Brain and Language 175 (2017) 47–56

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Sex steroid hormones and sex hormone binding globulin levels, CYP17 MSP AI (−34 T:C) and CYP19 codon 39 (Trp:Arg) variants in children with developmental stuttering ⁎

MARK

⁎

Hiwa Mohammadia, Mohammad Taghi Joghataeib,a, , Zohreh Rahimic, , Faezeh Faghihib, Habibolah Khazaied, Hashem Farhangdooste, Masoud Mehrpoura a

Department of Neuroscience, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran Cellular and Molecular Research Center, Iran University of Medical Sciences, Tehran, Iran Medical Biology Research Center, Kermanshah University of Medical Sciences, Kermanshah, Iran d Sleep Disorders Research Center, Department of Psychiatry, Kermanshah University of Medical Sciences, Kermanshah, Iran e Department of Speech Therapy, University of Social Welfare and Rehabilitation Sciences, Tehran, Iran b c

A R T I C L E I N F O

A B S T R A C T

Keywords: Stuttering Steroid hormones Polymorphism CYP17 −34 T:C (MSP AI) CYP19 T:C (Trp:Arg)

Developmental stuttering is known to be a sexually dimorphic and male-biased speech motor control disorder. In the present case-control study, we investigated the relationship between developmental stuttering and steroid hormones. Serum levels of testosterone, dihydrotestosterone (DHT), dehydroepiandrosterone (DHEA), oestradiol, progesterone, cortisol, and sex hormone binding globulin (SHBG), as well as the 2nd/4th digit ratio (2D:4D), an indicator of prenatal testosterone level, were compared between children who stutter (CWS) and children who do not stutter (CWNS). Moreover, two SNPs (CYP17 −34 T:C (MSP AI) and CYP19 T:C (Trp:Arg)) of cytochrome P450, which is involved in steroid metabolism pathways, were analysed between the groups. Our results showed significantly higher levels of testosterone, DHT, and oestradiol in CWS in comparison with CWNS. The severity of stuttering was positively correlated with the serum levels of testosterone, DHEA, and cortisol, whereas no association was seen between the stuttering and digit ratio, progesterone, or SHBG. The CYP17CC genotype was significantly associated with the disorder.

1. Introduction Developmental stuttering is a common speech motor control disorder in which the flow of speech is disrupted by involuntary blocks, frequent repetitions, and prolonged speech sounds. The onset of this disorder is usually observed in children aged 2–5 years (Bloodstein & Ratner, 2008). Generally, 5% of children are affected by this disorder with a male to female ratio of 2:1 (Månsson, 2000; Yairi & Ambrose, 1999). Since the approximate rate of children’s recovery is 80%, the overall prevalence of this disorder decreases to 1% in adult populations (Månsson, 2000). Because females are more likely to recover from this disorder than males, the male to female ratio for stuttering changes to 4:1 in adults (Bloodstein & Ratner, 2008; Yairi & Ambrose, 2005). Although the role of sexual dimorphism in the onset of stuttering and the high rate of recovery in females have been addressed in many studies to date, the underlying physiological mechanisms of this

gender-dependent disorder have not been well elucidated. This gender dependency of developmental stuttering suggests a role for androgens, the main male sex hormones, in the pathogenesis of the disorder. The role of testosterone in phenomena such as left-handedness (Hampson & Sankar, 2012) and some neurodevelopmental disorders such as attention deficit-hyperactivity disorder (ADHD) (Martel & Roberts, 2014; Roberts & Martel, 2013), autism spectrum disorder (ASD) (Baron-Cohen et al., 2015), and Tourette syndrome (TS) (Bortolato et al., 2013; James, 1995) has been reported. Controversies remain regarding the effects of testosterone on brain functions and disorders. A recent study by Papadatou-Pastou et al. did not reveal any association between salivary testosterone and handedness or cerebral lateralization for language (Papadatou-Pastou, Martin, & Mohr, 2017). Additionally, the role of prenatal testosterone exposure in the aetiology of ADHD has been challenged by some studies (Lemiere, Boets, & Danckaerts, 2010). Nevertheless, it has been suggested that some sex steroids, such as testosterone and oestrogen, contribute to the

⁎ Corresponding authors at: Department of Anatomy, Faculty of Allied Medical Sciences, Iran University of Medical Sciences, Tehran, Iran (M.T. Joghataei). Medical Biology Research Center, School of Medicine, Kermanshah University of Medical Sciences, Daneshgah Avenue, P.O. Box: 67148-69914, Kermanshah, Iran (Z. Rahimi). E-mail addresses: [email protected] (M.T. Joghataei), [email protected] (Z. Rahimi).

http://dx.doi.org/10.1016/j.bandl.2017.09.004 Received 5 February 2017; Received in revised form 8 September 2017; Accepted 24 September 2017 0093-934X/ © 2017 Published by Elsevier Inc.

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Selcuk et al. reported high serum testosterone levels in CWS aged 7–12 years compared to CWNS. They also showed that there is a remarkable positive correlation between the severity of stuttering and the testosterone level (Selcuk, Erbay, Ozcan, Kartalci, & Batcioglu, 2015). Beside the role of sex steroids in brain language processing in children, the effect of prenatal exposure to testosterone on brain development and functions should not be ignored. Notably, the foetal testosterone level has been directly investigated in autism spectrum disorders (ASD), and the effect of this parameter on autistic traits has been confirmed (Auyeung et al., 2009). However, due to methodological problems in foetal blood sampling, the postnatal 2nd to 4th digit ratio (2D:4D) is used as an indirect indicator of the prenatal testosterone exposure; it is lower in males than in females (Manning, Scutt, Wilson, & Lewis-Jones, 1998). This method has been used to evaluate the correlation between prenatal exposure to testosterone and some neurodevelopmental conditions such as autism (Al-Zaid, Alhader, & AlAyadhi, 2015; Guyatt, Heron, Knight Ble, Golding, & Rai, 2015). The only study of the relationship between 2D:4D and stuttering was that published by Montag et al.; no significant difference was reported in the digit ratio between adults who stutter and the controls. The authors concluded that prenatal testosterone level was not significantly different between the studied groups; however, they reported a negative correlation between the left digit ratio and negative experiences of people who stuttered, using the Overall Assessment of the Speaker’s Experience of Stuttering (OASES), suggesting that higher prenatal testosterone is linked to more negative experiences due to stuttering (Montag et al., 2015). The potential link between prenatal testosterone and stuttering still remains unclear. Moreover, the expression of sex steroids is controlled by specific enzymes involved in the androgen metabolism pathways that may indirectly control stuttering. The cytochrome P450 (CYP) family of enzymes is among the more important regulators of sex steroid synthesis. CYP17 and CYP19, as members of the cytochrome P450 supergene family, were the focus of this study. The enzyme CYP17 plays a key role in the synthesis of sex steroids by steroid 17α-hydroxylation and by 17, 20-lyase activity, which converts 17 α-hydroxypregnenolone to dehydroepiandrosterone (DHEA) and 17α-hydroxyprogesterone to androstenedione, precursors of testosterone and oestrogens, respectively (Weston et al., 1998). In addition, the cytochrome P450 family 19 gene (CYP19), located on chromosome 15, controls three consecutive hydroxylation reactions that convert C19 androgens to aromatic C18 oestrogenic steroids. The enzyme CYP19 converts androstenedione and testosterone to oestrone and oestradiol, respectively (Kitawaki et al., 1999). Any genetic variations at the locus of this gene may alter the aromatase activity of this enzyme and thereby the levels of subsequent hormones (Surekha et al., 2014). In the present study, we investigated the role of the CYP17 −34 T:C (MSP AI) and the CYP19 T:C (Trp:Arg) single nucleotide polymorphisms in susceptibility to stuttering. A substitution of C for T at −34 bp in the 5′ promoter region of the CYP17 gene leads to increased gene expression and increases the oestradiol level in pre-menopausal women (Small et al., 2005). A polymorphism in the 3′ UTR of CYP19 increases mRNA expression (Kravitz, Meyer, Seeman, Greendale, & Sowers, 2006) resulting in an increased level of oestradiol and a decreased level of testosterone (Olson, Bandera, & Orlow, 2007). The CYP17 −34 T:C (MSP AI) polymorphism has been well studied in the context of breast and reproductive system conditions such as endometrial (Olson et al., 2007; Xu, Lin, Zhu, Zhang, & Yang, 2013), breast (Ebrahimi, Sabokbar, Eskandarieh, Peyghambari, & Shirkoohi, 2017; Haiman et al., 1999; Ye & Parry, 2002), and prostate cancers (Madigan et al., 2003; Ntais, Polycarpou, & Ioannidis, 2003). Additionally, the CYP19 T:C (Trp:Arg) polymorphism has been associated with the risk of breast cancer (Tuzuner et al., 2010). Due to the important role of sex steroids in brain development, associations between these SNPs and brain functions such as personality traits and cognition have been investigated. CYP19 genotypes have been related to cognitive functions in women in midlife

sex-biased distribution of stuttering (Geschwind & Galaburda, 1985). The comorbidity and association of stuttering with weak laterality (Kushner, 2012; Mohammadi, Khazaie, Rezaei, & Joghataei, 2016), ADHD (Donaher & Richels, 2012), and TS (Abwender et al., 1998; De Nil, Sasisekaran, Van Lieshout, & Sandor, 2005) may support the role of sex steroids in the pathogenesis of this disorder. During speech and language processing, the left hemisphere of the human brain is more active in fluently speaking people; however, this left dominance is disrupted in people who stutter (Sato et al., 2011). The effects of prenatal testosterone on laterality have been explained by three hypotheses, and each hypothesis has proposed different directions and degrees of lateralization in relation to foetal testosterone exposure. According to the classic Geschwind-Behan-Galaburda hypothesis, foetal exposure to testosterone affects the development of the cerebral hemispheres. Therefore, more testosterone leads to greater dominance of the right hemisphere for language and handedness by either preventing the development of the left hemisphere (Galaburda, Corsiglia, Rosen, & Sherman, 1987; Geschwind & Galaburda, 1985) or enhancing the development of the right hemisphere (Rosen, Sherman, & Galaburda, 1991). The second hypothesis, named the corpus callosum hypothesis, proposed that the testosterone-mediated pruning of callosal neurons during pre- and post-natal development of the brain affects cerebral lateralization. According to this hypothesis, foetal testosterone enhances neural pruning in the corpus callosum, diminishes connectivity between the hemispheres and reinforces cerebral lateralization. In contrast with the Geschwind-Behan-Galaburda hypothesis, low but not high foetal testosterone would contribute to weak laterality by reducing neural pruning in the corpus callosum. This hypothesis proposed the effect of prenatal testosterone on the degree but not the direction of cerebral lateralization (Witelson, 1991; Witelson & Nowakowski, 1991). The final perspective, known as the sexual differentiation hypothesis, linked sexual dimorphism in laterality to testosterone-mediated sexual differentiation in the foetus. According to this hypothesis, higher exposure to testosterone in the foetus changes the degree of right-handedness or even leads to left-handedness but does not change the direction of cerebral language dominance and only influences the degree of language lateralization (Hines & Shipley, 1984). Taking this information into consideration, a role for sex steroids in brain language processing and related disorders is suggested. According to Schaadt et al., brain exposure to testosterone and oestradiol in early infancy may delay language development (Schaadt, Hesse, & Friederici, 2015). Additionally, there is an association between testosterone and cerebral laterality for language processing in male subjects (PapadatouPastou & Martin, 2017). An association between stuttering and brain language processing (Connally, Ward, Howell, & Watkins, 2014; Wymbs, Ingham, Ingham, Paolini, & Grafton, 2013), as well as brain laterality for language (Code, Lincoln, & Dredge, 2005; Foundas et al., 2003), and the effect of sex steroids on brain language processing and laterality reinforces the probable association between sex steroids and stuttering; nevertheless, data regarding this important relationship are rare. Moreover, the dopaminergic dysfunction in stuttering (Wu et al., 1997) together with the influence of steroid-dopamine interactions on brain functions and disorders (Purves-Tyson et al., 2014; Sanchez, Bourque, Morissette, & Di Paolo, 2010) strengthen the possible role of sex steroids in the neuropathology of stuttering. Fluctuations in the level of sex hormones during the menstrual cycle affect the severity of tics in women with TS, a disorder of basal ganglia circuits (Bortolato et al., 2013). Previous studies revealed more dysfluencies during the premenstrual period in women both with and without stuttering; this may confirm the role of steroids in stuttering pathology (Silverman, 1975; Silverman, Zimmer, & Silverman, 1974). Similarly, Kartalci et al., reported that after testosterone was injected into a 14-year-old boy with hypogonadism, he started stuttering despite lacking any previous speech disorder (Kartalcı, Özcan, Yüksel, & Ünal, 2012). Additionally, 48

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purposes (Riley, 1994; Sawyer & Yairi, 2006). The percentage of syllables stuttered (SS%) was computed by dividing the stuttered syllables by the total syllables and multiplying by 100. The SS% value in each speech sample represents the percentage of syllables that contains unambiguous stuttering (Jones et al., 2005). Children with an SS% value higher than 3 were diagnosed as CWS (Yairi & Ambrose, 2005).

(Kravitz et al., 2006). In addition, effects of CYP19 on personality traits (Matsumoto et al., 2009) and sexual behaviours (Jones, Boon, Proietto, & Simpson, 2006) have been reported. The T allele of a CYP19 polymorphism led to lower scores of harm avoidance in male but not in female subjects (Matsumoto et al., 2009). Personality traits such as novelty seeking, cooperativeness, and self-transcendence were positively affected by the C allele of a CYP17 polymorphism in males but not in females (Matsumoto et al., 2008). Additionally, polymorphisms in maternal CYP19 and CYP17 have been associated with behavioural problems such as poorer adaptive skills and attention problems in male children (Miodovnik et al., 2012). The CYP17 polymorphism was also found to be associated with Alzheimer’s disease (AD) particularly in males; subjects who carried the A2 (C) allele had an increased risk for AD (Corbo, Gambina, Broggio, Scarabino, & Scacchi, 2014). Studying the potential role of sex steroids during the time period close to the onset of stuttering would be useful to understand the pathogenesis of this developmental disorder and might aid further treatment of the disorder. Therefore, the present study focused on the serum levels of sex steroids and the 2D:4D digit ratio (as an indirect index for prenatal testosterone exposure) to determine if any associations exist between sex hormone levels and stuttering. Moreover, we investigated the association between CYP17 −34 T:C (MSP AI) and CYP19 T:C (Trp:Arg) single nucleotide polymorphisms involved in androgen metabolism pathways and stuttering.

2.3. Biochemical analysis Five millilitres of venous blood was collected at 9 AM under standard conditions. The samples were divided into two separate tubes. Three millilitres of the blood was centrifuged, and the serum was stored at −20 °C for subsequent analyses, according to standard protocols. Two millilitres of each blood sample was treated with EDTA to be used for DNA extraction and further genetic analysis. Serum levels of testosterone (Ref 2P13, ABBL311/R07), SHBG (Ref 8K26, G3-0703/R05), DHEA (Ref 8K27, G2-6144/R06), progesterone (Ref 7K77, G2-7955/R05), and cortisol (Ref 8D15, JL840685/R06) were measured based on the chemiluminescent microparticles immunoassay (CMIA) technique by using an ARCHITECT Immunoassay Analyzer (ARCHITECT plus i1000SR, Abbott Diagnostic, USA). To determine the free testosterone level, the total testosterone level was divided by the SHBG level. Serum DHT and oestradiol were measured using enzyme-linked immunosorbent assay kits, CAN-DHT-280, Version: 0.5 (DBC-Diagnostics Biochem Canada, Canada) and DK0003 (DiaMetra, Italy), respectively. The absorbance level was read using an Awareness Technology STAT FAX 2100 Microplate Reader (Awareness Technology, USA). Hormone levels were calibrated according to the standard calibration curve of each hormone in the kits. Data are presented using the following units: pg/ml for DHT and oestradiol, μg/dl for DHEA and cortisol, nmol/l for SHBG, and ng/dl for total and free testosterone.

2. Materials and methods 2.1. Participants A total of 92 Kurdish CWS, including 22 females (23.90%) aged 3–9 years (6.42 ± 1.83), were recruited from speech therapy centres of Kermanshah province in the west of Iran. Ninety one CWNS from a similar ethnic background and matched for both age, 3–9 years (6.44 ± 1.70; p = 0.94), and sex, including 27 females (29.70%) (p = 0.23), were included in this study as the control group. Controls were recruited from primary schools and kindergartens of Kermanshah. Moreover, a public announcement was made on social network media such as Facebook and Telegram. The protocol of this study was approved by the Ethics Committee of the Iran University of Medical Sciences. Detailed informed written consent was obtained from all the children’s parents. All participants and their parents were carefully interviewed by an expert speech therapist and a psychiatrist. Children with any speechlanguage disorder or delay other than stuttering in the CWS group, and those with concomitant psychological, psychiatric, or neurological disorders, as well as those who had used any medication in the recent past months, were excluded from the study. Of all the participants, 20 (23.50%) and 35 (41.20%) participants in the CWS group were established to have had stuttering in the history of their first- and seconddegree relatives, respectively. Based on our exclusion criteria, CWNS comprised individuals without any family history of stuttering. Collectively, 7 male CWS and 6 CWNS, 3 females and 3 males, were excluded according to the exclusion criteria; so 85 participants from each group were studied.

2.4. Digit ratio measurement The lengths of the index (2D) and the ring (4D) fingers of both hands in CWS and CWNS were measured by a digital calliper. The length of each finger was defined as the distance between the midpoints of the first line in the basal crease to the tip of the finger. Measurements were performed twice for each participant by two trained examiners who were blind to the group details and other measurements. The mean of the two measurements was used to calculate the digit ratio for each hand, which was calculated by dividing the 2D length by the 4D length. This value was calculated for the right hand, left hand, and the mean of both hands of each participant. In addition, the difference between the 2D:4D of the right hand and the 2D:4D of the left hand (DR-L) was considered for the analysis. There were no broken fingers among the participants. The inter-rater reliability was acceptable between the two measurements for the left and right 2D and 4D fingers; the correlations between measurements were 0.70 for the left 2D:4D ratio and 0.63 for the right 2D:4D ratio (p < 0.001). 2.5. Genotyping DNA was extracted from peripheral leucocytes using the phenolchloroform protocol (Rahimi, Rahimi, Shahsavandi, Bidoki, & Rezaei, 2013). Genotyping of CYP19 and CYP17 was done by allele-specificpolymerase chain reaction (AS-PCR) and PCR-restriction fragment length polymorphism (PCR-RFLP), respectively. The CYP19 T:C (Trp:Arg) polymorphism, rs2236722, was identified by AS-PCR using two specific primer pairs (Hamajima et al., 2000). Genomic DNA (100 ng) and 25 pmol of each primer, F1: 5′-ATC TGT ACT GTA CAG CAC C-3′, R1: 5′-ATG TGC CCT CAT AAT TCC G-3′, F2: 5′-GGC CTT TTT CTC TTG GTG T-3′, and R2: 5′-CTC CAA GTC CTC ATT TGC T-3′, were added to 12.5 µl of CinnaGen PCR Master Mix (CAT. NO.: PR901638, SinaClon, Tehran, Iran) containing 0.08 units/μl Taq DNA polymerase

2.2. Stuttering diagnosis and severity rating In all admitted participants of the CWS group, the presence of stuttering was identified by their parents, their speech therapists, and the principal investigator. In addition, a 40-min interview from each CWS was videotaped using conversation and storytelling to collect adequate spontaneous speech samples. For each participant, 500 syllables from storytelling were transcribed and analysed by an experienced speech therapist blind to the research questions who counted the total and stuttered syllables. According to previous reports, 500 syllables are adequate for evaluating stuttering for research and clinical 49

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Fig. 2. Agarose gel electrophoresis of RFLP products of the CYP17 −34 T:C polymorphism. From left to right, lane 1 is the 50-bp DNA molecular marker. Lanes 2 and 4 are the CT genotype. Lane 3 is the CC genotype. Lane 5 is the TT genotype.

Fig. 1. Agarose gel electrophoresis of the CYP19 polymorphism. From left to right, lane 1 is 50-bp DNA molecular marker. Lanes 2 and 3 are the TC genotype. Lane 3 is the TT genotype.

transformed to normal distributions and were then compared between CWS and CWNS by independent sample t-tests. Later, the data were back transformed for presentation. However, the distribution of progesterone did not change to normal; hence, we compared its median between groups using the nonparametric Mann-Whitney U test. The correlations between severity of stuttering and levels of steroids along with digit ratios in the CWS group were analysed by the Pearson and the Spearman correlation coefficients. Genotype and allele frequencies were compared between groups using the chi-square test. Odds ratios and 95% confidence intervals (95% CIs) were estimated using multivariate conditional logistic regression analysis to calculate the relative risk of each genotype in the development of stuttering. Additionally, the hormone levels and digit ratios were compared among different genotypes by ANOVA. The statistical package for social sciences (SPSS) (SPSS, Inc., Chicago, IL) version 16.0 was used for the statistical analyses.

in reaction buffer, 3 mM MgCl2, 0.4 mM dATP, 0.4 mM dCTP, 0.4 mM dGTP, and 0.4 mM dTTP. The reaction volume was increased to 25 µl by adding 7.5 µl sterile deionized water to each reaction. Thermocycling conditions included: 5-min initial denaturation at 95 °C; followed by 35 cycles of 95 °C for 45 s, 54 °C for 45 s, and 72 °C for 45 s; and then a final extension at 72 °C for 7 min. The amplified DNA was visualized on a 2.5% agarose gel with GelRed staining solution. The homozygous arginine allele (C) was identified by the presence of fragments with 427 and 264 bp; the homozygous tryptophan allele (T) was detected by the presence of 427- and 200-bp fragments, and the heterozygous state (CT) was identified by the presence of 427-, 264-, and 200-bp fragments (Fig. 1). The CYP17 −34 T:C (MSP AI) polymorphism (rs743572) in the 5′untranslated region, 34 bp from the initiation point of translation, was identified by using modification of a specific PCR-RFLP method described by Carey et al. (1994). A 414-bp fragment was amplified using these primers F: 5′-CAT TCG CAC TCT GGA GTC-3′ and R: 5′-AGG CTC TTG GGG TAC TTG-3′. Using a touch-down PCR protocol, the following thermocycling protocol was performed: an initial denaturing step of 95 °C for 5 min; followed by 10 cycles of denaturation at 95 °C for 45 s, annealing at 64 °C for 45 s, and extension at 72 °C for 45 s; followed by 30 cycles of denaturation at 95 °C for 45 s, annealing at 61 °C for 45 s, and extension at 72 °C for 45 s; followed by a final extension at 72 °C for 10 min. Finally, the amplified PCR product was digested with 3 units of the restriction endonuclease MspAI (R7021, Promega, Madison, WI) at 37 °C overnight and electrophoresed on a 2% agarose gel stained by GelRed. The A1, or TT, genotype was identified by the presence of a 414-bp fragment; the A1/A2, or TC, genotype was detected by the presence of 414-, 290-, and 124-bp fragments, and the A2, or CC, genotype was identified by the presence of fragments of 290 and 124 bp (Fig. 2).

3. Results 3.1. Biochemical findings Comparative analysis of sex steroids between CWS and CWNS displayed significantly higher levels of total testosterone (6.02 ± 1.85 ng/dl), free testosterone (0.05 ± 0.16 ng/dl), DHT (3.16 ± 7.39 pg/ml), and oestradiol (4.92 ± 2.87 pg/ml) in CWS than in the controls (5.04 ± 1.63 ng/dl, 0.04 ± 0.067 ng/dl, 0.85 ± 6.80 pg/ml, and 2.79 ± 2.87 pg/ml, respectively). Although higher levels of DHEA and cortisol were observed in CWS than in CWNS, the differences were not statistically significant. No significant difference between the two groups was observed for the progesterone level (Table 1). Biochemical analysis in each sex subgroup showed no significant difference in the total testosterone level between CWS and CWNS (male: p = 0.15; female: p = 0.09). Notably, the free testosterone level was significantly higher in CWS than in CWNS (p = 0.034); however, when the results were analysed by sex, a statistically significant difference was observed only between females with stuttering (0.08 ± 0.27 ng/dl) and females without stuttering (0.04 ± 0.02 ng/ dl; p = 0.006). Interestingly, the oestradiol level was significantly higher in females with stuttering (7.78 ± 1.78 pg/ml) than in females without stuttering (3.38 ± 3.49 pg/ml; p = 0.025). The DHT level was

2.6. Statistical analysis According to the Kolmogorov-Smirnov test, the distributions of age, cortisol, and SHBG levels, as well as digit ratios were normal, and the parameters were directly compared between CWS and CWNS groups by independent sample t-tests. By using logarithmic transformation, we transformed the non-normally distributed data to normal distributions. The distributions of testosterone, DHT, DHEA, and oestradiol were 50

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Table 1 Biochemical parameters and digit ratio in CWS and CWNS. Variables

CWS, n = 85 Mean ± SD

CWNS, n = 85 Mean ± SD

p

Biochemical Parameters

Total testosterone (ng/dl) SHBG (nmol/l) Free testosterone (ng/dl) DHT (pg/ml) Oestradiol (pg/ml) DHEA (μg/dl) Cortisol (μg/dl) Progesterone (ng/ml)

6.02 ± 1.85 110.28 ± 38.22 0.05 ± 2.53 3.16 ± 7.39 4.92 ± 2.88 19.56 ± 3.25 8.44 ± 3.34 0.13 ± 0.10

5.04 ± 1.63 118.95 ± 37.36 0.04 ± 2 0.85 ± 6.97 2.79 ± 2.87 17.64 ± 2.60 8.25 ± 4.33 0.13 ± 0.15

0.042a 0.13a 0.034a < 0.001a 0.017a 0.53a 0.74a 0.65b

Digit ratio

Right hand Left hand Both hands DR-L

0.98 ± 0.03 0.98 ± 0.03 0.98 ± 0.03 −0.0031 ± 0.02

0.98 ± 0.03 0.98 ± 0.03 0.98 ± 0.02 −0.0008 ± 0.03

0.98a 0.61a 0.77a 0.60a

Data were compared by athe independent sample T-test or bthe Mann-Whitney U test. CWS; children who stutter. CWNS; children who do not stutter. Table 2 Comparison of biochemical parameters and digit ratios between CWS and CWNS in male and female subgroups. Male

p

Case, n = 63 Mean ± SD

Control, n = 61 Mean ± SD

Female

p

Case, n = 22 Mean ± SD

Control, n = 24 Mean ± SD

Biochemical parameters

Total testosterone (ng/dl) SHBG (nmol/l) Free testosterone (ng/dl) DHT (pg/ml) Oestradiol (pg/ml) DHEA (μg/dl) Cortisol (μg/dl) Progesterone (ng/ml)

5.727 ± 1.84 115.47 ± 36.04 0.05 ± 2.35 3.26 ± 10.90 4.06 ± 3.17 19.10 ± 3.24 8.97 ± 3.19 0.13 ± 0.10

4.95 ± 1.68 112.81 ± 35.44 0.04 ± 2.14 0.98 ± 6.88 2.58 ± 2.67 18.33 ± 2.73 8.75 ± 4.70 0.14 ± 0.17

0.15 0.68a 0.42a < 0.001a 0.12a 0.83a 0.75a 0.90b

6.94 ± 1.88 95.40 ± 41.20 0.08 ± 2.89 2.90 ± 10.90 7.78 ± 1.78 20.92 ± 3.36 6.93 ± 3.34 0.14 ± 0.10

5.30 ± 1.52 134.50 ± 38.30 0.04 ± 1.62 0.98 ± 6.93 3.38 ± 3.49 16.05 ± 2.28 6.93 ± 2.85 0.10 ± 0.08

0.09a 0.002a 0.006a 0.10a 0.025a 0.38a 0.90a 0.19b

Digit ratio

Right hand Left hand Both hands DR-L

0.97 ± 0.98 ± 0.97 ± -0.0033

0.98 ± 0.03 0.98 ± 0.02 0.98 ± 0.02 0.0006 ± 0.02

0.57a 0.90a 0.75a 0.47a

0.99 ± 1.00 ± 0.99 ± -0.0027

0.98 ± 0.99 ± 0.98 ± -0.0041

0.25a 0.35a 0.24a 0.88a

0.03 0.03 0.03 ± 0.03

0.02 0.03 0.02 ± 0.02

0.03 0.03 0.03 ± 0.03

Data were compared by athe independent sample T-test or bthe Mann-Whitney U test.

significant (p = 0.4).

significantly increased in males with stuttering (3.26 ± 10.9) compared to males in the control group (0.98 ± 6.88; p < 0.001) (Table 2). Using the Pearson correlation coefficient, we investigated the relationship between the severity of stuttering (SS%) and the levels of hormones. There was a significant positive correlation between SS% and the serum total testosterone (r = 0.29, p = 0.007), free testosterone (r = 0.26, p = 0.016), DHEA (r = 0.24, p = 0.026), and cortisol (r = 0.22, p = 0.044) levels. No significant correlation was observed between SS% and the levels of DHT or oestradiol (Table 3). We also compared SS% between male and female individuals of the CWS group. The SS% was higher in males (13.23 ± 5.90) than in females (12.10 ± 4.70); however, the difference was not statistically

3.2. Digit ratio We compared the 2D:4D ratio of the right, the left, and both hands, as well as the DR-L between CWS and CWNS. As shown in Table 1, no significant difference was detected in the digit ratios between the two groups. In addition, no significant difference was observed between CWS and CWNS in both male and female subgroups (Table 2). No significant correlation was observed between the left, the right, or both hands digit ratios and SS% (all p > 0.39). Moreover, no significant correlation was observed between the digit ratios and the serum testosterone level (all p > 0.13). In addition, no significant correlation was observed between DR-L and SS% (p = 0.39). Furthermore, the correlation between DR-L and the testosterone level was not significant (p = 0.93). The right 2D:4D ratio in females (0.991 ± 0.031) was significantly higher than in males (0.97 ± 0.03; p = 0.03). Similarly, the left digit ratio was significantly higher in the female group (0.99 ± 0.03) than in the males (0.98 ± 0.03; p = 0.008). No significant difference was observed between the right (0.98 ± 0.03) and the left (0.98 ± 0.03) digit ratios (p = 0.39).

Table 3 Correlation between severity of stuttering (SS%) and hormones.

Total testosterone Free testosterone (ng/dl) SHBG (nmol/l) DHT (pg/ml) Oestradiol (pg/ml) DHEA (μg/dl) Cortisol (μg/dl) Progesterone (ng/ml)

Correlation coefficient

p

0.29 0.26 −0.11 0.08 0.03 0.24 0.22 0.06

0.007a 0.016a 0.30a 0.40a 0.70a 0.026a 0.044a 0.50b

3.3. Genetic findings The distribution of the CYP17 −34 T:C genotypes was in

Results obtained by athe Pearson or bthe Spearman correlation coefficient.

51

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Table 4 Distribution of CYP17 −34 T:C and CYP19 T:C genotypes and alleles in CWS and CWNS.

CYP17

Genotype

Allele CYP19

Genotype

Allele

CWS, n = 85 n (%)

CWNS, n = 85 n (%)

p (χ2)

OR (95% CI, p)

TT TC CC T C

21 44 20 86 84

25 (24.90) 55 (64.70) 5 (5.90) 105 (61.80) 65 (38.20)

0.005 (10.57)

Reference 0.95 (0.47–1.92, 0.89) 4.76 (1.52–14.87, 0.007) Reference 1.57 (1.02–2.42, 0.038)

TT TC CC T C

1 (1.20) 84 (98.80) 0 86 (50.60) 84 (49.40)

(24.70) (51.80) (23.50) (50.60) (49.40)

0.024 (4.31)

2 (2.40) 83 (97.60) 0 87 (51.20) 83 (48.80)

0.50 (0.34)

Reference 2.02 (0.18–22.75, 0.56)

0.90 (0.01)

Reference 1.02 (0.66–1.56, 0.91)

No significant difference was observed in the testosterone level among three different genotypes in CWS (F = 3.07, p = 0.05). Comparison of testosterone and oestradiol among different genotypes of CYP17 was done for the male and female subgroups in CWS, in CWNS, and in all the subjects from the combined groups (Table 5). No significant difference in the testosterone level was detected among CYP17 genotypes in either male or female subgroups. The oestradiol level was significantly different among three genotypes in females (p = 0.03) but not in males without stuttering. The oestradiol level was significantly different among three genotypes in males with stuttering (p = 0.02) (Table 5). Tukey’s post hoc analysis in the CWS group indicated that there were significant differences in the testosterone and oestradiol levels between the CYP17 TC and the CYP17 CC genotypes (Table 6).

accordance with Hardy-Weinberg equilibrium in the CWS group (χ2 = 0.11; p > 0.1). The distributions of the CYP17 −34 T:C and the CYT19 T:C genotypes and the related alleles are presented in Table 4. The frequency of the −34 CC genotype was significantly higher in CWS (23.5%) than in the controls (5.9%; p = 0.005). Moreover, the frequency of the −34 C allele was remarkably higher in CWS (49.4%) than in the controls (38.2%; p = 0.024). The presence of the CYP17 −34 CC genotype increased the risk of stuttering by 4.76 times (p = 0.007). In addition, the risk of stuttering increased to 1.57-fold in the presence of the CYP17 C allele (p = 0.038) (Table 4). The CYP19 CC genotype was not detected in this study. The CYP19 TC genotype was detected in 98.8% of CWS and in 97.60% of the controls (p = 0.50). We did not observe the rare genotype of CYP19 CC. There were only two heterozygote TC genotypes in CWNS and one TC genotype in CWS. The frequency of the CYP19 C allele was 49.4% in CWS and 48.8% in the controls (p = 0.90) (Table 4). Digit ratios and serum levels of testosterone, free testosterone, cortisol, DHEA, DHT, oestradiol, progesterone, and SHBG were compared among CYP17 and CYP19 genotypes using ANOVA and the Tukey post hoc test. No significant difference was observed in digit ratios, SHBG, cortisol, DHEA, progesterone, DHT, or free testosterone levels among various CYP17 genotypes in both CWS and CWNS groups. As illustrated in Table 5, no significant difference was observed in the levels of oestradiol and testosterone among various genotypes in CWNS; however, there was a remarkable difference in the oestradiol level among various CYP17 genotypes among CWS (F = 3.76, p = 0.029).

4. Discussion Developmental stuttering is a sexually dimorphic and male-biased speech motor control disorder. Recent studies suggested the role of steroid hormones in the pathogenesis of this disorder. The present study focused on serum levels of sex steroids and 2D:4D digit ratio (as an indirect index for prenatal testosterone exposure) to determine if any associations exist between sex hormones and stuttering. Moreover, we investigated the association between the CYP17 −34 T:C (MSP AI) and the CYP19 T:C (Trp:Arg) single nucleotide polymorphisms involved in androgen metabolism pathways and stuttering. To the best of our

Table 5 Comparison of testosterone and oestradiol levels in various CYP17 genotypes. Group

Genotype

Testosterone (ng/dl) CWNS TT TC CC CWS TT TC CC Both groups TT TC CC Oestradiol (pg/ml) CWNS

CWS

Both groups

TT TC CC TT TC CC TT TC CC

Both sexes

Male

Female

Mean ± SD

F

p

Mean ± SD

F

p

Mean ± SD

F

p

5.08 4.88 6.96 6.05 6.81 4.55 5.50 5.66 4.95

± ± ± ± ± ± ± ± ±

1.60 1.65 1.60 1.89 1.89 1.71 1.74 1.77 1.72

1.19

0.30

0.05

0.71

0.95

0.38

1.44 1.52 2.04 2.13 1.86 1.91 1.70 1.74 1.84

0.34

0.57

± ± ± ± ± ± ± ± ±

0.47

3.06

5.65 4.94 7.15 6.19 7.71 6.05 5.87 6.03 6.35

0.77

0.55

1.67 1.70 1.55 1.87 1.85 1.62 1.77 1.80 1.65

0.57

0.05

± ± ± ± ± ± ± ± ±

0.60

3.07

4.88 4.86 6.84 6.01 6.51 4.14 5.38 5.53 4.50

0.04

0.95

1.63 3.70 3.08 4.46 6.75 2.90 2.93 5.41 2.92

± ± ± ± ± ± ± ± ±

3.93 2.31 1.32 3.56 2.20 3.13 3.97 2.34 2.84

2.09

0.14

0.02

0.95

3.15

0.05

5.35 1.64 1.41 1.78 1.53 2.70 4.52 1.55 2.41

0.045

0.024

± ± ± ± ± ± ± ± ±

0.03

4.07

0.58 6.88 3.34 7.68 8.10 7.26 3.68 7.64 5.60

6.67

3.89

3.58 2.30 1.00 4.38 2.45 2.86 3.90 2.56 2.73

0.78

0.029

± ± ± ± ± ± ± ± ±

0.25

3.76

2.11 2.92 2.62 3.31 6.27 2.14 2.68 4.72 2.17

1.50

0.24

52

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previous finding that confirmed male-female dimorphism in the 2D:4D ratio (McFadden & Shubel, 2002; McIntyre, Cohn, & Ellison, 2006; Peters, Mackenzie, & Bryden, 2002). Moreover, similarly to the results reported by Robertson et al. (2008), we could not detect any significant difference between right and left digit ratios. Our results, along with two previous reports (Kartalcı et al., 2012; Selcuk et al., 2015), confirmed the association between high postnatal testosterone level and stuttering. High testosterone level could change the cerebral laterality for language processing (PapadatouPastou & Martin, 2017) and influence language development (Schaadt et al., 2015) in a way that disrupts speech motor control and leads to stuttering. Steroids can regulate development, differentiation, and function of the brain by altering the gene expression through interaction with appropriate nuclear receptors (Brunton, 2015; Melcangi & Panzica, 2014). Moreover, it has been reported that steroids can bind to synaptic membrane receptors temporarily and rapidly regulate neurotransmission and electrophysiological properties of the cells (Melcangi & Panzica, 2014). Interaction of testosterone and its metabolites with synaptic membrane receptors may rapidly modify the dopaminergic pathways and lead to hyper-dopaminergic activity in stuttering. Subsequent dysregulation in brain circuits responsible for speech motor control could ultimately lead to dysfluency in CWS. The steroid-dependent, short-term regulation of brain dopaminergic pathways appropriately explains the dynamic nature of stuttering. As a dynamic motor control disorder, the severity of stuttering is changed dynamically according to the communication situations and environments (Ludlow & Loucks, 2003). Future work should consider the effects of steroids on stuttering in activational (e.g., in the different phases of the menstrual cycle) vs. organizational (e.g., the more permanent effects that start to occur in the embryonic period of life) perspectives. From the activational point of view, occur by short-term regulation of neurotransmission, future studies should investigate the effect of fluctuations in the level of sex steroids and changes in stuttering severity during the menstrual cycle in women with stuttering. Previous rare evidence supports this activational effect (Silverman, 1975), and future studies should systematically investigate fluency fluctuations among women with stuttering during different phases of the menstrual cycle and after menopause with specific attention to the role of sex steroids. In contrast to the short-term, activational effect of steroids, the organizational effect has more permanent effect on brain networking by regulating gene expression through interaction with appropriate nuclear receptors. This effect begins during the embryonic period and regulates development, differentiation and organization of the brain. On one hand, longitudinal studies to investigate prenatal steroids and their effect on future brain development and disorders, specifically on language development and language disorders, by measuring hormones in amniotic fluid could shed light on organizational effects of steroids in stuttering. On the other hand, it has been reported that the brain is able to modify the direct effect of steroids by extra metabolism of hormones in the production of neurosteroids (Melcangi & Panzica, 2014). Investigation of steroid-brain interactions and the role of neurosteroids in stuttering neuropathology is necessary. Moreover, involvement of the basal ganglia-thalamocortical motor circuits (Alm, 2004) and the brain dopaminergic system in stuttering neuropathology (Wu et al., 1997) may clarify the role of testosterone or its metabolites. The effect of androgens on the brain dopamine-signalling pathway has been documented (Sinclair, Purves-Tyson, Allen, & Weickert, 2014). Studies have revealed that in animal models, testosterone and DHT increase dopamine transporter and vesicular monoamine transporter (VMAT) mRNAs in the substantia nigra (Purves-Tyson et al., 2012). In addition, testosterone increases the accumulation of DAT protein in cell bodies. Androgens increase the dopamine D2 receptor mRNA and decrease D3 mRNA in the substantia nigra and/or the striatum (Purves-Tyson et al., 2014). The C957 T polymorphism (rs6277) in the dopamine D2 receptor, DRD2, on chromosome 11q23 changes the DRD2 availability in the human striatum

Table 6 Tukey post hoc analysis for evaluation of testosterone and oestradiol level in various CYP17 genotypes in CWS. Genotype Testosterone (ng/dl) TT TC Oestradiol (pg/ml) TT TC

Mean difference

p

CC TC CC

1.33 0.88 1.49

0.29 0.74 0.04

CC TC CC

1.54 0.66 2.32

0.47 0.42 0.02

knowledge, this is the first study with a large sample size that focused on the levels of different sex steroid hormones in CWS. In this casecontrol study, we compared serum levels of testosterone, DHT, DHEA, oestradiol, progesterone, SHBG, and cortisol, as well as the digit ratio as an indirect index of prenatal testosterone in a group of CWS in comparison with sex- and age-matched CWNS. Moreover, the occurrence of two SNPs of cytochrome P450, CYP 17 −34 T:C (MSP AI) and CYP19 T:C (Trp:Arg), were analysed in these children. Our biochemical analysis revealed that there are significantly higher levels of testosterone and its metabolites, including DHT and oestradiol, in CWS than in sex- and age-matched CWNS. Moreover, the level of free testosterone, the active form of the hormone, was significantly higher in CWS than in the controls. Furthermore, the testosterone level showed a significant positive correlation with stuttering severity. Similarly, Selcuk et al. also reported a high testosterone level in people with stuttering (Selcuk et al., 2015). They recruited older children within the age range of 7–10 years, whereas we studied children aged 3–9 years that were at the average age of stuttering onset. The Selcuk et al. study had some limitations, including low sample size and lack of confirmed objective stuttering assessment methods such as stuttered syllables (SS %) (Yairi & Ambrose, 2005) and stuttering severity instrument (Movsessian, 2005; Riley, 1972). Instead, it used subjective assessment methods such as the clinical global impression-severity scale (CGI-S). In our study, a significant difference was not seen in the digit ratios between CWS and CWNS, and no correlation was observed between the digit ratio and SS%. Likewise, Montag et al. did not find any significant difference in the digit ratios between adults with stuttering and the control group. They also reported a negative correlation between the left digit ratio and OASES only in females, suggesting that higher prenatal testosterone is linked to higher negative experiences due to stuttering (Montag et al., 2015). Unlike Montag et al., we used SS% to calculate the severity of stuttering. Similarly, they used indirect measurement techniques to calculate the finger lengths, but we directly measured the finger length by using a digital calliper. The inter-rater reliabilities for the 2D:4D scans in our study are at the lower end of acceptability and digit ratio finding should be interpreted based on this limitation. Although the application of different methods may influence the consistency of results, it seems that measuring the prenatal testosterone indexed by digit ratio does not precisely determine the direct effect of foetal testosterone on stuttering. Some previous studies in both human (Berenbaum, Bryk, Nowak, Quigley, & Moffat, 2009; Hickey et al., 2010) and animal subjects (Yan, Bunning, Wahlsten, & Hurd, 2009) demonstrated that the 2D:4D ratio might not be a good indicator of prenatal testosterone. Although 2D:4D ratio as an index for foetal exposure to testosterone is not associated with developmental stuttering, high testosterone metabolism in infancy may play an important role in this disorder. According to Papadatou-Pastou, the cerebral laterality for language processing among adult male subjects was associated with salivary testosterone but not with the 2D:4D digit ratio (Papadatou-Pastou & Martin, 2017). Our 2D:4D digit ratio analyses revealed that both right and left digit ratios were remarkably lower in males rather than in females. This result was in accordance with the 53

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by other enzymes. Testosterone can be converted to DHT by 5 α-reductase in target tissues such as the brain. In the present study, the CYP17 −34 T:C polymorphism was significantly associated with stuttering. The CYP17 CC genotype frequency was higher in the stuttering group than in the controls. Moreover, the CC genotype was associated with susceptibility to stuttering (OR = 4.76; 95% CI, 1.52–14.87; p = 0.007). Furthermore, we could detect higher levels of testosterone and oestradiol in CWS in the presence of the TC compared to the TT genotype. Consequently, the high testosterone and DHT levels in the stuttering group might be due to the high frequency of the CYP17 C allele in CWS compared to the controls. The presence of this polymorphism (the C allele) was hypothesized to create an additional Sp1 binding site with enhanced promoter activity and increased gene expression; however, experimental studies have yet to confirm this (Ambrosone et al., 2003; Piller, Verla-Tebit, WangGohrke, Linseisen, & Chang-Claude, 2006). It has been suggested that the presence of the C (Arg) allele of CYP19 T:C (Trp:Arg) might reduce enzyme activity and lower the production of oestrogens (Miyoshi, Iwao, Ikeda, Egawa, & Noguchi, 2000); however, we did not observe any significant association between CYP19 T:C genotype and sex hormones or SHBG. Moreover, no association was found between the CYP19 T:C polymorphism and susceptibility to stuttering. The CYP19 T:C (Trp:Arg) is a rare polymorphism as we did not find the homozygous CC genotype. In similar recent results, the rare CC genotype was not observed in Indian (Samson et al., 2009; Surekha et al., 2014) or Turkish populations (Tuzuner et al., 2010). Additionally, it has been reported in only few Hawaiians (2.1%) and Japanese (2.9%) (Samson et al., 2009). Nevertheless, it should be noted that our sample size is rather small from a genetics perspective. Future studies with more participants from stuttering and non-stuttering populations are recommended.

and extrastriate regions in vivo (Hirvonen, Laakso et al., 2009; Hirvonen, Lumme et al., 2009). An association between stuttering and rs6277 has been reported in the Chinese population. It has been reported that the C allele of DRD2 C957 T was a significant risk factor for stuttering, and the frequencies of both the C allele and the CC genotype were significantly higher in PWS than in the controls (Lan et al., 2009); however, subsequent studies with both Brazilian and western Caucasian populations did not show any association between DRD2 C957 T and stuttering (Kang et al., 2011; Montag, Bleek, Faber, & Reuter, 2012). Simultaneously, Montag et al. reported that the C957 T polymorphism was associated with neuroticism among PWS such that participants with the CC and CT genotypes had higher neuroticism scores (Montag et al., 2012). With the variability of the stuttering phenotype and the role of personality traits in the disorder (Jafari, Baziar, Bleek, Reuter, & Montag, 2015), it might be worthwhile to include personality measures in future studies of the biological basis of stuttering, which may shed light on biology-behaviour interaction in this neurodevelopmental disorder. In any case, high levels of testosterone and DHT in children with stuttering may increase the dopaminergic activity in basal ganglia circuits, leading to speech motor dysregulation. The role of androgens in the pathology of TS, a disease with similar characteristics to stuttering, has been reported. Exogenous androgen could intensify tics in males with TS, whereas antitestosterone drugs diminished tics in these patients (Leckman & Scahill, 1990). In our study, the level of cortisol, an important stress biomarker, was approximately the same in the CWS and the CWNS groups; however, it was positively correlated with stuttering severity. It seems that both groups had similar physiological stress profiles when encountering a stressful condition such as blood sampling. Our findings confirm the results of a previous study in which no stress difference was observed between individuals with and without stuttering (Alm, 2014). According to our results, high testosterone level in CWS did not arise from stress conditions via activation of the hypothalamic-pituitaryadrenal axis. However, some studies reported a high level of cortisol during stressful conditions in adults and children with stuttering (Blood, Blood, Frederick, Wertz, & Simpson, 1997; Ortega & Ambrose, 2011). Despite a non-significant difference between CWS and CWNS, a significant positive correlation between cortisol and stuttering severity suggests a role for stress as a modulating factor in determining the severity of stuttering. Our findings did not indicate significant differences in progesterone or DHEA between CWS and CWNS. It seems that the initial metabolites in sex steroid synthetic pathways do not differ between CWS and CWNS. The data indicated a positive correlation between DHEA and stuttering severity in the CWS group. DHEA could be metabolized to testosterone, and high testosterone and particularly its active form, free testosterone, could be a precursor for synthesis of DHT and oestradiol by 5α-reductase and aromatase enzymes, respectively. The hypothalamic-pituitary-gonadal/adrenal axes regulate biosynthesis of steroid hormones. In children, the main androgens are produced by the adrenal cortex. Conversion of cholesterol to pregnenolone, and subsequently to androgen and progesterone, are the main synthesis route in steroid metabolism. Important reactions involved in sex steroid metabolism are catalysed by the CYP17 enzyme, which has dual bioactivities as a 17α-hydroxylase and a 17,20-lyase. The 17α-hydroxylase converts pregnenolone to 17α-hydroxypregnenolone and progesterone to 17αhydroxyprogesterone. Alternatively, C17,20-lyase converts 17α-hydroxypregnenolone to DHEA and 17α–hydroxyprogesterone to androstenedione (Brock & Waterman, 1999; Fevold et al., 1989). The human form of CYP17 has a significantly lower affinity for 17α-hydroxyprogesterone than for 17α-hydroxypregnenolone (Nakajin, Shinoda, Haniu, Shively, & Hall, 1984). Therefore, the metabolic route of testosterone formation in humans favours pregnenolone as the starting precursor rather than progesterone (Brock & Waterman, 1999). DHEA and androstenedione may be subsequently transformed to testosterone

5. Conclusion In conclusion, the present study showed that serum levels of testosterone and its metabolites, DHT and oestradiol, were significantly higher in CWS compared to CWNS. We also found a significant positive correlation between testosterone, DHEA and cortisol levels and stuttering severity. In addition, the frequency of an allele of the CYP17 −34 T:C polymorphism was associated with susceptibility to stuttering. Furthermore, we could find higher levels of testosterone and oestradiol in CWS in the presence of the TC compared to the TT genotype. Further studies are needed to investigate more detailed mechanisms underlying the effect of steroids on the pathophysiology of stuttering. Conflict of interest The authors of this article have no conflicts of interest to declare. Statement of significance The data shed light on biological bases of developmental stuttering. The results emphasis the role of steroids in speech fluency and language organization in the brain. For the first time, we introduce the cytochrome P450 (CYP) 17 −34 T:C (MSP AI) as an important polymorphism related to developmental stuttering. Acknowledgements This work was performed in partial fulfillment of the requirements for PhD degree of Dr. Hiwa Mohammadi and was financially supported by the Iran University of Medical Sciences, Tehran, Iran (grant no: 9404-87-27001). The authors appreciate the staff members of the Medical Biology Research Center at Kermanshah University of Medical Sciences for their cooperation. The authors also are grateful for the Iran National Science Foundation (INSF). 54

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