Longitudinal clinical course in patients with 5α-reductase type 2 deficiency treated with testosterone and dihydrotestosterone during infancy and puberty

Abstract

5α-reductase type 2 (5αRD2) deficiency is a 46,XY disorder of sex development caused by impaired conversion of testosterone (T) to dihydrotestosterone (DHT). Penile enlargement therapy is important for male patients with 46,XY 5αRD2 deficiency who have undermasculinized external genitalia, such as severe micropenis. High-dose T and percutaneous DHT replacement are reportedly efficacious for penile enlargement in patients with this disorder. We presented herein the longitudinal course of four patients with 46,XY 5αRD2 deficiency who received T and DHT. T replacement therapy during infancy increased the stretched penile length (SPL) in three of the patients but was ineffective in one patient. DHT was administered to the three patients after T replacement therapy and further increased the SPL. During and after puberty, two patients asked for and received T replacement therapy, which contributed to increasing their SPL. A semen test in one patient with T replacement therapy at age 27 years revealed cryptozoospermia despite normal testicular volume. The clinical course of our patients during infancy indicated that DHT therapy may be preferrable to T replacement therapy for penile enlargement in patients with 5αRD2 deficiency. During and after puberty, T replacement therapy promoted penile enlargement possibly because of increased conversion of T to DHT via increased 5α-reductase type 1 activity even in patients in whom it was ineffective during infancy. In conclusion, DHT is effective for penile enlargement during infancy in patients with 5αRD2 deficiency while T replacement therapy is a viable option during puberty.

5α-REDUCTASE TYPE 2 (5αRD2) deficiency is an autosomal recessive 46,XY disorder of sex development (DSD) involving impaired conversion of testosterone (T) to dihydrotestosterone (DHT) [1]. Patients with 5αRD2 deficiency harbor biallelic pathogenic variants of the SRD5A2 gene encoding the 5αRD2 enzyme.

Patients with 46,XY 5αRD2 deficiency have disrupted DHT production, resulting in a broad spectrum of undermasculinization of the external genitalia, ranging from isolated micropenis to nearly female external genitalia with mild clitoromegaly, often leading clinicians to assign these patients female sex at birth [2]. However, male sex assignment is strongly recommended in patients with this disorder regardless of the severity of undermasculinization of the external genitalia at birth [3] because T secretion is sufficient for male psychosexual development in the fetal period [4]. Indeed, 60% of female patients with 46,XY 5αRD2 deficiency self-identify as male [3]; some studies have reported that more than 50% of such individuals undergo female-to-male sex reassignment [4]. Another reason for male sex assignment at birth is that the patients can become biologically fertile males with the aid of assisted reproduction techniques, and some patients are even naturally fertile [2, 5]. Thus, penile enlargement therapy has a particular significance for patients with this disorder and is critical for various, related surgical treatments, such as the surgical correction of hypospadias, as well as for the patients’ psychological well-being.

Percutaneous DHT replacement therapy is used for penile enlargement in patients with 5αRD2 deficiency even when their external genitalia are feminized [6-12]. Sasaki et al. reported that percutaneous DHT 12.5–25 mg/day for 8–12 weeks had a greater effect on penile enlargement before, than after, puberty onset [10]. DHT has two benefits: as supplementation therapy it is reasonable to administer DHT to patients with 5αRD2 deficiency, and DHT is not aromatized to estradiol to cause epiphyseal fusion or gynecomastia [13, 14]. However, the long-term efficacy and safety of DHT therapy during puberty and adulthood and the optimal treatment dosage have not been established although several, empirical dosages have been reported [6-11].

Interestingly, a pharmacological dosage of testosterone (T) was also effective for penile enlargement in some post-pubertal patients with 5αRD2 deficiency who had a normal, or even elevated, pretreatment serum T level [15]. Price et al. reported that high dose T therapy (500 mg via intramuscular injection weekly for several months) during puberty in patients with 5αRD2 deficiency was efficacious for penile enlargement, suggesting that it is a viable treatment option [15]. They also stated that enlargement of the penile shaft occurred in all four patients, but longitudinal data on penile size and testicular volume (TV) were not collected [15]. The optimal dosage, target serum concentration, and long-term efficacy and safety of T replacement therapy in patients with other forms of hypogonadism have been already established. However, large dosages of T may cause loss of final height due to premature epiphyseal fusion as well as impairment of spermatogenesis.

Herein we described the longitudinal data on androgen dosages, stretched penile length (SPL), and TV in four patients with 46,XY 5αRD2 deficiency who received T and DHT therapy, which have not been extensively reported in previous studies [6-11, 15].

Subjects and Methods

Four patients with 5αRD2 deficiency, raised as males, were included in this study. All of them were born to nonconsanguineous parents, their delivery was uneventful, and abnormalities in their external genitalia were noted within several days postpartum. All the patients had severe micropenis. Other genital anomalies included hypospadias and cryptorchidism (Table 1).

Table 1 Characteristics of Cases 1–4. Nationality, genital abnormalities other than micropenis, SRD5A2 gene variants, T/DHT ratio, and the clinically important SPL values are shown.

		case 1					case 2					case 3			case 4
nationality		Japanese					Japanese					Japanese			Chinese
remarks		penoscrotal hypospadias, left cryptorchidism					retractile testis					penoscrotal hypospadias			hypospadias, bilateral cryptorchidism
SRD5A2 variants		Val82del/Arg227Gln					Glu38Glufs*89/Arg227Gln					Gln6*/Arg227Gln			Ala217Glu/Ala217Glu
T/DHT		21.7					24.0					17.2			19.3
		prepubertal			pubertal		prepubertal		pubertal			prepubertal			prepubertal
		pre	post T	post DHT	pre	post T	pre	post T	pre		post T	pre	post T	post DHT	pre	post T	post DHT
chronological age		1y2m	1y8m	3y0m	11y11m	15y11m	1y3m	3y8m	6y8m	12y3m	22y4m	1m	8m	1y9m	day5	5m	10m
SPL	(mm)	13	13	30	27	50	23	27	35	33	60	10	20	30	14	24	37
	(SD)	–5.0	–5.0	–0.7	–1.9		–2.5	–1.5	–1.6	–1.5		–5.2	–2.8	–0.7	–3.9	–2.7	0.4

SPL was defined as the distance from the pubic ramus to the tip of the glans penis manually measured with traction along the penile shaft to the point of increased resistance [16]. The data on SPL were evaluated using Japanese reference SPL values for patients in Cases 1 to 3 [17, 18] and Chinese reference values for the patient in Case 4 [19]. TV was measured by the primary physicians using an orchidometer in an outpatient clinic. T was administered in the form of intramuscular injections of testosterone enanthate once a month. For prepubertal boys, three doses of T 25 mg per month were administered as previously reported [20]. For pubertal boys, T therapy was begun at 25 mg per month, then gradually increased at the discretion of each patient’s primary physician. Since DHT was not commercially available in Japan, special approval for its use was obtained from the institutional review board (H30a-13). In Case 1, two different concentrations (1% and 5%) of DHT gel were used at the discretion of the primary physician depending on the DHT dosage. The details are described in the Results section. In Cases 3 and 4, only 10% DHT gel was prescribed once a month. The gel was apportioned into four to five containers by the hospital pharmacist, and the patients’ parents were trained to administer the entire content of one container each week via percutaneous application to the penis, scrotum, and lower abdomen.

The SRD5A2 gene analysis was performed by Sanger sequencing in Cases 1 to 3 and by targeted next generation sequencing with a platform of 11 DSD-related genes (AR, SRD5A2, WT1, FGF8, FGFR1, CHD7, NR5A1, HSD17B3, HSD3B2, ANOS1, and SRY) in Case 4. In the latter, a variant of the SRD5A2 gene (p.Ala217Glu) was confirmed by Sanger sequencing.

Serum LH and FSH concentrations were determined by immunofluorometric assay. In Cases 1 and 2, the concentration of T and DHT was assessed by radioimmunoassay (RIA) while in Case 3 it was assessed by RIA and electrochemiluminescence immunoassay. In Case 4, the T and DHT concentration was measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The T/DHT ratio was calculated by an hCG stimulation test (3,000 U/m² for three consecutive days with blood sampling on Days 5 and 6) in Cases 1 to 3 and by the basal values in Case 4. The urinary steroid profile in Cases 3 and 4 was analyzed using gas chromatography/mass spectrometry by technicians in the Department of Laboratory at Keio University School of Medicine. The 5α-tetrahydrocorticosterone (5αTHE) to 5β-tetrahydrocorticosterone (5βTHE) ratio was used to evaluate 5α reductase activity.

The present study complied with the guidelines for human studies, was conducted in accordance with the Declaration of Helsinki, and was approved by the institutional ethics committee (2021-b95; Tokyo Metropolitan Children’s Medical Center). Informed consent was obtained from the patients and their parents for the publication of their data, including all the accompanying images.

Results

The four patients in this study were diagnosed as 5αRD2 deficiency by elevated serum T/DHT ratios and SRD5A2 gene variants in all cases (Table 1), and decreased urinary 5αTHE/5βTHE ratios in Cases 3 and 4 (data not shown). The p.Arg227Gln variant common to Cases 1 to 3 derives from a founder effect in the Japanese and Chinese populations and is associated with some residual enzymatic activity (3.2%) [21]. p.Gln6* in Case 3 was a previously reported, nonsense variant [22]. p.Glu38Glufs*89 in Case 2 was novel and involved a 23 bp deletion in exon 1, leading to the generation of a presumably non-functional, premature termination codon. p.Val82del in Case 1 was novel, and p.Ala217Glu in Case 4 was reported previously [23, 24]. Although these variants were not proven to be pathogenic, strictly speaking, p.Val82 and p.Ala217 are highly conserved amino-acid residues among vertebrates. Furthermore, p.Val82del and p.Ala217Glu are not registered in the general population database (gnomAD, TogoVar, and 1,000 genomes), and in silico analysis has suggested that both p.Val82del (PROVEAN: deleterious) and p.Ala217Glu (PolyPhen-2:1.00) variants decrease 5αRD2 enzymatic activity. Inheritance of SRD5A2 gene variants from both parents was confirmed in all the present patients using Sanger sequencing.

Case 1 was born to Japanese parents. Severe micropenis, penoscrotal hypospadias, and left cryptorchidism were found at birth. The SPL was 13 mm (–5.0 SD), and three doses of T 25 mg per month as per the standard treatment were administered intramuscularly but failed to increase the SPL (Fig. 1a). At age 1 year 8 months, a hCG stimulation test revealed an intact T response (5.87 ng/mL) and low DHT (0.27 ng/mL; T/DHT ratio: 21.7). At age 2 years, a genetic diagnosis was made by analysis of the SRD5A2 gene, prompting administration of DHT therapy, which was begun at a low dose (13 mg per month administered once weekly using a 1% DHT gel) and was gradually increased to 320 mg per month (administered five days per week using a 5% formulation), resulting in an increase in the SPL to 30 mm (–0.7 SD) (Figs. 1b, 2a, and Table 1). However, its efficacy plateaued after about six months, and a higher dosage (640 mg per month administered twice daily five days per week using the 5% formulation) and longer therapy failed to increase the SPL any further (Fig. 2a). DHT was discontinued at age 3 years, and the left cryptorchidism and hypospadias were corrected at age 2 years 7 months and 3 years 8 months, respectively. DHT therapy was begun at a low dosage on the assumption that T 25 mg by injection corresponded to DHT 12.5 mg via percutaneous administration because DHT has a four-fold higher affinity than T to androgen receptor [25], and the percutaneous absorption rate of gel applied to the external genitalia is reportedly about 50% [26]. However, the DHT dosage was increased to 320 mg per month in line with a previous report to increase the SPL [27]. The serum DHT concentration was evaluated on an outpatient basis, but no positive correlation between dosage and DHT concentration was detected, presumably because the interval between DHT gel administration and blood sampling was not constant (data not shown). At age 12 years 11 months, the patient chose T instead of DHT therapy for androgen supplementation, and T replacement therapy was begun at 25 mg per month, then was increased steadily. His SPL increased to 47 mm (–4.5 SD) at age 13 years 11 months before plateauing. Thereafter, neither increasing the T dosage nor extending the treatment period resulted in any further penile enlargement (Fig. 2a). T replacement therapy was continued according to the patient’s wishes. His final SPL was 50 mm (–2.8 SD) at age 17 years 2 months. Gonadotropins showed no suppression at age 16 years 6 months (LH 5.02 mIU/mL and FSH 6.11 mIU/mL).

Fig. 1

Photographs of external genitalia in Cases 1–4.

Fig. 1a, b. Images of Case 1. (a) Before DHT therapy at age 1 year 11 months before the second surgery. SPL: 13 mm. (b) After DHT treatment at age 3 years 8 months. SPL: 30 mm.

Fig. 1c, d. Images of Case 2. (c) Before T replacement therapy at age 11 years 9 months. SPL: 27 mm. (d) After T replacement therapy at age 13 years 11 months. SPL: 60 mm.

Fig. 1e. Image of Case 3 before DHT therapy at age 8 months. SPL: 20 mm.

Fig. 1f, g. Images of Case 4. (f) Before T replacement therapy at age 2 months. SPL: 15 mm. (g) After DHT therapy at age 10 months. SPL: 37 mm.

Fig. 2

Longitudinal course of Case 1 (a), Case 2 (b), Case 3 (c), and Case 4 (d), including SPL, testicular volume (TV), DHT, T dosage, and gonadotropin value. The vertical axis represents the SPL and TV, and the horizontal axis represents the chronological age. The closed circles represent the SPL, and the open triangles represent the TV. The TV was the mean of the values for the left and right testicles. In (b), the gonadotropin levels indicate values immediately before T administration. The arrows indicate the times when the photos of the external genitalia in Fig. 1 were taken.

Case 2 was born to Japanese parents. Micropenis was diagnosed at birth. At age 1 year 5 months, T 25 mg per month was administered intramuscularly three times and increased the SPL from 23 mm (–2.5 SD) to 27 mm (–1.5 SD). A hCG stimulation test at age 6 years 8 months demonstrated a high T/DHT ratio of 24 (Table 1), suggesting that the patient had 5αRD2 deficiency. However, analysis of the SRD5A2 gene revealed no pathological variants. Another hCG stimulation test at age 12 years 3 months demonstrated a moderately high T/DHT ratio of 13.7, leading us again to perform a genetic analysis, which identified SRD5A2 gene variants. The patient chose T for his androgen replacement therapy. At age 12 years 3 months, one month before beginning his T replacement therapy, his testosterone, LH, and FSH level was 0.6 ng/mL, 1.49 mIU/mL, and 2.43 mIU/mL, respectively, indicating pubertal status. His SPL increased to 60 mm (–0.4 SD) (Fig. 1c and 1d) but plateaued at age 13 years 4 months (Fig. 2b). The patient was receiving T replacement therapy until several months prior to this writing (when DHT therapy was begun as described below), but the final SPL remained 60 mm (–2.4 SD). At age 17 years, his TV was 15 mL (left) and 20 mL (right) and did not decrease thereafter, indicating normal testicular development. The T dosage was reduced to 125 mg at age 22 because FSH showed suppression to 1.4 mIU/mL. At age 25 years, his LH and FSH was 1.66 mIU/mL and 3.48 mIU/mL, respectively. However, semen testing at age 27 years revealed cryptozoospermia, i.e., only one, mobile sperm was detected per one microscopic field after concentrating the seminal fluid. His treatment was recently switched to physiological replacement using DHT, given the possibility that pharmacological doses of testosterone were inhibiting spermatogenesis. Cryogenically stored sperm obtained via testicular sperm extraction may be used if his partner fails to conceive naturally.

Case 3, also born to Japanese parents, showed severe micropenis and penoscrotal hypospadias at birth. Laboratory data at age 1 month revealed an intact T response (9.14 ng/mL) and low DHT (0.53 ng/mL, T/DHT ratio 17.2) on a hCG stimulation test and a low 5αTHE/5βTHE ratio in his urinary steroid profile, indicating the possibility of 5αRD2 deficiency. T was administered from age 3 months to 5 months, and the SPL increased from 10 mm (–5.2 SD) to 20 mm (–2.8 SD) (Figs. 1e and 2c). Following the diagnosis of 5αRD2 deficiency by genetic analysis (Table 1), DHT gel treatment was administered from age 1 year to age 3 years 3 months. DHT 50 mg per month was begun and gradually increased to 320 mg per month. As a result, his SPL increased to 30 mm (–0.7 SD) at age 2 years 4 months before plateauing (Fig. 2c).

Case 4 was born to Chinese parents and was thought to be female at birth until his mother noticed clitoromegaly and masses in the bilateral labia majora before submitting the infant’s birth certificate. G-banding showed 46,XY, and neither a uterus nor a vagina was detected by ultrasonography. At age 1 month, a high serum basal T/DHT ratio (19.5; T 2.9 ng/mL; DHT 0.15 ng/mL) and a low urine 5αTHE/5βTHE ratio (data not shown) led to the diagnosis of 5αRD2 deficiency, and the patient was assigned male sex. Genetic analysis revealed a homozygous SRD5A2 variant, which was previous reported by one of the authors of the current study as pathogenic [23]. The SPL increased from 15 mm (–3.9 SD) (Fig. 1f) to 24 mm (–2.7 SD) after T administration at age 2 months to 4 months. DHT therapy (300 mg per month) was begun at age 5 months, and the SPL increased to 37 mm (+0.4 SD) after about five months. DHT therapy was stopped when the patient was aged 7 months because he achieved normal penile length. At age 8 months, a urologist recommended DHT therapy to promote penile enlargement before urethroplasty. DHT was discontinued because little penile enlargement was observed at age 10 months. At age 1 year 3 months DHT administration was resumed before the second surgery at an increased dosage but it was discontinued on the emergence of pubic hair at age 1 year 6 months (Figs. 1g and 2d).

Discussion

The present study examined the clinical course and therapeutic effect of T and DHT replacement therapy in four male patients with 5αRD2 deficiency. In line with previous reports, our study demonstrated that T was less effective for penile enlargement than DHT in infancy, but had an efficacy comparable to that of DHT during puberty [10, 15]. The novelty of our study lies in the detailed data on the clinical course provided, including androgen dosages, SPL increases, and gonadotropin levels.

In infants with 5αRD2 deficiency, DHT was apparently more effective than T in increasing the SPL, as seen in the patients in Cases 1, 3, and 4, who either had no response (Case 1) or only a partial response (Cases 3 and 4) to T replacement therapy. These findings indicate that in infants with 5αRD2 deficiency, T has limited efficacy in enlarging the penis even in instances where some residual 5αRD2 activity might be present; thus, DHT therapy is recommended as an alternative. However, DHT therapy in the neonatal period or early infancy is difficult for two reasons: 1) for a definitive diagnosis of 5αRD2 deficiency, genetic analysis is required, but this may be difficult to perform depending on the institution or country; and 2) DHT gel is not commercially available in certain countries, including Japan. Thus, the authors believe that short-term T therapy consisting of three, 25-mg, intramuscular injections is a practical option in some situations, initially to achieve some increase in SPL even if there is a high index of suspicion of 5αRD2 deficiency.

There are only two reports of androgen (T or DHT) replacement therapy in pubertal and post-pubertal patients with 5αRD2 deficiency. In one report, high-dose T replacement therapy was effective for penile enlargement in post-pubertal patients with 5αRD2 deficiency [15]. Another study demonstrated that DHT was effective for penile enlargement in both the pre- and post-pubertal periods, but compared to the former period, increases in the SPL were apparently retarded after puberty [10]. The fact that a 46,XY female patient with no 5αRD2 activity due to SRD5A2 bi-allelic nonsense variants (p.Gln6*/Gln6*) achieved clitoral enlargement up to 50 mm without any treatment [22] indicates that increased T production during puberty can cause genital masculinization without 5αRD2 activity. This can be explained by increased conversion of T to DHT via increased 5α-reductase type 1 (5αRD1) activity in the skin and liver during puberty [1, 28]. Similarly, in the present study, the patients in Cases 1 and 2 received T instead of DHT during puberty, and their final SPL (50 mm and 60 mm, respectively) was comparable with that of previously reported patients who received DHT [10]. It is unclear whether it was endogenous or exogenous T, which contributed to a greater SPL increase in Cases 1 and 2 because the penile enlargement in these cases coincided with puberty. However, theoretically, pharmacological or exogenous T contributes to increased DHT production via increased 5αRD1 activity; thus, we believe that T replacement therapy for penile development is a viable treatment option during puberty.

T therapy has the well-known adverse effect of decreasing spermatogenesis by suppressing gonadotropins. In the present study, T was administered in Case 2 at a dosage of 125–250 mg per month for 15 years, and the LH and FSH levels occasionally fell to the low-normal or below-normal range (Fig. 2b), possibly accounting for the patient’s cryptozoospermia. However, the degree to which the T replacement therapy contributed to the disruption of spermatogenesis is unclear because his testicular volume was normal at age 17 years while most patients with 5αRD2 deficiency have absent or profoundly impaired spermatogenesis regardless of the therapy received [1]. Another risk associated with T replacement therapy is premature bone maturation and loss of adult height caused by the conversion of T to E2 via aromatization. However, in the present study, the adult height of the patients in Cases 1 (170.0 cm) and 2 (175.5 cm) was within the target range based on parental height [29], suggesting that the risk of this adverse effect was low in our patients.

DHT therapy also carries the risk of suppressing gonadotropins, as previously reported [10, 30]. The difference in the contribution of T and DHT to gonadotropin suppression is unknown because no studies have compared the effect of exogenous T and DHT on gonadotropin secretion in patients with 5αRD2 deficiency. Another concern in DHT therapy is prostatic hypertrophy as the prostate is a DHT-specific target organ. However, this may not pose a serious problem because the intraprostatic DHT concentration is tightly regulated by other, as of yet unknown mechanisms that are independent of the serum DHT concentration [31].

DHT therapy has certain disadvantages in that the optimal location for application, dosages, and treatment duration have not been established, and the safety of long-term treatment is still unknown. In our experience, a DHT dosage of around 300 mg per month appeared to be effective in increasing SPL in patients with 5αRD2, but the patient pool was too small to determine an optimal DHT dosage and formulate a treatment strategy. In practice, starting with a small dosage and increasing gradually until penile enlargement occurs may be the best policy at present. Terminating T or DHT replacement therapy once penile length plateaus may also be an option. Further investigation is necessary to establish the safety parameters for DHT therapy in patients with 5αRD2 deficiency.

Several, previous reports and the present study have shown that the final SPL in patients with 5αRD2 deficiency were below the normal range even though the condition was correctly diagnosed in early infancy and the appropriate treatment was administered [2, 10, 32]. This fact indicates two possibilities: 1) most of the penile growth potential is probably determined during the fetal period, possibly accounting for the plateau in penile growth observed in patients with 5αRD2 deficiency despite treatment; and 2) in situ production of DHT, i.e., the conversion of T to DHT by 5αRD2 in penile shaft tissue (corpus cavernosum penis, corpus cavernosum urethrae, and genital skin) contributes more than circulating DHT to penile enlargement, as evidenced by a recent finding that DHT is almost undetectable in fetal testes and fetal serum samples in the second trimester when the genital tubercle develops into the penis and penile enlargement occurs [33]. From this perspective, exogenous T might increase in situ DHT production described above in certain patients with 5αRD2 deficiency who have residual enzymatic activity.

In conclusion, we reported four, male patients with 5αRD2 deficiency. During infancy, DHT appeared to be more effective than T for penile enlargement. During puberty, T replacement therapy may be used instead by taking advantage of increased 5αRD1 activity.

Acknowledgments

We are indebted to Dr. Hironori Shibata and Dr. Kenji Nanao for their useful discussion and to Mr. James R. Valera for his assistance in editing this manuscript.

Disclosures

The authors have no conflicts of interest to declare.

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