Characteristic external genitalia in male neonates with 5α-reductase deficiency

Abstract

There have been no reports comparing neonatal external genitalia of 5α-reductase deficiency (5αRD) with those of other 46,XY differences of sex differentiation (DSD). This study enrolled 31 Japanese cases of 46,XY DSD whose external genitalia was examined during the neonatal period; four were diagnosed as 5αRD and 15 were defined as non-5αRD by genetic analysis of SRD5A2 or urinary steroid metabolites. We compared the following characteristics between 5αRD and non-5αRD groups, adjusting the severity of undermasculinization of the external genitalia: stretched penile length (SPL), glans width, location of the external urethral opening, and proportion of undescended testis. The external genitalia of all the 5αRD cases were Quigley classification grade 2 or 3. We compared the phenotypes between the four 5αRD cases and 11 non-5αRD cases with grade 2 or 3. The median (range) of SPL in the 5αRD group (14 mm [11-16]) was significantly lower than that in the non-5αRD group (22 mm [15-29]) (p = 0.003). An SPL cut-off value of <15 mm yielded a sensitivity of 50% (95% confidence interval [CI]; 7–93%) and specificity of 100% (95% CI, 72–100%) for discriminating between the groups. The median glans width, location of the external urethral opening, and proportion of undescended testis were not significantly different between the groups. The SPL of 5αRD in Quigley classification grade 2 or 3 was significantly shorter than that of other 46,XY DSDs with the equivalent grade.

 Introduction

5α-reductase deficiency (5αRD) is an autosomal recessive disorder caused by impaired conversion of testosterone (T) to dihydrotestosterone (DHT). Compared to T, DHT has a 2- to 5-fold higher affinity for the androgen receptor (AR) and activates AR signal transduction 10-fold [1]. As a more potent androgen, DHT induces masculinization of the external genitalia of the fetus. The phenotype of undermasculinization of the external genitalia in 5αRD varies from complete female external genitalia to micropenis and overlaps that in other conditions of 46,XY differences of sex development (DSD).

Early recognition of external genital phenotype of 5αRD during the neonatal period will improve clinical management of patients with 5αRD, including gender assignment or treatment strategy. Considering spontaneous masculinization at puberty, preserved spermatogenesis, and potential male gender identity, 46,XY neonates with 5αRD are recommended to raise as male [2]. It is also helpful to choose a treatment option to increase the penile size. In 5αRD, the application of transdermal DHT is effective to increase penile length and avoid difficulty in voiding in a standing position and urethroplasty in cases with hypospadias. Thus, 5αRD is better evaluated in every neonate with undermasculinization and no detectable Mullerian derivative, if other etiologies are clinically or biochemically not likely.

To our best knowledge, there have been no reports comparing neonatal external genital phenotype of 5αRD with that of other 46,XY DSDs. The present study defined characteristic features of the external genitalia of 5αRD during the neonatal period among those of other 46,XY DSDs.

 Materials and Methods

A total of 149 infants with DSD were observed at the Department of Pediatrics, Keio University Hospital, Tokyo, from January 1, 2012, through December 31, 2020. The study enrolled 31 Japanese cases with 46,XY DSD whose external genitalia were examined and photographed during the neonatal period. This study excluded 12 cases because they had neither genetic analysis of SRD5A2 nor gas chromatograph-mass spectrometry (GC-MS) analysis at the age of ≥3 months [3]. Among the remaining 19 cases, four cases were diagnosed as 5αRD by either analysis, and 15 cases were defined as non-5αRD. Two non-5αRD cases were excluded because of the presence of anorectal malformation. We performed the genetic analysis of AR in all cases of 5αRD and seven cases of non-5αRD.

The external genital phenotype of 5αRD (N = 4) and non-5αRD (N = 13) was analyzed retrospectively using medical records and external genitalia images. We compared the following characteristics between 5αRD and non-5αRD groups, adjusting the severity of undermasculinization based on the Quigley classification and the External Masculinization Score (EMS) [4] of the external genitalia: 1) patients background, 2) stretched penile length (SPL), 3) glans width, 4) location of the external urethral opening, and 5) proportion of undescended testis.

Pediatric endocrinologists or urologists examined the external genitalia. The SPL was determined as a distance from the pubic ramus to the tip of the glans with a measuring tape [5]. Microphallus is defined as a penis with hypospadias and SPL of <2.5 standard deviations below the mean for age [6]. SPLs were measured after 24 h of birth in all cases, except three cases in the non-5αRD group whose SPLs were measured within 24 h of birth. The glans width was defined as the maximum transverse diameter of the glans when the foreskin was inverted as possible. The location of the external urethral opening was evaluated by Meatus scores 1–4 [7].

The Mann–Whitney U test was used to compare the medians of EMS, SPL, glans width, and Meatus score. The χ-square test was used to compare the proportions of undescended testis; p < 0.05 was considered statistically significant.

This study was conducted in accordance with the Declaration of Helsinki and the Ethical Guidelines for Life Science and Medical Research Involving Human Subjects issued by the Ministry of Health, Labour and Welfare. This study was approved by the Ethical Committee of Keio University School of Medicine (#20150104 and #20170130). Written informed consent was obtained from the parents for genetic analysis (#20170130) but was not required for the retrospective observational study (#20150104).

 Results

Patient  background

All four 5αRD cases were raised as male and had hypospadias and rugated scrotums. Based on the Quigley classification, the external genitalia were defined as grade 3 in three cases and grade 2 in one case. As for non-5αRD cases, the genitalia were grade 4 in two cases, grade 3 in nine, grade 2/3 in one, and grade 2 in one. Thus, we compared the phenotype between four cases of 5αRD and 11 cases of non-5αRD with grade 2 or 3. EMS in the 5αRD and non-5αRD groups ranged from 2.5–8 and 3–8, respectively. The median EMS was not significantly different between the 5αRD and the non-5αRD groups (4.5 vs. 6.0, p = 0.753). All four cases of 5αRD were born full-term. Two cases of non-5αRD were born preterm. The clinical and laboratory features of the cases were presented in Table 1.

Table 1 Clinical and laboratory features in subjects of 5α-reductase deficiency (5αRD) and non-5αRD group

Case	Quigley classification	EMS	Age at penile measurement (days)	SPL (mm)	Glans width (mm)	Meatus score	Undescended testis		SRD5A2 genotype	AR genotype	Urinary 5α/5β steroid metabolite ratios			Serum gonadotropin and androgen levels					Others
							Left	Right			An/Et	THF	THB	Age at blood test	LH/FSH (mIU/mL)	T (ng/mL)	After hCG stimulation
																	T (ng/mL)	DHT (ng/mL)
5αRD group
1	3	2.5	5	16	7	4	inguinal	—	p.[Gln6*]†;[Arg246Gln]†	wild	0.319 (2.287)	0.010 (2.105)	0.541 (2.991)	1 week	NA	NA	3.01	0.20
2	3	3	25	11	10	4	—	—	p.[Tyr26*]†;[Arg227Gln]†	wild	NA	NA	NA	1 week	NA	NA	7.44	NA
3	2	8	2	15	8	2	—	—	p.[Arg227Gln]†;[Gly21Arg]	wild	0.829 (1.257)	0.049 (2.217)	0.459 (4.054)	1 week	NA	NA	4.07	0.19
4	3	6	10	13	7	4	—	—	p.[Asn193Ser]†;[p.Leu185Ser]	wild	<0.167 (0.460)	0.059 (0.869)	0.377 (1.280)	NA	NA	NA	NA	NA
non-5αRD group
1	3	3	0	20	12	4	—	—	wild;wild	wild	NA	NA	NA	5 years	<0.3/1.2	0.05	4.19	NA
2	3	6	0	24	NA	4	—	—	NA	NA	2.643 (0.460)	1.768 (0.869)	8.207 (1.280)	1 year	<0.3/1.0	<0.04	4.91	NA
3	3	5.5	13	23	12	4	—	inguinal	wild;wild	wild	NA	NA	NA	2 weeks	6.0/7.5	1.22	3.03	NA	preterm birth at 36 weeks of GA
4	3	6	2	20	10	4	—	—	wild;wild	wild	NA	NA	NA	5 months	0.4/3.4	0.19	NA	NA	NR5A1 pathogenic variant
5	3	3	12	22	11	4	—	—	NA	NA	11.466 (1.257)	4.615 (2.217)	9.088 (4.054)	3 months	8.4/3.4	3.46	NA	NA	preterm birth at 32 weeks of GA
6	3	3	1	15	7	4	—	—	wild;wild	wild	NA	NA	NA	9 months	0.8/2.6	0.11	7.45	NA
7	3	3	1	22	8	4	—	—	NA	NA	5.185 (0.460)	3.323 (0.869)	4.113 (1.280)	4 months	<0.3/3.6	0.05	NA	NA	NR5A1 pathogenic variant
8	3	6	1	27	13	4	—	—	wild;wild	wild	NA	NA	NA	3 months	2.5/2.7	1.80	3.46	NA
9	2/3	7	2	25	NA	4	—	—	wild;wild	wild	NA	NA	NA	3 months	2.2/2.2	2.40	NA	NA	NR5A1 pathogenic variant
10	2	8	0	22	11	2	—	—	NA	NA	5.054 (1.257)	1.850 (2.217)	2.038 (4.054)	NA	NA	NA	NA	NA
11	3	6	24	29	10	4	—	—	wild;wild	wild	NA	NA	NA	1 week	NA	2.34	5.57	NA

The urinary steroid metabolites were analyzed at three months of age or older. The values in the parenthesis represent age-matched 2.5th percentile in our institution.

In hCG stimulation, hCG (3,000 U/m²) was injected once a day for three consecutive days, and blood samples were taken on day 4.

^† Previously reported pathogenic variants. EMS = External masculinization score; SPL = Stretched penile length; An = Androsterone; Et =Etiocholanolone; THF = Tetrahydrocortisol; THB = Tetrahydrocorticosterone; GA = gestational age; T = testosterone; DHT = dihydrotestosterone; NA = not available.

Cases 1, 3, and 4 of the 5αRD group were diagnosed by reduced ratios of urinary 5α/5β steroid metabolites. Genetic analysis confirmed that all cases of the 5αRD group were compound heterozygous for SRD5A2 variants. Cases 1 and 2 were compound heterozygous for previously reported pathogenic variants. These variants were classified as pathogenic according to the American College of Medical Genetics and Genomics (ACMG) guideline. In cases 3 and 4, only one variant was previously reported as pathogenic. p.Gly21Arg in case 3 and p.Leu185Ser in case 4 were not reported to be pathogenic and classified as a variant of uncertain significance according to the ACMG guideline. p.Gly21Arg in case 3 has been previously reported in a single case without functional analysis [8]. p.Gly21Arg was not identified in the Japanese population database (54KJPN, https://jmorp.megabank.tohoku.ac.jp) and observed at a minor allele frequency of 0.0000016 in global population database (gnomAD v4.0.0, https://gnomad.broadinstitute.org/). The Glycine residue at codon 21 is conserved among species, such as rhesus macaques, mice, and dogs (PhyloP score: 1.293) (https://genome.ucsc.edu/index.html). In silico analysis of p.Gly21Arg showed consistent results, suggesting pathogenicity (Polyphen2: PROBABLY DAMAGING 1.00, CADD score: 22.0). p.Leu185Ser in case 4 was reported only in ClinVar. p.Leu185Ser was observed at a minor allele frequency of 0.000157 in 54 KJPN and 0.0000128 in gnomAD v4.0.0. The Leucine residue at codon 185 is highly conserved (PhyloP score: 9.176). In silico analysis of p.Leu185Ser showed consistent results, suggesting pathogenicity (Polyphen2: PROBABLY DAMAGING 1.00, CADD score: 25.8). Genetic analysis of AR revealed no pathological variants in all four cases of 5αRD.

The non-5αRD group included three cases with heterozygous pathogenic NR5A1 variants and eight cases of unknown etiology. Of the eight patients with unknown etiology, five patients underwent targeted exome sequencing for 36 candidate genes related to 46,XY DSD. The remaining three patients underwent only GC-MS analysis. All the ratios of urinary androsterone/etiocholanolone, 5α/5β tetrahydrocortisol, and 5α/5β tetrahydrocorticosterone in the non-5αRD group were greater than those in the 5αRD group (Table 1). Seven of the 11 patients underwent genetic analysis for AR, and no pathological variants were identified.

Characteristic  features of the external genitalia

Fig. 1 shows the SPL, glans width, location of the external urethral opening, and presence of an undescended testis in the 5αRD and non-5αRD groups. In the 5αRD group, SPLs ranged from 11–16 mm, indicating that all the cases had microphallus. In the non-5αRD group, SPLs ranged from 15–29 mm, and seven cases had microphallus. The median of SPL in the 5αRD group (14 mm) was significantly lower than that in the non-5αRD group (22 mm) (p = 0.003). An SPL cut-off value of <15 mm yielded a sensitivity of 50% (95% confidence interval [95% CI], 7–93%) and specificity of 100% (95% CI, 72–100%) for discriminating 5αRD and non-5αRD groups. Glans widths in the 5αRD and non-5αRD (N = 9) groups ranged from 7–10 mm and 7–13 mm, respectively. The median glans width in the 5αRD group (7.5 mm) was not significantly lower than that in the non-5αRD group (11 mm) (p = 0.050). In comparing the location of the external urethral opening, Meatus scores were not significantly different between both groups (p = 0.661). There was one case of unilateral inguinal undescended testis in each group, and the proportions of undescended testis were not significantly different between both groups (25.0% vs. 9.1%, p = 0.467).

Fig. 1  Comparison of SPL (a), glans width (b), and location of urethral opening (c) between 5αRD and non-5αRD cases with Quigley classification grade 2 or 3. Dotted line in (a) shows 24 mm (–2.5 standard deviation of the mean). *p < 0.05; ns = not significant.

 Discussion

We compared the genital phenotypes between the 5αRD and non-5αRD groups, adjusting the severity of undermasculinization of the external genitalia. We revealed that the SPL of 5αRD was significantly shorter than that of non-5αRD. No significant differences were found in the glans width, location of the external urethral opening, and proportion of undescended testis. One of the strengths of this study is that we compared the phenotypes of 5αRD and non-5αRD cases all of whom underwent either genetic testing or urinary GC-MS.

The SPL of 5αRD is significantly shorter than that of non-5αRD with the equivalent undermasculinization. This study included neonates with insufficient masculinization who required laboratory examinations for gender assignment. The SPL of 5αRD was significantly short even among 46,XY DSDs with such insufficient masculinization. The median difference of 8 mm in SPL between 5αRD and non-5αRD groups is clinically significant, since it is larger than an interobserver variation of infantile SPL (95% limits of agreement ranging from –4.9–4.2 mm) [9]. Thus, short SPL can be helpful to suspect 5αRD if we examine DSDs with Quigley classification grade 2 or 3. The non-5αRD group of unknown etiology may be caused by various etiologies. However, there was a difference in the degree of DHT function between the two groups. Thus, the degree of DHT function may affect the differences in the SPL. These results suggested that the growth of penile length is more sensitive to DHT deficiency than the growth of glans width, urethral formation, and descent of the testis.

5αRD can be suspected for every neonate with 46,XY DSD whose Quigley classification is grade 2 or 3 and SPL is <15 mm. The penis of 5αRD case is so short that it can be reared as female without consideration of gender assessment. Historically, 57.2% of 46,XY patients with 5αRD have been assigned as females since 1999 [10]. However, Maimoun et al. [11] showed that 12.5% (5/40) of patients raised as females switched to males due to gender dysphoria, whereas none of the patients oriented as male switched to female. If physicians consider short SPL as a clue for 5αRD diagnosis, more neonates with 5αRD can be diagnosed and do not miss an opportunity for the gender assessment. Thus, an SPL cutoff value of 15 mm can be clinically useful for Japanese 46,XY DSD neonates without detectable Müllerian derivatives.

No significant difference in glans width between 5αRD and non-5αRD groups may be biologically plausible. The corpus cavernosum is the main component of the penile shaft, whereas stromal cells account for most of the glans. The expression density of AR has been reported to be lower in glans stromal cells than in the penile corpus cavernosum at 12–14 and 16–20 gestational weeks [12]. Therefore, the degree of DHT function may have less impact on the glans, resulting in no significant difference in the glans width between both groups.

There are some limitations in this study. First, we investigated only cases with Quigley classification grade 2 or 3. Whether the SPL of 5αRD, other than grade 2 or 3, is significantly shorter than that of non-5αRD with the equivalent grade is unclear. Second, this study only enrolled Japanese patients with 46,XY DSD. Ethnic background may have affected the study results. Third, urinary GC-MS may not be a definitive method for diagnosing 5αRD as genetic testing. However, previous studies indicated that the urinary GC-MS is reliable in diagnosing or excluding 5αRD in infants aged ≥3 months [13, 14]. Fourth, the etiology of DSD may vary among non-5αRD cases. We did not perform a genetic test in four of 11 non-5αRD cases. In those cases, we did not exclude partial AIS that may share similar features as 5αRD. Further study is needed to compare genital phenotypes of 5αRD with those of other 46,XY DSD. Fifth, some neonates in the non-5αRD group were born preterm or measured for SPL within 24 h of birth. We speculated that the SPL of preterm neonates was shorter than that of full-term neonates. SPL within 12 h of birth is significantly shorter than that of 12 h after birth [9]. If we had compared SPLs measured only 24 h after birth in full-term neonates, the difference in SPL between both groups could have been larger. Sixth, accurate measurement of the glans width is difficult because they cannot be fully exposed during the neonatal period. The interobserver variation in the glans width could be larger than that of the SPL. Despite these limitations, our results addressed hitherto undescribed characteristics of 5αRD.

In conclusion, the external genitalia of male neonates with 5αRD in this study were Quigley classification grade 2 or 3. The SPL of 5αRD in grades 2 and 3 was significantly shorter than that of other 46,XY DSDs with the equivalent grades. This characteristic feature in 5αRD will help early diagnosis and proper management, including gender assignment and treatment strategies during the neonatal period.

 Acknowledgments

We appreciate Ph.D. Yuhei Koyama (Roche Diagnostics Japan) for the helpful discussions.

 Disclosure

Tomonobu Hasegawa is a member of Endocrine Journal’s Editorial Board.

 Funding

This work was supported by a Grant-in-Aid from the Ministry of Health, Labour, and Welfare, Japan (23FC1040 to T.I.).

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