Increased production of sex hormones in normal female subjects during adrenarche and gonadarche induces growth of the larynx, causing a rapid decrease in vocal pitch. Despite knowledge of this phenomenon, little is known regarding the sensitivity of the female larynx to androgen exposure during the pre-pubertal stage. This knowledge gap is primarily due to the absence of F0 analysis not only in the rare cases of childhood ACT 
, but also in the few cases of childhood virilization caused by topical exposure (usually accidental) to testosterone in the form of gels or patches 
. To our knowledge, this study is the first long-term analysis of F0 in female patients exposed to high levels of androgens at a very young age. We examined the effect of the timing of this exposure, specifically whether androgen exposure causes significant virilization during the pre-pubertal period (ranging from few months after birth to adrenarche).
Due to the fact that survivors of multiple ACT recurrence were excluded from the study because of prolonged androgen exposure, as were smokers and patients who presented with signs of inflammatory throat lesions, as well as the existence of a low ACT survival rate 
and follow-up losses, only 19 ACT patients were eligible for study participation. Nevertheless, the foremost research goal–determination of the impact of androgen exposure leading to the development of remarkable pre-pubertal virilizing signs on long-term (adult or adolescent) female F0–was achieved. All the subjects (19/19) demonstrated a good understanding of ACT signs and symptoms, especially due to their participation in previous studies 
. The variables of dose, timing, and duration of androgen exposure were all found to be determinants of virilization in the subjects, of whom 18 had experienced virilization during the pre-pubertal stage and 1 during puberty. Virilization was remarkable because of different degrees of pubic hair growth (19/19, 100%) and its association with clitoral enlargement in the majority of the subjects (14/19, 74%). All the subjects reported that their medical history had been uneventful after ACT treatment, and most reported that their clitoris had partially returned to normal size, although this parameter was not reevaluated in the most recent physical exam.
Although all the subjects had experienced a relatively long period (2 to 30 months) of androgen exposure in childhood, the exposure had not been sufficiently strong to cause loss of TH in any of the subjects. Furthermore, the observed decrease in F0 in 45% (5/11) of the subjects within 0.9 to 7.4 years after tumor resection, i.e., after normalization of androgen levels 
, had subsequently recovered to normal values in 3 cases, and only 18% (2/11; subjects 7 and 10) presented with partial virilization of F0 (165 and 169 Hz, respectively) and mild virilization of pitch. Interestingly, 1 of these 2 cases (1/11) had presented with a typical adult male F0 in childhood, suggesting that F0 may also be partially recovered (from 111 Hz to 169 Hz) in the absence of any treatment. Observation of mild or moderate virilization of pitch observed in 4 participants was consistent with the lowest detected F0, which ranged from 189 to 132 Hz. However, as expected, the method used to measure F0 was more reliable (and quantitative) than that used to evaluate pitch.
Although F0 has previously been investigated in female subjects with CAH 
, these subjects may have been periodically exposed to increased levels of androgens due to suboptimal treatment with glucocorticoids 
, making it difficult to identify the timing and duration of androgenic effects and making these subjects less than ideal models. describes the timing and degrees of androgen exposure (Tanner stage) as a reflection of the intensity of the accumulated androgen effect. The determinants of laryngeal tissue sensitivity to androgens and extent of differentiation shown in were defined on the basis of the results of this study and previous studies 
. Lack of analysis regarding early androgen exposure, particularly during the initial development of ACT symptoms, combined with differences found among the types of androgens examined has prevented systematic comparison of hormone levels. Despite this challenge, the findings of this study reveal the differences between the impact of ACT and CAH, specifically that androgen exposure during early childhood is less likely to result in an irreversibly virilized F0, and any significant reductions in F0 at an early age are likely to be partially reversed by increases to a normal F0 between childhood and adulthood.
A flow chart summarizing the results of this and other studies investigating the postnatal periods of lowest and greatest larynx sensitivity to androgen exposure and the mechanism of vocal development in response to this exposure.
Previous authors have proposed the existence of an androgenic effect that includes the development of increased mass in laryngeal tissue. Current knowledge of the mechanism by which steroids induce sexual differentiation of vocal production at the brain level is limited to animal models, particularly songbirds 
and female African clawed frogs (Xenopus laevis
. The female laryngeal muscle differs from the male laryngeal muscle 
, as most female laryngeal neuromuscular synapses are stronger than the corresponding male synapses 
. At the CNS level, these differences are responsible for generating sexually dimorphic vocalizations, which are recorded in motor neurons 
The study data shown in support the hypothesis that a period of low sensitivity in laryngeal tissue exists between birth and adrenarche. The experience of subject 14, who had developed ACT at the onset of or before adrenarche, with her parents first reporting virilizing signs when she was less than 6 years old, provides particular evidence of this phenomenon. Moreover, subject 14 had also been exposed to particularly high levels of androgens for a prolonged period lasting until close to the onset of adrenarche, which, being a period of tissue maturation in the larynx, had increased the probability that she would experience vocal virilization.
As shown in , 3 subjects presented with CS, as confirmed by measurement of mild to modest elevations in cortisol levels. However, it is unclear whether the elevation in serum cortisol concentration impacts the effects of androgen on laryngeal tissue. Therefore, further studies are necessary to evaluate the effects of the concentration, duration, and timing of cortisol exposure on the F0 of children with CS.
In conclusion, the findings of this study support the hypothesis that female laryngeal tissue is less sensitive to androgen exposure between birth and adrenarche. Particularly compelling was the finding of F0 virilization in only 1 subject, who had experienced androgen exposure immediately before the period at which adrenarche typically begins and for a longer duration (30 months) than that in the other subjects. Although a subject who had experienced androgen exposure during adrenarche had subsequently developed a normal female F0, she had also experienced elevated cortisol levels during the same period (8 months before ACT diagnosis) based on the reported clinical signs. Thus, the possibility that cortisol production had exerted an inhibitory effect on androgen exposure cannot be ruled out in this case. Furthermore, it was not possible to define the exact contribution of ACT androgens, which most likely acted during the final stages of adrenarche. The findings also indicate that differential larynx sensitivity to androgens exposure in childhood and F0 irreversibility later in adulthood are age-, androgen concentration-, duration-, and timing-dependent events that may also be affected by exposure to inhibitory or stimulatory hormones, such as glucocorticoids. Further studies are now required to better characterize each of these factors.