Introduction

Congenital adrenal hyperplasia (CAH) describes a group of autosomal recessive disorders of cortisol biosynthesis with varying levels of severity [1]. Indeed, the clinical phenotype is typically classified as a classic form (the most severe one), and non-classic that is mild or late onset. Disorders of sex development (DSD) depend on the affected gene that cause CAH but also on sex proband. The presence of salt-wasting, postnatal virilization, sex steroid deficiency, hypertension, and other features, such as skeletal defects, are caused by the specific gene that is mutated [1]. Among the long-term complications of CAH, infertility is frequent in both female and male patients [2,3,4,5,6,7,8]. Male patients may experience the growth of testicular adrenal rest tumors (TARTs), which can cause intra-testicular compression of efferent seminiferous tubules in turn affecting spermatogenesis [9].

CAHs have a specific hormonal pattern based on the enzymatic dysfunction that causes it. The CYP21A2 genotype is the main, but not the only, determinant of the phenotype in patients with 21α-hydroxylase deficiency (21α-OHD) [10]. In some patients suspected of having a 21α-OHD, no pathogenic variants were found in one or both alleles even after complete sequencing of the CYP21A2 gene [11]. Other patients have pathogenetic variants of the CYP21A2 gene, but the genotype does not fully match the phenotype. These apparent discrepancies between genotype and phenotype suggest the presence of other genetic factors including modifier genes that can modulate the clinical expression of some aspects of 21α-OHD [10].

In humans, P450 oxidoreductase (POR) deficiency (PORD) causes an unusual and rare form of CAH, whose exact incidence is not known [12]. POR is an 82-kDa membrane-bound protein containing 680 residues encoded by a 32-kb gene containing 15 exons mapping on chromosome 7q11.2 [13]. It is necessary for the metabolic activity of P450 cytochrome enzymes including CYP17A1, CYP21A2, CYP19A1, and CYP51A1 [14]. Consequently, PORD can affect the function of these enzymes with different phenotype based on the residual enzymatic activity. As an example, studies on the CYP17A1, the steroidogenic enzyme that catalyzes both 17α-hydroxylase and 17,20 lyase activities [15], show that the levels of some P450 activities are determined, at least in part, by the stereochemistry of the interaction of POR with the cytochrome P450 [13]. In the case of CYP17A1, the 17,20 lyase reaction, but not the 17α-hydroxylase reaction, is very sensitive to this stereochemistry, as shown by three lines of evidence [13]. First, variants of basic residues in the redox-partner binding site of CYP17A1 selectively reduce the 17,20 lyase activity [16]. Second, cytochrome b5 acts as an allosteric factor to promote the interaction of P450c17 with POR, selectively increasing 17,20 lyase activity [17,18,19]. Third, phosphorylation of CYP17A1 selectively increases the 17,20 lyase activity of CYP17A1 [19,20,21].

PORD phenotype is characterized by DSD in both sexes and is often associated with skeletal defects [13, 22]. The latter, known as Antley–Bixler syndrome (ABS), is characterized by craniosynostosis, brachycephaly, radio-ulnar or radio-humeral synostosis, bowed femora, arachnodactyly, midface hypoplasia, proptosis, and choanal stenosis. ABS is transmitted with an autosomal recessive mechanism by POR pathogenic variants and with an autosomal dominant mechanism by gain-of-function variants of the fibroblast growth factor receptor 2 (FGFR2) gene [23]. No definitive data are available on TARTs and fertility in male patients with recessive PORD.

Therefore, this study aimed to systematically review the literature to gather all the available information on gender and genotype-related prevalence of CAH, DSD, TARTs, and fertility in patients with heterozygous or homozygous POR gene variants. In the resulting database, we added also the case of a male patient with CAH and TARTs but without DSD. He resulted in being a carrier of a combined heterozygous POR pathogenic variant and achieved fatherhood spontaneously.

Systematic review of the literature

Methods

Sources

Data for the systematic review were independently extracted by C.G. and R.C. A systematic search was performed through PubMed, MEDLINE, Cochrane, Academic One Files, Google Scholar, and Scopus databases, from the beginning of each database through May 22, 2021. The search strategy was based on the combination of the following Medical Subjects Headings (MeSH) terms and keywords, using “AND” between each MeSH search term: “P450 oxidoreductase” AND “congenital adrenal hyperplasia”, “P450 oxidoreductase” AND “DSD”, “P450 oxidoreductase” AND “homozygosity”, “P450 oxidoreductase” AND “heterozygosity”, “P450 oxidoreductase” AND “pregnancy”. Additional manual searches were made using the reference lists of relevant studies. Only articles available in English full-text have been included.

Study selection

All studies that reported the clinical phenotype of patients with POR gene variants were included. In particular, we focused on the presence of CAH, DSD (defined as virilization in female patients, under-masculinization in male patients, and abnormalities of the reproductive system in both sexes), TARTs, and fertility. Review articles and studies on experimental animals were excluded.

Description of the proband

We added to our database the case of a patient not previously described in the literature who resulted in a carrier of compound heterozygous c.1891G > A, p. (Val631Ile) and c.516G > A variant. He had classical salt-wasting CAH, TARTs (Fig. 1), infertility, abnormal sperm parameters (oligoasthenoteratozoospermia, OAT), and extremely elevated ACTH serum levels. He achieved spontaneous paternity after adding dexamethasone 0.25 mg/day to his daily cortisol replacement therapy. This therapeutic arrangement led to the normalization of ACTH serum levels.

Fig. 1
figure 1

Ultrasound pictures of the testicular adrenal rest tumors. Upper panels: longitudinal (A) and transversal (B) scans of the right testis. Lower panels: longitudinal (C) and transversal (D) scans of the left testis

Full size image

Results

Using the above-mentioned search terms, we found 246 articles. After the exclusion of 34 duplicated records, 212 articles were screened. Thirty-one articles were excluded because the English full-text was not available. Of the remaining articles, 119 were excluded after having read their title and abstract, since they did not satisfy the inclusion criteria. In particular, 10 studies were excluded because performed in vitro and/or on animals, and 24 were excluded because they were reviews. The remaining 62 full-texts were carefully read. Finally, 48 articles matched the inclusion criteria [14, 22, 24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69]. These studies included 119 patients (46 males and 73 females) with PORD (Fig. 2). They had homozygous, combined, or heterozygous POR gene variants in 58.0% (69/119), 32.8% (39/119), and 9.2% (11/119), respectively. These patients are worldwide distributed. The main features of the included studies are summarized in Table 1.

Fig. 2
figure 2

Flowchart of the included studies

Full size image
Table 1 Summary of the studies included in this systematic review
Full size table

Congenital adrenal hyperplasia

The results showed that all the 119 patients with PORD had CAH. All of them showed increased serum 17α-hydroxyprogesterone levels at baseline or after the ACTH-stimulation test. Hence, no difference in the prevalence of CAH among homozygous, combined, or heterozygous POR gene pathogenetic variant was found (Table 1).

Disorder of sex development

DSD was described in 85 out of the 119 patients (71.4%) but there was a different genotype (homozygous or heterozygous) and gender-related distribution. DSD was found in 65.2% (45/69) of patients with compound heterozygous variants, 82.1% (32/39) of patients with homozygous variants, and 72.7% (8/11) of those with monoallelic heterozygous variants. Moreover, DSD occurred more frequently in females affecting 79.5% (58/73) of them and 58.7% (27/46) male patients (p < 0.05).

Testicular adrenal rest tumors

Out of 119 patients retrieved in the literature, ultrasound evaluation was performed only in 15 male probands without evidence of TARTs. Considering the case reported here, the prevalence of TARTs in PORD is, therefore, 6.6% (1/15), taking into account only patients who have undergone testicular ultrasound screening.

Fertility

Considering the higher prevalence of pediatric probands found in the literature, we found an important limitation in the assessment of fertility in patients with PORD. Of these, only 27 probands were over the age of 18 and, therefore, eligible for fertility research. No studies had focused on fertility in patients with PORD until 2017. To date, only five studies of female patients with PORD who have successfully delivered after ART-induced pregnancy have been published [57, 62, 63, 69].

Discussion

By reviewing data from 48 articles including 119 patients from around the world, the present study aims to show the gender- and genotype-related prevalence of CAH, DSD, TARTs, and infertility in patients with heterozygous or homozygous POR gene variants. Genotype–phenotype correlation is sometimes a complex and attempted association and is still a matter of research, considering the most recent studies that have focused on mutations of the CYP21A2 gene [70, 71]. This review allows a better understanding of the PORD phenotype. Our results show that CAH can be caused by both homozygous and heterozygous POR gene variants. Among 119 patients DSD was found in 85 (71.4%), respectively, in 65.2% (45/69) of patients with compound heterozygous variants, 82.1% (32/39) of patients with homozygous variants (82.1%), and 72.7% (8/11) of those with monoallelic heterozygous variants. Furthermore, DSD had a higher frequency in females affecting 79.5% (58/73) and 58.7% (27/46) male patients (p < 0.05), indicating that DSD affects both homozygous and heterozygous carrier patients, although with a higher prevalence in the former.

Specifically analyzing the individual variants, the most frequent are c.859G > C (p.A287P), typically with a higher prevalence of DSD when the heterozygous mutation is found in association with another monoallelic variant (DSD present in 81% of probands with compound heterozygous variants); c.1370G > A(p.R457H), that shows a higher prevalence of DSD when the homozygous variants are found (79%); c.1697G > A(p.G539R), with no difference between homozygous or heterozygous pathogenic variants (DSD present in 100% of probands) (Table 2). All the other detected variants, including the one found in our proband, were found in 1–2 patients (for each variant), so statistical analysis could not be applied (Table 3). According to a recent publication on POR polymorphisms, variants of this gene can influence CAH phenotypical expression acting as a genetic modifier of CYP21A2 defects [72]. Although the literature has so far focused on different allelic mutations on a single gene, we should now consider the coexistence of POR variants and polymorphisms in CAH patients carrying CYP21A2, CYP11B1, CYP17A1, HSD3B2, StAR, or CYP11A1 variants.

Table 2 Prevalence of disorders of sex development (DSD) in homozygous and heterozygous pathogenetic variants
Full size table
Table 3 Single pathogenetic variant associations of P450 oxidoreductase-associated congenital adrenal hyperplasia
Full size table

This is the first article reporting the presence of TARTs in a patient with CAH due to PORD. So far, TARTs have been described in patients with 21α-OHD and 11ß-hydroxylase deficiency [73, 74]. It is thought that poor hormonal control, leading to high blood levels of ACTH, is an important factor in the pathogenesis of TARTs by inducing hypertrophy and hyperplasia of these adrenal-like cells within the testis [75, 76]. Accordingly, the case herein reported showed that TARTs tend to grow when ACTH levels are elevated. However, TARTs are also found in properly treated patients, whereas some poorly controlled CAH patients never develop TARTs despite they are chronically exposed to elevated ACTH levels [77, 78]. The most plausible explanation for this observation is that in the embryological period aberrant adrenal cells do not nestle in the testes in all males, so the presence of these aberrant adrenal cells within the testis is a prerequisite for the development of TARTs [73]. This ectopic migration does not seem to be related to a specific genotype as so far described. However, the presence of TARTs in the patients described in this article allows us to speculate that also POR gene variants, can cause TART development. Articles on PORD patients do not report the presence of TARTs in any of the patients described.

The present study shows, for the first time, spontaneous fatherhood in a patient with CAH and TARTs due to variants of the POR gene, although paternity must to be genetically proven once the child is born. Female patients, on the other side, may have infertility due to increased androgen secretion or impaired sex steroid production [79], but few cases of successful births by ART-induced pregnancy have been reported [57, 62, 63, 69]. This observation suggests that women with PORD must undergo ART programs to achieve pregnancy.

In conclusion, the unknown incidence of POR gene variants and the poorness of fertility-investigating reports enlighten that it is still unclear whether PORD is associated with human infertility since most of the cases reported so far did not focus on patients’ fertility. Further studies exploring the relationship between POR genotype and fertility are needed. Evidence from the male proband herein reported suggests that spontaneous fatherhood can occur in male patients with PORD but proper management of CAH is needed to reach spontaneous fertility without the need to undergo ART.