2.1. 3βHSD2 Deficiency: General Frame of Approach

3βHSD2 deficiency, the rarest form of CAH, is part of the non-21OH-related CAH cluster which overall counts for less than 5–10% of CAHs (generally, 5–8% for 11-beta hydroxylase deficiency, and less than 1% for 17-hydroxylase deficiency in association with 3βHSD2 deficiency) [22,23]. As mentioned, some exceptions of a relatively higher incidence are registered in areas like Old Order Amish of North America due to a c.35GA founder mutation [22,24,25,26].

There are two types of CAH underling 3βHSD2 deficiency: SW due to associated aldosterone deficiency, and non-SW that involves a normal mineralocorticoids production. DSDs are positive in any of these forms, while non-SW also includes a milder presentation which may be predominantly expressed as hirsutism and menstrual cycle anomalies [27]. Other authors consider that we should discuss of 3 distinct forms: SW, non-SW, and non-classical type as a different entity with pubertal onset especially in females [27,28]. Generally, 95% of cases diagnosed with paediatric adrenal insufficiency are caused by CAH, but 3βHSD2 deficiency has a modest epidemiological impact opposite to 21OH deficiency [29]. In teenagers and young adults, the most frequent cause of adrenal insufficiency is Addison’s disease followed by CAH, overall chronic adrenal failure having more than 30 aetiologies [30].

3βHSD is an enzyme that converts 5-steroids: pregnenolone (Preg), 17-hydroxypregnenolone (17OH-Preg), dehydroepiandrosterone (DHEA), and androstenediol to the corresponding 4-steroids, meaning progesterone, 17-hydroxyprogesterone (17OHP), androstenedione, and testosterone [5].

In the human genome, there are two active 3βHSD genes located on chromosome 1p13.1; these encode two 3βHSD isoenzymes [5,31,32]. The 3βHSD type 1 isoenzyme is found in several tissues such as placenta, brain, liver, and breast, skin, and prostate while the type II enzyme is identified at the level of adrenals and gonads [5,31,32,33]. In contrast with the type II isoenzyme, which acts only on intra-glandular steroids, 3βHSD type 1 is able to act on low levels of circulating steroids, due to its low Michaelis–Menten constant [5,31,32,33]. For instance, this enables the liver isoenzyme to convert some of the elevated 17OH-Preg into 17OHP in cases with 3βHSD type II deficiency [5,31,32,34]. There have not been reported mutations of the 3βHSD type I gene, presumably because such mutation would prevent progesterone synthesis in the placenta, leading to spontaneous abortion [33,34].

The advance of molecular and genetic diagnosis is mostly useful in different types of CAH, starting with the most frequent “mutation”, namely 21OH deficiency, since a level of genotype–phenotype correlation is expected. However, in 3βHSD2 deficiency the current level of knowledge makes this association less predictable due to the paucity of studies [29].

The most significant hormonal changes associated with 3βHSD deficiency are high ratios of the Δ5-steroids (Preg, 17OH-Preg, DHEA, and androstenediol) to corresponding Δ4-steroids (progesterone, 17OHP, androstenedione, and testosterone), especially high 17OH-Preg/17OHP, elevated serum levels of 17OH-Preg, DHEA and dehydroepiandrosterone sulfate (DHEA-S) and increased urinary levels of their corresponding metabolites [5,35,36].

A useful diagnostic (dynamic) test is the ACTH stimulation test: 17OH-Preg, cortisol, 17OHP, DHEA-S, and androstenedione are measured before and one hour after administration of 250 µg of synthetic ACTH (Cosyntropin) [35]. In patients with 3βHSD2 deficiency, 17OH-Preg is expected to be >150 nmol/L after stimulation, while 17OH-Preg to 17OHP ratio is expected to become very high [37].

In the absence of treatment, SW forms, manifesting through vomiting and dehydration, may cause hypoglycemia, hyperkalemic acidosis, and hemodynamic instability including death [37]. Severe 3βHSD2 deficiency may lead to disorders of sex development (DSD) with ambiguous genitalia at birth (46,XY or 46,XX individuals); DSDs may accompany SW or non-SW type. Milder forms display females with virilization in association with menstrual cycle anomalies, while males have incomplete masculinization (non-classical phenotype) [37].

Management of 3βHSD2 deficiency is similar to other types of CAH, with glucocorticoid and mineralocorticoid replacement therapy in cases with severe enzyme deficiency. Concerning the paediatric population, treatment with hydrocortisone 10–15 mg/m²/day is preferred, while in adulthood long-acting glucocorticoids can be used. Mineralocorticoid replacement consists of therapy with fludrocortisone 0.1 mg/day, adjusted depending on plasma renin activity levels. Females with androgen excess (non-classical type) are candidates for oral contraceptives and anti-androgenic drugs in cases with severe hirsutism and menstrual cycle disturbances [35].

2.2. Clinical Presentation of Patients Confirmed with 3βHSD Type II Deficiency

We synthetized the studies published according to the mentioned methodology in Table 1 [5,22,25,26,27,31,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61].

Most articles introduce cases with SW forms, which seem like a common presentation due to its severity. The majority of patients were diagnosed after a SW crisis (vomiting, diarrhoea, hyponatremia, hyperkaliaemia, and hypoglycaemia) early during childhood or soon after birth [22,25,26,31,38,40,41,43,44,47,50,53,55,57,58]. Other signs and symptoms that are described under these circumstances are a lack of sweating, malnutrition, and developmental delay [31,50]. Some patients with SW associated DSD with males (mostly 46,XY males and mild virilisation in some 46,XX females; these generally are less affected concerning the spectrum of DSDs). Most cases of ambiguous genitalia were found in male patients who were affected by undermasculinization and presented with hypospadias, embedded penis, micropenis, scrotum bifidum, cryptorchidism, and vaginal introitus [22,25,26,31,38,39,40,43,44,46,47,48,49,53,56,57,58,59]. Female patients had either normal genitalia or were mildly virilised (for instance, clitoromegaly) [22,25,26,31,38,40,43,44,46,47,48,49,53,56,57,58,59,61]. Other elements of clinical spectrum include: premature pubarche, gynecomastia, obesity, intellectual and developmental delay, and neurological sequelae (irritability, convulsions, etc. [22,31,38,46,47,51,57,61]. We mention the study of Ladjouze A. et al., in 14 Algerian patients with SW type who associated a median Prader stage of 2 for females (N = 8/14) at median age of 20 days without posterior labial fusion, while males had a median external masculinization score of 6 (N = 6/14). During follow-up, the same population showed, according to the Tanner system: premature pubarche (N = 4/14; female/male ratio of 3/1), spontaneous puberty (N = 6/14, female/male ratio of 5/1) at the age of 11 y respective for boys, respective 11.5 y for girls; overall 4/8 females reached menarche by the age of 14.3 y. A median IQ (intelligence quotient) of 90 was identified (2/14 patients had an IQ > 100 and 3/14 of studied individuals with an IQ < 70) [38].

The largest study on 3βHSD2 deficiency was a cross-sectional, nine-centred, Turkish cohort on 31 patients (average age of 6.6 ± 5.1 y, female/male ratio of 12/19). The authors identified SW and non-SW forms with a heterogeneous spectrum: homozygous pathogenic missense mutations of >5% wild type-associated non-SW phenotype; all males (19/31) had ambiguous genitalia at birth, while one female (1/12) had virilised genitalia. Premature puberty was confirmed in 80% of cases, despite the subjects of the study being on hydrocortisone therapy; through teenager years, females displayed irregular menstrual cycles, ultrasound aspects of polycystic ovaries, while males had hypogonadism and TARTs (2 brothers) [26]. Overall, the researchers concluded that intact mineralocorticoid production and unvirilized genitalia in 46,XX females represent the major pitfalls of missing the diagnosis of 3βHSD2 deficiency, while the particular constellation of an androgens profile might be attributed to other forms of CAH [26].

2.3. Biochemical/Hormonal Assessments in 3βHSD Type II Deficiency

Most relevant endocrine assays are displayed in Table 2 (some of the patients were undergoing hormonal replacement) [22,25,26,27,31,38,39,40,41,43,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59].

The diagnosis of 3βHSD2 deficiency was established during neonatal screening for 21OH deficiency due to increased 17OHP levels [22,38,52,54,58]. High-risk molecular screening for patients with a family history of CAH was also performed in some patients [22,38].

In terms of overall hormonal picture, as expected, patients had high levels of 17OH-Preg, ACTH, DHEA, DHEA-S, 17OHP, renin, testosterone, androstenedione, and elevated ratios of 17OH-Preg to 17OHP ratio, respective of the 17OH-Preg to cortisol ratio [62]. The ACTH stimulation test was performed in 2 studies and showed an increase of 17OH-Preg (to a maximum of 1979 ng/dL), as well as a high 17OH-Preg to 17OHP ratio of 35.3 [48,51].

Ladjouze A. et al., showed that, unless the patients were under glucocorticoid replacement therapy, 17OHP levels were above upper limit in each patient. A value of 17OH-Preg above 90 nmol/L was found to be suggestive for the diagnosis of 3βHSD2 deficiency (a median of 139.7 nmol/L). DHEA-S revealed a wide area of values, between 9.4 and 5441.3 nmol/L (a median of 501.2 nmol/L). ACTH was abnormally high in 4/14 patients depending on the severity of genetic defect and the disease control achieved through daily hydrocortisone [38].

Li Z. et al., published a 5-case Chinese study on CAH (non-21OHD deficiency) and identified a 9-year-old boy with 3βHSD2 deficiency. Liquid chromatography tandem–mass spectrometry (LC-MS/MS) confirmed high levels of 17OHP and testosterone and very high values of DHEAS and androstenedione. High testosterone caused an enlargement of the testes within the last 5 years of surveillance in addition to accelerated height growth (+1.36 SD for his age), while his older brother sharing the same genetic defect had a final adult height deficiency of −3.71 SD [27]. Also, Guran T. et al. performed an LC-MS/MS analysis in 19/31 patients with 3βHSD2 deficiency; this showed an improvement of hormonal panel under adequate adrenocortical hormonal therapy; subjects who were 8 years and older showed higher levels of DHEAS and testosterone than patients below this cut off of age [26]. LC-MS/MS unequivocally sustained the diagnosis based on increased baseline 17OH-Preg to cortisol ratio as well as 11-oxyandrogen levels [26].

As mentioned by Fanis P. et al., the “pivotal role” of using gas chromatography-mass spectrometry in order to perform the urinary assays concerning steroids metabolomics finds its place in 3βHSD2 deficiency. In this case, the method revealed a rather late diagnosis of CAH, an 8.5-year-old boy (46,XY) with precocious puberty due to high testosterone (a 4-y advance of bone age versus his biological age) who was born with ambiguous genitalia and associated neonatal SW crisis, followed by gynecomastia, adrenarche and relapse of SW—related clinical elements by the age of 3.5 y [40].

Alternatively, liquid chromatography/electrospray ionization tandem mass spectrometry was used to assess urinary panel of cholesterol metabolites as a potential metabolomics approach to 3βHSD2 deficiency [63].

2.4. Genetic Background: HSD3B2

HSD3B2 gene sequencing was provided in 26 articles; around 20 mutations were described as a “novel pathogenic mutation” [5,22,25,26,27,31,38,39,40,41,43,44,45,46,47,48,49,50,52,53,54,57,58,59,60,61] (Table 3).

There are over 40 pathogenic variants of the HSD3B2 gene [64]. Loss of function mutations such as frameshift, in-frame deletions and nonsense mutations are associated with SW, while missense mutations generally lead to non-SW and non-classical forms of the disease [24,64]. Homozygous status leads to a more severe clinical manifestation; many of these patients had a parental history of consanguinity with parents often being first- or second- degree cousins [25,38,40,46,48,50,52,57]. This can be attributed to the fact that consanguinity may increase the risk of any autosomal recessive diseases, including 3βHSD2 deficiency [38,65]. We prior mentioned a single centric longitudinal study from Algeria which was published in 2022 on 14 patients (female/male ratio of 8/6), between 2007 and 2021. The subjects came from 10 families, and 8 of them were consanguineous, all individuals displaying p.Pro222Gln mutation (based on Sanger sequencing), except for two sisters with a novel mutation—a deletion of 3βHSD2 gene at the level of c.453_464del or p. (Thr152_Pro155del). Ladjouze A. et al., performed this genetic analysis at a median age of 20 days (between 0 and 390 days) while all subjects presented SW form (N = 14/14) in addition to DSD in majority cases (N = 10/14) [38].

Sanger sequencing also showed 11 homozygous pathogenic mutations that involved 6 novel mutations, including a c0.967A > G (p.N323D) which was positive in 14/31 patients coming from 11 Turkish families (Black Sea area), and homozygous c0.652T > C (p.S218P) mutation in 4 subjects originating from 4 families of Anatolian origin [26]. Another two studies presented patients from non-consanguineous families, with CAH due to 3βHSD2 deficiency caused by uniparental isodisomy [41,49]. As seen in other disorders, this is a condition characterised by two homologous chromosomes or regions which are inherited from the same parent [66].

Several case reports provided gene sequencing, as well. For instance, c.674T > A (p.V225D) mutation was confirmed on a 9-year-old Chinese boy with maternal and paternal inheritance of the genetic defect (the condition affected his older brother, too) [27]. Yu L. et al. described two novel mutations on a male subject: one with paternal inheritance—C.776 C > T—and the other from his mother—C.674 T > A —which might be responsible for the presence of bilateral TARTs [39]. Fanis P. et al., revealed a novel nonsense mutation—p.Lys36Ter (homozygosity, consanguineous parents)—in addition to a heterozygous status for CYP21A2 gene—p.Val281Leu [40]. Teasdale SL. and Morton A. identified a novel mutation on a 46,XY subject with ambiguous genitalia who was first considered as having androgen insensitivity syndrome and became clinically relevant as CAH after the onset of the adrenarche 931C > T(p.Gln311) variant in addition to a previously diagnosed c.244G > A (p.Ala82Thr) mutation [47]. In 2017, a novel mutation of homozygous type was reported—p.W355R (c.763 T > C)—on two brothers who presented TARTs diagnosed during the first, respective the fourth year of life [48]. A 46,XX female was confirmed with a novel nonsense homozygous mutation Q334X of HSD3B2 gene; the subject was born without DSD, but presented an SW crisis at the age of 13 days; 17OHP screening assays were positive, and, despite initially being considered as having 21OH deficiency, CYP21A2 gene showed no mutations, thus the patient was re-assessed [60]. One series of 3 cases identified 2 novel pathogenic variants c.154_162delinsTCCTGTT and c.674T > A [31]. The first mutation in Italy was described in 2016 on a 46,XY boy coming from a non-consanguineous family admitted for DSD and SW: a novel homozygous frameshift mutation—a single nucleotide deletion at codon 319 (GTC(Val)x2192;GC) [53]. Of note, this patient who presented perineal hypospadias and microphallus in association with biochemistry anomalies due to SW crisis like hyponatremia was also included in a larger study concerning acute manifestations due to aldosterone deficiency (but we registered only the first report) [67]. Also, a relatively large study on different types of CAH identified 6 cases of 3βHSD2 deficiency introducing 4 novel mutations in Anatolian population [44].

2.5. Fertility Issues

Data regarding fertility are limited. Spermogram was analysed in several articles [25,45,48]. One case report showed a normal sperm count and percentage of sperm with typical morphology, but subnormal vitality of 41% (normal > 58%) in a 24-year-old male diagnosed with a SW form due to a 3βHSD2 deficiency caused by a c.687del27 homozygous mutation, as revealed by gene sequencing. The 46,XY male patient experienced neonatal SW crisis in addition to micropenis, hypospadias and bilateral intra-scrotal testicles, followed by normal puberty by the age of 15 years and current apparently optimal fertility potential, as opposite to majority of the other published data [25]. Another case showed an unlikely fertile male with 3βHSD2 deficiency due to a c.687del27 deletion, whose testis histology in late puberty revealed few tubules with arrested spermatogenesis. He showed no neoplastic changes and was therefore considered low risk for malignancy [57]. Another two case reports showed azoospermia; however, in these cases genetic testing was not performed [45,56].

Fertility in people affected by CAH is influenced by the severity of the condition, with most cases of infertility occurring in people with SW forms [68]. Generally, infertility in CAH appears to be connected with multiple factors, such as ambiguous genitalia and virilization of the external genitalia in females, excessive androgen secretion leading to impaired hypothalamic-pituitary-ovarian axis function, and various psychosocial factors [68,69].

We generally know that females with non-severe CAH might display anomalies of menstrual cycle and rarely conceive, but the level of statistical evidence remains very low regarding 3βHSD2 deficiency, and no particular study specifically addressed the fertility profile in females with non-classical form according to our mentioned methodology.

Concerning pre-implantation diagnosis and an associated particular approach, we mention a case study that introduced a healthy boy born via in vitro fertilization after pre-implantation diagnosis and identification of a healthy heterozygote. The child’s parents had heterogeneous mutations causing 3βHSD deficiency (c.690G > A) and a history of affected children [42].

2.6. Complications & Outcome

3βHSD3 deficiency may cause a heterogonous picture of complications: life threatening acute adrenal insufficiency manifested as adrenal crisis, hypoglycemia, and hyperkalemic acidosis; failure to thrive or advanced skeletal maturation; neurological impairments following perinatal asphyxia, etc. [22,55,57]. Mild non-SW forms might be misdiagnosed as having POS due to hirsutism, acne, and menstrual cycle anomalies (non-classical CAH) [22,38]. Ultrasound and computed tomography sometimes show adrenal hyperplasia [38,50]. Orchialgia and testicular masses were described in patients with testicular adrenal rest tumours (TARTs) [22,38,39,48,56].

Associated tumours like ovarian adrenal rest tumours (OART), respective TARTs (ectopic adrenal tissue at testes) were identified in 9 patients [22,38,39,45,48,56].

Genetic mutations associated with TARTs were c.35G > A, Cys-72-Arg in homozygote form or as compound heterozygous with deletion, c.674T > A, c.776 C > T, p.W355R (c.763 T > C), p.(Pro222Gln) [22,26,38,39,45,48,57]. Non-malignant Sertoli-only cell tumour (which is not part of TARTs) was associated with a homozygote c.687del27 of deletion type on a 46,XY male with ambiguous genitalia (SW form). He also proved at puberty some signs of virilisation due to testes and peripheral sources of testosterone as well as breast enlargement and high oestrogens levels, probably through peripheral production [57]. The first adult report of TARTs with extra-testicular (perirenal) location was introduced in 2012 fn a 23-year-old male with suboptimal replacement therapy (homozygous 694C > G mutation within the HSD3B2 gene) [5].

The distinction between TART and Leydig cell tumours is sometimes difficult. We mention the case of a 34-year-old adult who, despite early treatment with glucocorticoids and mineralocorticoids (the first SW crisis was at the age of 1 month), was diagnosed with bilateral testicular lumps by the age of 13. An open testicular biopsy raised the question of a malignancy, and due to the tumour-related local accuses (masses of 4 cm) and azoospermia, bilateral orchiectomy was decided upon and later confirmed TARTs [45]. Interestingly, increased DHEAS seemed resistant to adequate adrenocortical hormonal replacement before surgery, while testosterone was within normal limits; thus, the decision of radical intervention versus testes-sparing surgery is even more challenging [45]. Another case of a 33-year-old male presented contra-lateral TARTs after a history of surgery for apparently a Leydig cell tumour of the testes. However, adrenal insufficiency due to a mild form of CAH was eventually recognized and both tumours were reconsidered as TARTs [56]. Testicular biopsy may be performed at young ages; this might add value regarding CAH evolution and hormonal control, for instance, to take into consideration the switch to another glucocorticoid formula or regime. We mention two siblings of 3.5 y and, respective, of 22 months who were diagnosed with TARTs through a testes biopsy; dexamethasone was offered and contributed to the tumour regression, but accelerated their growth thus a high dose of hydrocortisone was administered (>20 mg/m²/day) [48].

As mentioned, Ladjouze A. et al. identified 3/6 boys with TARTs, respective 3/8 females with adrenal masses (that required removal in two cases), in addition to one case of OART [38]. Yu L. et al., introduced the case of a Chinese boy who was admitted by the age of 14 y for a giant bilateral TARTs of 8.3 cm, respective 7.4 cm maximum diameter, that required surgical removal (after initial responsiveness to glucocorticoid replacement, TARTs enlarged because of non-compliance to therapy amid COVID-19 pandemic) [39].

2.7. Management

The mentioned studies included patients who were treated with glucocorticoid replacement therapy (hydrocortisone, dexamethasone, and prednisolone; hydrocortisone equivalent doses between 8 mg/m²/day and 100 mg/m²/day) in addition with mineralocorticoid replacement like fludrocortisone (doses between 0.05 mg/day and 0.25 mg/day) in SW forms [22,26,31,46,48,57]. One retrospective study on 16 individuals (mean age of 7.2 ± 6.4 y; female/male ratio of 10/6) from Lancaster, Pennsylvania showed that, despite high-dose of hydrocortisone, the abnormal hormonal assays are persistent, and that seems to be a distinctive feature of 3βHSD2 deficiency, a correction profile of hormones values more resistant to therapy compared to 21OH deficiency (persistent high ACTH was 10 times above normal, 17OH-Preg was 20 times as normal, and DHEA was 20 times as normal; in this study, the standard dose of hydrocortisone versus high-dose was 15.4 ± 4.9 mg/m²/day versus 37.8 ± 15.4 mg/m²/day, p < 0.0001) [22]. Additionally, some male patients received testosterone replacement therapy [26,31,45]. Urogenital anomalies were surgically corrected with hypospadias repair and orchidopexy [26,31,46,48,57]. Also, clitoral reduction was performed [47]. Tumours were surgically removed or followed up for a while depending on disease control methods [39,45,48,56].