A compromised function of POR will disrupt the cytochrome P450 system, leading to failures in steroid synthesis, skeletal development and ambiguous genitalia. PORD is rare but usually exhibits a variety of clinical manifestations, such as oligomenorrhea and ovarian cysts that resemble polycystic ovary syndrome (PCOS), elevated 17OHP that resembles 21-OHD, and skeletal deformities that resemble ABS. Notably, PORD does not reveal hyperandrogenism, which is a departure from PCOS and 21-OHD. These seven 46 XX patients in this cohort exhibited consistent clinical hormone profiles, mimicking combinations of 21OHD and 17OHD. In addition, their masculinized genitalia, malformation of the skeleton and classical genetic mutations also correspond to the signature PORD.
Genotype-Phenotype Analysis
Although we describe cases particularly in 46 XX females, PORD involves both chromosomal sexes at a roughly equal rate, and the majority are of European or Japanese descent[7]. In line with the autosomal recessive pattern of inheritance, biallelic mutations in the POR gene were detected in all seven cases. The POR gene contains 16 exons. Exons 1 to 15 encode the POR enzyme containing 680 amino acids, while an extra exon does not encode any protein[8]. Spread is the flavin mononucleotide domain of POR directly transferring electrons for P450 enzymes, while most mutations are clustered in the NADPH-binding domain serving as the electron donor. Nevertheless, data from three large PORD cohorts have reached only a poor genotype–phenotype correlation[9–11]. Among all seven patients, exon 11 of POR harbours at least one mutation (c.1370G > A), which leads to a conversion of arginine at amino acid position 457 to histidine. This POR mutation, p.Arg457His, is most widely reported in Japanese people [4, 7], and it is also the most prevalent in our cohort, present in 13 out of 14 alleles. However, in patients of Caucasian origin, the p.Ala287Pro is the most frequently reported[10]. Previously, an in vitro study found that p.Ala287Pro does not impair aromatase activity but that p.Arg457His abolishes it; both mutations lead to 46 XX virilization if present in the homozygous state[12]. Therefore, the p.Arg457His is reported to be associated with virilization in 46,XX patients but likely results in normal male genital development[6]. In addition, because cholesterol production in skeletal tissues occurs in a simple one-way manner without alternative reactions, the skeletal phenotype is supposed to depend obviously on the p.Arg457His dosage, reflecting the residual activity of POR[6]. As a result, the threshold for the development of severe skeletal phenotypes resides between a single copy and two copies of the p.Arg457His residual activity. In Case 7, we reported a newly discovered null mutation c.1684dupG (p. Glu562Gly fs*13). Since the frameshift variance contributes to a premature termination of peptide chain, leaving almost 100 amino acids unattached, the mutated enzyme is presumably to be loss-of-function. No PORD-affected live-born individual has been found to carry this kind of null mutation on both alleles thus far. This suggests that such a genotype is incompatible and lethal to postnatal life, which is in accordance with the observation of early intrauterine foetal death in the Por deletion murine model[13].
Endocrine characteristics
Most newly discovered PORD cases are infants or even foetuses, while adolescents and adults account for only 24%[7]. The most prevalent clinical manifestations were an aberrant hormonal profile at birth or developmental delay since puberty (89.2%), ABS-like skeletal deformity (82.7%), disorders of sex development or hermaphroditism (75%), nonclassical CAH-related symptoms (74.6%), ovarian multilocular morphology in females (46.7%) and maternal virilization during pregnancy (40.8%)[7]. These figures refer to PORD as a typical endocrinological issue. The low serum E2 levels (21.7–40.7 pg/mL) of our seven cases are attributed to decreased ovarian androgen production (except Case 2) and its subsequent aromatization[14]. The hypogonadism is partially responsible for the negative feedback of high FSH. This could be mixed with ovarian insufficiency, particularly when patients have menstrual problems and low AMH (Cases 5, 6 & 7). Low-to-normal AMH can exist in these patients, and this scenario is commonly seen in other CAH subtypes, such as 17 OHD[15]. Since patients with severe POR mutations suffer from severe P450 enzymatic defects, their follicular reservoir can undergo premature failure due to impaired granular cell steroid metabolism; mild forms of mutation that leave partial secretive function of follicles contribute to the residual ovarian follicles’ response to the persistent stimulation of raised FSH. Interestingly, regardless of the level of AMH or FSH, all patients are found to have ovarian multilocular cysts, and these cysts are frequently seen in compromised function of POR[16]. The underlying cause is that the relatively young age of these patients is distinguished from the postmenopausal exhaustion of the ovarian reserve. Even a limited number of remaining follicles will respond to FSH recruitment and grow into a multilocular ovarian morphology. In addition to the accumulation of P as a steroid precursor, excessive LH will mediate luteinization of these cysts and further promote P production in these PORD-CAH patients[17]. All affected individuals from our cohort exhibited a simultaneous increase in serum 17OHP and P, which misleadingly suggested nonclassical (NC) 21OHD. Moreover, impaired catalytic activity of 17,20-lyase is responsible for decreased circulating androgen levels, which mimics the isolated 17,20-lyase (ILD) form of 17OHD, but differs from 21OHD and PCOS[16]. Surprisingly, 75% of PORD patients with 46 XX are commonly accompanied by ambiguous genitalia[7]. Four of our cohorts (4/7, 57.1%, Cases 2, 3, 5 & 6) underwent virilization of external genitalia. This is an apparent dichotomy, given that DHEA and androstenedione production in the foetus should be disturbed due to impaired 17,20-lyase activity. Even the modest amount of androgens produced could be accumulated due to the deficiency of placental aromatase affected by PORD[18]. In addition, elevated 17OHP, which cannot be efficiently metabolized via P450c21 or P450c17 activities, seeks an alternative pathway via 5α-reduction and is ultimately converted to DHT[19].
Skeletal deformities are associated with lanosterol 14α demethylase (CYP51A1), which is involved in cholesterol biosynthesis[20] and retinoic acid metabolism by microsomal CYP26 enzymes[21]. Compound heterozygous and homozygous mutations in the POR gene are associated with more serious skeletal deformities, resulting from the effect of various mutations on the activity of related enzymes[10]. Cases 1 to 6 harbour the same homozygous mutation but have various skeletal deformities, illustrating the heterogeneity of this symptom in PORD.
Ovarian cysts and fertility outcomes
Ovarian cysts can trigger significant morbidity ranging from mild abdominal tenderness to acute ovarian cyst rupture or torsion. All patients from our cohort had fluid-filling luteinized cysts of at least one ovary. Cases 2, 6 & 7 have even previously undergone ovarian cystectomy, and their pathologies confirmed the diagnosis of luteinized cysts. All patients are prescribed COC after the operation, and this provides effective conservative management for this type of recurrent cyst. Large cysts shrink soon after COC administration in most patients by suppressing gonadotropin stimulation from the hypophysis; however, ovarian cysts may reappear after the cessation of COC treatment, and it is still effective to use it again to repress the cyst[22]. Their ovarian tumour markers were negative at the beginning. However, if ovarian cysts persist after COC intervention, the possibility of nonfunctional tumours should be excluded. In addition, recurrent functional ovarian cysts in 46XX PORD must be differentiated from other endocrine-related diseases, such as 46 XX partial 17OHD, Van Wyk–Grumbach (VWG) syndrome, and functional gonadotroph adenomas (FGAs) [23].
Fertility preservation is a challenging issue for 46 XX patients with PORD. Natural conception has not been reported in these females, and almost all patients of reproductive age encounter primary infertility[16]. PORD will implicate CYP51A1, an enzyme responsible for the conversion of lanosterol to meiosis-activating sterols, which in turn cause defective oocyte maturation[24]. For patients with severe mutations of POR leading to the complete loss of enzymatic function, the premature depleted ovarian reservoir offers them a rare chance to conceive[16]. For patients still equipped with remaining primordial follicles and diagnosed with a partial loss of POR activity, endocrinological elements lead to a barrier to natural fertility. The main contributing factor is excessive P levels without cyclic change, which in turn exert an anti-gonadotropic effect on the hypothalamus and pituitary gland. Without the physical periodic fluctuation of gonadotropin, even in those cases with oligomenorrhea, ovulation seldom occurs spontaneously. This is presumably due to the blockage of LH positive feedback on rising E2 by high P levels. Persistently high circulating P levels reverse the endometrial receptivity synchronized with embryo development[25] and alter the cervical mucus. Moreover, anovulation could also be blamed for defective follicular maturation due to steroidogenesis failure, particularly androgen deficiency[26]. Thanks to the advancement of assisted reproductive technology (ART), pregnancy can be achieved by controlled ovarian stimulation (COS) and in vitro fertilization[22] while suppressing P with potent corticosteroids and creating an artificial menstrual cycle with hormone replacement for frozen-thawed embryo transfer[27]. Bosch et al revealed that serum P levels < 1.5 ng/ml over COS are associated with higher live pregnancy rates[28]. Consequently, pregnancy can be achieved in females through ART if PORD is correctly diagnosed as early as possible and if the ovaries are well protected by avoiding unnecessary operation due to the spontaneous disappearance of cysts following the commencement of COC[22]. This is essential for dealing with multilocular cysts and protecting the diminished ovarian reserve in 46 XX PORD patients, maximizing their chance for future ART.
Limitations
Several limitations exist in our study. Primarily, since PORD is a rare type of CAH, the sample size is very limited, but we are working on the collection and updating of new cases. Second, some laboratory studies can be modified without consideration of cost and accessibility in our hospital. The pathogenicity of the newly identified mutation (c.1684dupG) was not evaluated by an in vitro functional study. The skeletal evaluation was based on physical examination and plain radiographs in some cases; peripheral quantitative computed tomography for the study of bone lesions might be a better option. In addition, the assessment of adrenal function by serum steroid variation with ACTH stimulation is more precise and solid[29]. Given that PORD affects multiple systems, its management is challenging and should encompass transdisciplinary cooperation, including paediatric endocrinologists and orthopaedists. Herein, we provide management experience dominantly from the perspectives of gynaecological endocrinology and reproduction and hope to promote patients’ sexual-developmental and fertility outcomes.