associated with genetic variation in FSH action variants in the FSH-Receptor (FSHR) gene. 46) The length of the uterus grows until day 7 and then decreases during the first 3 months, before stabilizing in size until the second year of life. 42) This leads to controversy regarding the effect of estradiol on the uterus.
Elevated sex steroids in minipuberty may also influence other physiological endpoints such as body composition and linear growth. During the first years of life, thyroid hormones along with insulin and glucocorticoids, and hereafter growth hormone, and insulin-like growth factors influence linear growth. In puberty, sex steroids are important for the pubertal growth spurt, but importantly, sex steroids also play a role for linear growth in minipuberty. Thus, testosterone levels are high in boys during minipuberty, which may partly explain the higher growth velocity observed in boys compared to girls. Interestingly, the maximum growth velocity difference between boys and girls is observed at 1 month of age, when male testosterone serum levels are at their peak. 47,48) In addition, boys with congenital hypogonadotropic hypogonadism (CHH), thus lacking the infant testosterone surge, have decreased growth rate during minipuberty compared to controls. 49) The hormonal differences between boys and girls during minipuberty appears to be important for sex differences in body composition, with males accumulating more lean mass and ultimately gaining a lower percent fat mass than females. 50)

Minipuberty in different species
The transition from one life-history stage to the next is initiated by switch mechanisms such as the rise in hormones during the minipuberty. Evolutionary theories try to explain the variations in these life stages explored both among and within species. Humans differ from other species by being born immature and helpless and having a relatively short period of infancy followed by a long childhood period. 51) Previous studies investigated the postnatal hormonal levels in several animal species such as mouse, rat, and horse as well as newborn boys and found a postnatal surge of reproductive hormones of mammals in general, but these studies were limited by the less precise assays for measurement of hormones available at that time. 52) However, in other higher primates, minipuberty has been discovered with activation of the pulsatile GnRH release during infancy followed by the quiescent period until the onset of puberty in these species. In vitro studies revealed that embryonic GnRH neurons from rhesus monkeys have a pulsatile secretion and suggested that the pulsatile release of GnRH becomes functional during early fetal development. 53) Another study on rhesus monkeys indicated that the switch that turns off the GnRH pulsation during infancy was associated with a decline in the numbers of kisspeptin neurons in the arcuate nucleus in agonadal male monkeys. 54) In addition, recent rodent studies revealed that minipuberty also occurs in mice and that gonocyte transformation is influenced by the transient FSH signaling pathway. 55) Thus, minipuberty is not limited to the human neonate but is observed in other species. However, humans differ compared to other species in their much longer duration of the childhood period spanning the interval between minipuberty and puberty. The evolutionary role of this long hormonally quiescent period in humans compared to other species remains speculative, but the acquisition of skills to accommodate a complex society before being ready to reproduce appears a plausible explanation.

Minipuberty in various differences of sexual development conditions
The HPG axis can readily be evaluated in newborns suspected of differences of sexual development (DSD) conditions during minipuberty. A few studies on reproductive hormones in infants with DSD conditions exist.
Infants with CHH have subnormal FSH, LH, and inhibin B levels in minipuberty. 56) The postnatal surge of hormones is absent in boys with CHH, who often present with bilateral cryptorchidism with or without micropenis. Hypospadias rarely occur in CHH because first trimester placental hCG secretion ensure sufficient androgen production needed for early urethral closure, whereas testicular descent and penile growth are dependent on sufficient testosterone and INSL3 secretion in the second half of pregnancy. 29,56) In newborns with cryptorchidism reproductive hormones during minipuberty may be altered. Thus, higher FSH and www.e-apem.org LH levels, and reduced inhibin B and INSL3 have been demonstrated in cryptorchid boys. 29) Newborn boys with non-palpable testes remain a diagnostic challenge. Clinically it can be difficult to distinguish between boys with anorchia who do not have functional testicular tissue and boys with bilateral cryptorchidism who have intraabdominal testes. However, serum concentrations of LH, FSH, AMH, inhibin B, and testosterone hormone levels in minipuberty differ between the groups and can, therefore, be used as clinical tool to determine the presence of functional testicular tissue during minipuberty in infant boys. 57) Furthermore, an exaggerated biphasic gonadotropin pattern is present in boys with anorchia, with higher levels of FSH and LH as well as undetectable AMH and inhibin B levels during minipuberty compared to healthy boys. 57) Klinefelter syndrome (47,XXY) is characterized by primary testicular failure and infertility in adulthood, whereas data on the HPG axis during minipuberty are sparse and contradicting. [58][59][60] However, one recent study including 38 infants and measurement of testosterone by means of liquid chromatography/tandem mass spectrometry showed that the majority of infants presented with a testosterone concentration within the normal range, but the testosterone concentrations in the infants with Klinefelter syndrome were significantly lower as compared with controls. 61,62) Despite reduced testosterone, INSL3 and LH concentrations were normal in these infants. In addition, normal inhibin B and AMH concentrations were found, but significantly elevated FSH in minipuberty. 61,62) Turner syndrome (45,X) is characterized by primary ovarian failure, hypergonadotropic hypogonadism, lack of pubertal development, and infertility. A longitudinal study assessed the HPG axis during infancy in patients with Turner syndrome. FSH and LH demonstrate biphasic age patterns with peaks at infancy and expected puberty, similar to patterns seen in healthy girls, with no dependence of karyotype. Importantly, gonadotropins were highly elevated in minipuberty compared to healthy girls. 63) Children with X-linked adrenal hypoplasia congenita caused by NR0B1 mutations are characterized by adrenal failure and hypogonadotropic hypogonadism, which is not evident in infancy, as studies have suggested that reproductive hormones are normal during minipuberty. 64,65) Prader Willi syndrome (PWS) is caused by a deletion of a section of the paternal derived chromosome 15, which results in mental retardation, obesity, and hypothalamic dysfunction. At birth cryptorchidism, scrotal hypoplasia, and micropenis are frequently reported. By the time of puberty, many of the boys have hypogonadotropic hypogonadism due to their hypothalamic dysfunction, but some of the boys may have hypergonadotropic hypogonadism due to their cryptorchidism. However, in infant PWS boys, levels of LH, FSH, and testosterone during minipuberty were all within reference ranges of healthy boys. 66,67) Infants with AR mutations resulting in complete androgen insensitivity syndrome (AIS) have lowered LH and testosterone levels, and male LH/FSH ratio in minipuberty, 27) whereas infants with partial AIS have normal or high LH and testosterone levels, 68) suggesting some physiological role of androgen signaling in the HPG axis activation during minipuberty.
Infants with aromatase deficiency due to CYP19 mutations which results in a lack of estradiol, present with elevated FSH and LH levels during minipuberty in girls, which may suggest that estrogen feedback on the HPG axis activation in minipuberty is more important in girls compared to boys. 69,70)

Treatment of hypogonadism in minipuberty
Minipuberty also represents a "window of opportunity" for treatment in conditions with an impaired HPG axis. Treatment with testosterone is effective with regards to penile growth, and a short course of testosterone treatment of micropenis in otherwise healthy boys is widely accepted. Such treatment during minipuberty is effective and well-tolerated without virilization or disturbances of growth. 71,72) Few reports of infants with hypogonadotropic hypogonadism treated with gonadotropins during minipuberty exist. [73][74][75][76] These indicate that short-term treatment with recombinant FSH and hCG may mimic the physiological minipuberty by stimulating the production of testosterone, inhibin B, and AMH and potentially stimulating the proliferation of Sertoli cells (for review, see 77) ).

Minipuberty -a "window of opportunity"
Minipuberty may represent a "window of opportunity" to evaluate the HPG axis by measuring the basal hormone concentrations without stimulation tests in infants with suspected of reproductive disorders. We have a unique opportunity to evaluate the spontaneous function of the HPG hormone axis during minipuberty, which is lost hereafter for another 10 years until the HPG axis is reactivated in puberty. It is therefore recommended that serum FSH, LH, and testosterone are measured in minipuberty by reliable and ultrasensitive hormone assays in infants with DSD or suspected of CHH. 78)

Conflicts of interest
No potential conflict of interest relevant to this article was reported.