The authors have declared that no competing interests exist.
Conceived and designed the experiments: BCF. Performed the experiments: LG MLL MIRG FP. Analyzed the data: BCF CAL. Contributed reagents/materials/analysis tools: ERS GC IPSZ CAL. Wrote the paper: LG BCF GC CAL.
The impact of early postnatal androgen exposure on female laryngeal tissue may depend on certain characteristics of this exposure. We assessed the impact of the dose, duration, and timing of early androgen exposure on the vocal development of female subjects who had been treated for adrenocortical tumor (ACT) in childhood.
The long-term effects of androgen exposure on the fundamental vocal frequency (F0), vocal pitch, and final height and the presence of virilizing signs were examined in 9 adult (age, 18.4 to 33.5 years) and 10 adolescent (13.6 to 17.8 years) female ACT patients. We also compared the current values with values obtained 0.9 years to 7.4 years after these subjects had undergone ACT surgery, a period during which they had shown normal androgen levels.
Of the 19 subjects, 17 (89%) had been diagnosed with ACT before 4 years of age, 1 (5%) at 8.16 years, and 1 (5%) at 10.75 years. Androgen exposure (2 to 30 months) was sufficiently strong to cause pubic hair growth in all subjects and clitoromegaly in 74% (14/19) of the subjects, but did not reduce their height from the target value. Although androgen exposure induced a remarkable reduction in F0 (132 Hz) and moderate pitch virilization in 1 subject and partial F0 virilization, resulting in F0 of 165 and 169 Hz, in 2 subjects, the majority had normal F0 ranging from 189 to 245 Hz.
Female laryngeal tissue is less sensitive to androgen exposure between birth and adrenarche than during other periods. Differential larynx sensitivity to androgen exposure in childhood and F0 irreversibility in adulthood are age-, concentration-, duration-, and timing-dependent events that may also be affected by exposure to inhibitory or stimulatory hormones. Further studies are required to better characterize each of these factors.
Clinicians who are not voice specialists frequently examine vocal pitch as a proxy of perceptual vocal characteristics
The mean F0 of the mature voice of women in southern Brazil, the majority of whom are of European descent, is 211 Hz
Although the greatest F0 reduction in men occurs during puberty, male F0 may still continue to decrease until the fourth decade, and may subsequently increase during the sixth decade
The incidence of ACT in children younger than 15 years is at least 15 times higher in southern Brazil than that in the United States or Europe
Subject | Age (years) | Age at ACT diagnosis (years) | Length of virilizationbefore diagnosis (months) | Time betweenACT andstudy (years) | Tanner stageat ACT diagnosis | Clitoral enlargement? | Age at menarche (years) | Height (FS) | FS Z-score | Target height (TH) | TH Z-score | Initial F0 0.9 to 7.4y after ACT Dx/expected F0(Hz) | Present F0 11 to 15 (Hz) | Vocal pitch |
1 |
21.1 | 10.7 | 8 | 10.41 | P5 | Yes | 13 | 162.5 | −0.13 | 156.1 | −1.12 | 240/232 | 205 | F |
2 | 20.6 | 1.83 | 4 | 19.13 | P3 | Yes | 12 | 165 | 0.26 | 167 | 0.57 | 233/232 | 216 | F |
3 | 13.6 | 1.16 | 2 | 12.42 | P2 | Yes | 11 | 164.5 | 0.79 | 161 | 0.27 | 284/283 | 210 | F |
4 |
17.8 | 2.75 | 4 | 14.81 | P3 | Yes | 9 | 160.2 | −0.45 | 155 | −1.25 | 276/272 | 205 | F |
5 | 16.0 | 1.41 | 5 | 14.58 | P3 | Yes | 10 | 160 | −0.39 | 162 | −0.08 | 304/275 | 245 | F |
6 |
20.6 | 1.83 | 15 | 18.75 | P3 | Yes | 13 | 158 | −0.82 | 158.5 | −0.75 | 214/232 | 199 | F |
7 | 18.4 | 2.16 | 6 | 16.25 | P2 | Yes | 11 | 168.6 | 0.84 | 158 | −0.80 | 203/247 | 165 | M+ |
8 | 17.3 | 0.91 | 3 | 16.41 | P3 | No | 12 | 164.1 | 0.17 | 165.3 | 0.36 | 228/275 | 238 | F |
9 | 15.1 | 1.5 | 3 | 13.51 | P3 | Yes | 12 | 161.4 | −0.08 | 163.2 | 0.20 | 221/275 | 199 | F |
10 | 13.9 | 2.16 | 10 | 11.67 | P4 | Yes | 11 | 155 | −0.77 | 153.5 | −1.00 | 111/283 | 169 | M+ |
11 | 16.1 | 2.25 | 17 | 13.81 | P4 | Yes | 11 | 150.9 | −1.81 | 157.5 | −0.78 | 219/280 | 225 | F |
12 | 15.9 | 1.0 | 11 | 14.8 | P4 | Yes | 14 | 164 | 0.23 | 158 | −0.69 | NA | 217 | F |
13 | 21.9 | 3.16 | 6 | 18.74 | ? | ? | 11 | 166.4 | 0.47 | 153.5 | −1.51 | NA | 185 | F |
14 | 20.4 | 8.16 | 30 | 12.25 | ? | Yes | 9 | 152 | −1.74 | 160.5 | −0.44 | NA | 132 | M++ |
15 | 33.5 | 1.50 | 8 | 32.25 | ? | ? | 12 | 158 | −0.82 | 156.5 | −1.05 | NA | 217 | F |
16 | 15.1 | 2.50 | 13 | 12.66 | P2 | Yes | 14 | 165 | 0.48 | 165 | 0.48 | NA | 199 | F |
17 | 14.8 | 2.25 | 12 | 12.41 | P2 | ? | 13.5 | 158.5 | −0.48 | 160.2 | −0.22 | NA | 211 | F |
18 | 31.1 | 2.66 | 6 | 28.50 | ? | Yes | 12 | 170.8 | 1.16 | 162.4 | −0.14 | NA | 189 | M+ |
19 | 25.2 | 3.91 | 16 | 21.34 | P2 | No | 13 | 168 | 0.72 | 165 | 0.26 | NA | 216 | F |
Mean | 19.58 | 2.93 | 9.42 | 16.56 | 11.76 | 161.73 | −0.12 | 159.91 | −0.40 | − | 202 | |||
SD | 5.61 | 2.51 | 6.80 | 5.70 | 1.48 | 5.44 | 0.83 | 4.09 | 0.65 | − | 26 |
Cushing’s syndrome. F: female pitch; M+: mild virilization; M++: moderate virilization; NA: not available. Initial F0 was obtained several weeks and 4 years after ACT diagnosis, as reported by Leonel in her thesis in 2003
Subject | Age (years) | F0 | Vocal pitch |
1 | 21.5 | 215.3 | F |
2 | 23.9 | 202.4 | F |
3 | 17.5 | 219.7 | F |
4 | 26.5 | 244.5 | F |
5 | 26.1 | 231.2 | F |
6 | 21.2 | 207.3 | F |
Mean | 22.8 | 220.1 | |
SD | 3.3 | 15.6 |
Before the study initiation, approval for this study was obtained by the Ethics Committee of Pequeno Príncipe Hospital and from each subject or, if the subject was a minor, from a parent, by requesting the subject or her parent to read and sign the consent form. Thirty female patients who had completed more than 11.4 years of ACT treatment at the main reference hospitals of Curitiba, the capital of Paraná State, were invited to participate. Of the 22 volunteers who had ACT and agreed to participate, 19 met the inclusion criteria of (1) having experienced virilizing symptoms related to a previously treated adrenocortical tumor in the pre-pubertal stage, with 16 having been treated for adrenocortical carcinoma and 3 for adenoma; (2) being at least 13 years of age, and (3) having experienced menarche at least 2 years prior to study initiation. All 19 also met none of the exclusion criteria of (1) being a smoker, (2) having undergone vocal treatment, (3) currently experiencing edema of the vocal folds for other reasons, or (4) having experienced prolonged androgen exposure due to multiple ACT recurrence. Of the 19 subjects, whose age ranged from 13.6 to 33.5 years, 9 were adult females ranging from 18.4 to 33.5 years of age and 10 were adolescents ranging from 13.6 to 17.8 years of age. Comparison of actual height and predicted target height (TH) and vocal assessment were conducted for all subjects.
Pink curves correspond to female F0 spectra and blue curves to male F0 spectra. (1) Normal development of F0 differentiation in males and females. Adrenarche and activation of the hypothalamus–pituitary–gonadal axis (gonadarche) are the main events leading to attainment of adult vocal patterns. At puberty, the larynx, vocal folds, and vocal tract (e.g., the resonance tube up to the oral cavity) acquire increased mass, leading to variable degrees of F0 reduction in men at adrenarche and gonadarche and to partial reduction in women at adrenarche. F0 decreases and then stabilizes between the ages of 14 and 18 years. Mean F0, which is shown for normal male and female Brazilian subjects
Of the 19 subjects, 17 (89%) had been diagnosed with ACT before 4 years of age, 1 (5%) at 8.16 years, and 1 (5%) at 10.75 years. The majority (15/19, 78%) had participated in a molecular analysis of ACT patients in which they had tested positive for the germline R337H mutation in the
Vocal recordings were conducted in a speech pathology outpatient clinic room free of noise and echo. While seated with her feet on the ground, her posture upright, and a digital recorder (IMP Optimus Unidirectional Microphone 600Ω; Philippines) placed 10 cm from her mouth at a 90° angle from her chin, each participant was recorded continuously vocalizing the “e” vowel sound while counting from 1 to 20. In accordance with the observations of recording manufacturers and other authors, sustained vocalization of the “e” vowel sound was selected from among all possible vocalizations because it provides for the best possible capture
The F0 values were compared to the results of previous analyses of recordings of 11 subjects, which had been made from 0.9 years to 7.4 years after these subjects had undergone ACT surgery; during this period, these subjects had presented with normal androgen levels
Using reference population data from the National Center for Health Statistics (NCHS)
None of the subjects had experienced any pathological condition other than ACT or taken any anabolic steroids. At ACT diagnosis, all the subjects (19/19) presented with pubic hair growth and the majority (14/19, 74%) presented with clitoromegaly. In addition, 3 subjects, patients 1, 4, and 6, presented with signs of CS, specifically with virilizing signs, since 8, 4, and 15 months before diagnosis, respectively, and these signs had been confirmed to be CS indicators by subsequent measurement of mildly or modestly elevated cortisol levels. The height of all the subjects fell within the 50th and 75th percentiles, corresponding to their familial TH, including that of the 1 participant (subject 14) who presented with the lowest F0 (132 Hz) and moderate pitch virilization.
As previously described, 57% (11/19) of the participants had previously participated in an analysis of F0 values obtained from 0.9 years to 7.4 years after ACT surgery. At this early age, 54% (6/11) had presented with a normal F0 and 45% (5/11) had F0 significantly lower than the expected F0 for their chronological age. This reduction in F0 was found to be inversely correlated (p<0.05) with Tanner stage
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
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
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
Although F0 has previously been investigated in female subjects with CAH
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
The study data shown in
As shown in
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.
We thank Dr. Marcella Rabassi de Lima for her helpful technical assistance.