Prenatal diagnosis and in utero treatment of congenital adrenal hyperplasia: An up‐to‐date comprehensive review

Congenital adrenal hyperplasia (CAH) is a term that encompasses a wide range of conditions that affect the adrenals. Diagnosis and treatment before birth are important as irreparable birth defects can be avoided, decreasing the need for surgical intervention later in life, especially regarding genitalia anomalies. Although early implementation of dexamethasone in the prenatal treatment of CAH has been controversial, there is recent evidence that this treatment can reduce long‐term complications.

fourth gestational week when celomic epithelial cells and mesenchymal cells from the mesoderm migrate to form primary steroidsecreting tissues, and neural crest cells migrate to form the adrenal medulla. 7The process is completed by the ninth gestational week when the adrenal glands are seen as encapsulated organs separated from the renal system. 7 previously described, the adrenal glands have two distinct zones with independent functions: the adrenal cortex and the medulla.Once the organogenesis has ended, the fetal adrenal will be formed by two zones, the fetal and the definitive zones.During gestation, the fetal zone constitutes nearly 90% of the cortical structure and secretes androgen hormone, while the definitive zone primarily releases cortisol. 7An intermediate zone exists between the fetal and definitive zones, which secretes glucocorticoids, particularly towards the end of gestation. 7The definitive cortical zone will overtake the primitive fetal zone due to cellular hyperplasia and apoptosis of the fetal zone. 8e neural crest cells that eventually form the medulla remain scattered islands until birth, when they differentiate into chromaffin cells, producing catecholamines, epinephrine, and norepinephrine. 9e adrenal glands, located on the superior poles of the kidneys, are surrounded by vital organs and vessels around the level of the 12th rib. 1 Due to their extensive steroidogenesis activity, the blood supply to the glands is tightly controlled via paracrine and neuroendocrine mechanisms. 1 The three adrenal arteries supply the adrenal cortex and arise from different origins.The superior adrenal arteries branch off the inferior phrenic artery; the middle adrenal artery arises from the abdominal aorta, while the inferior adrenal artery originates from the renal arteries.1,9 The adrenal function is unique as it requires hormonal and nervous input for normal functioning.Nervous innervation of the gland is regulated either by sympathetic or parasympathetic ganglia, while hormonal stimulation arises from the release of adrenocorticotropic hormone (ACTH) from the anterior pituitary gland. 1

| The function of the adrenal cortical zones
The adrenal cortex consists of three major zones, from the outer to innermost, known as the zona glomerulosa (ZG), zona fasciculata (ZF), and zona reticularis (ZR). 10The ZG secretes hormones known as mineralocorticoids, the main one being aldosterone, which regulates the amount of fluid, sodium, and potassium in the body.This secretion is upregulated by serum levels of potassium and controlled by angiotensin II, which is produced in the lungs. 9,10This function enables aldosterone to maintain homeostasis and regulate blood pressure. 11e ZF emits glucocorticoids via the stimulation of ACTH secreted by the anterior pituitary gland. 9These corticosteroids regulate the metabolism of fats and carbohydrates and ensure proper immune and stress responses. 12Cortisol and other glucocorticoids are essential in regulating the hypothalamic-pituitary-adrenal (HPA) axis, as their levels will inhibit ACTH release from the anterior pituitary gland. 9nversely, the ZR produces androgen precursors such as dehydroepiandrosterone (DHEA) and androstenedione.These precursors can then create more potent androgen steroid hormones. 9

| Adrenal steroidogenesis
Adrenal steroidogenesis is a complex process that relies on cholesterol to form mineralocorticoids, glucocorticoids, and adrenal androgens under the stimulation of regulators such as the hormone ACTH. 12e cholesterol utilized for this process is derived from the outer mitochondrial membrane; the steroidogenic acute regulatory protein enzyme, known as StAR, can then transfer cholesterol to the inner mitochondrial membrane where steroidogenesis occurs. 13is inner membrane is where the rate-limiting step of the adrenal hormone synthesis begins; this is done by the side-cleaving enzyme CYP11A1, which will convert cholesterol to pregnenolone. 14neralocorticoids are synthesized in the ZG, which begins with the enzyme 3β-hydroxysteroid dehydrogenase that converts the pregnenolone to progesterone.Progesterone will be acted on by 21 hydroxylase to convert it to 11-deoxycorticosterone (DOC).Aldosterone synthase will convert DOC to aldosterone via three mechanisms: 11 β-hydroxylation, 18-hydroxylation, and finally 18-methyl oxidation. 13is zone is optimized for producing mineralocorticoids, as this is the only region with Aldosterone synthase and very little of the 17α hydroxylase, which drives the steroid synthase towards the production of cortisol and adrenal androgens. 13e ZF synthesizes cortisol under the direct regulation of the ACTH.As stated above, the ZF expresses a high level of 17α hydroxylase, which will convert pregnenolone to 17-OH pregnenolone; thus, it acts as a gatekeeper for cortisol synthesis. 13,14Like its neighboring zone, the ZF also expresses a high level of 3β-hydroxysteroid dehydrogenase and 21 hydroxylase, whose dual action eventually converts the 17-OH pregnenolone to 11 Deoxycortisol.The final enzyme in this pathway, 11 β -hydroxylase, will then act on this substrate to produce cortisol. 14e most abundant steroids produced in the ZR are DHEA and DHEA sulfate (DHEAS).This production is mediated by the 17α hydroxylase, which turns pregnenolone to 17-OH pregnenolone and eventually DHEA, sulfonation will then occur following the action of sulfotransferase. 13thogenic variants lead to monogenic disorders in the adrenal hormone synthesis, leading to the constellation of symptoms that make up CAH, which we will discuss below (Figure 1).corticotropin-releasing hormone and adrenocorticotrophic hormone ACTH that leads to accumulation of adrenal hormones precursor. 15is cascade of events will end in a constellation of congenital features, ranging from variation of sexual development (VSD) to metabolic syndromes. 15e different phenotypic presentations of CAH depend on the enzymatic defects involved.It has been demonstrated that deficiencies in the enzyme 21-hydroxylase account for nearly 95% of CAH, and this can be classified into three subtypes: nonclassical, classic salt wasting, and simple virilizing.Classic type affects approximately 1 in 16,000 live births, while nonclassical can affect 1 in 1000. 15though 95% of CAH cases are caused by 21-hydroxylase (OH) deficiency, genotype-to-phenotype characterization is dependent on the type of gene abnormality.21-hydroxylase (OH), a part of the cytochrome P450 family of enzymes, is coded by the gene CYP21A2.CYP21A2 is located 30 kb away from CYP21P, a homologous pseudogene, on chromosome 6; thus, 21-OH deficiency is caused by recombination events between the two genes. 16Most causes of the CYP21A2 pathogenic variants are inactivation of the gene caused by deletions, single-point mutations, and extensive gene conversions. 17e second most common enzyme deficiency leading to CAH is 11 ß-hydroxylase (11 ß-OH) deficiency, which makes up 5%-8% of CAH cases and affects 1 in 100,000 live births. 15,17Normally, 11 ß-OH is deputed to the production of cortisol in the ZG and ZF of the adrenal glands.11 ß-OH is a protein encoded by the CYP11B2 gene located on chromosome 8q21. 17CAH caused by 11 ß-OH deficiency may be secondary to nonsense, missense mutations, or too small deletions and splice-site mutations detected mainly in the Jewish population. 17Caucasian infants more frequently, the overall incidence ranges from 1:10,000 to 1:20,000. 18Classical CAH is associated with a significant loss of the enzyme 21-hydroxylase (21-OHD), an intracellular enzyme located in the endoplasmic reticulum that catalyzes the conversion of 17-hydroxy progesterone (17-OH-P) to 11deoxycortisol, a precursor of cortisol, and progesterone to deoxycorticosterone, a precursor of aldosterone, causing a shunting towards other pathways in cortical hormonal biosynthesis.This shunting allows CCAH to be further subdivided into salt-wasting or simple virilizing in female fetuses. 19e salt-wasting variant is considered a medical emergency as infants affected cannot synthesize essential mineralocorticoids, leading to inevitable hypotension, hyponatremia, and hyperkalemia. 19ath will occur within the first few weeks of life if left untreated or improperly diagnosed.The neonatal mortality rate of salt-wasting CCAH is notably higher in male infants due to their normalappearing genitalia. 19While postnatal screening performed via the newborn heel prick tests can measure the level of 17-OH-P, the accuracy is quite limited as enzyme measurement can be varied following delivery leading to false-positive results within the first few days of life. 20mple virilizing CAH is considered the other spectrum of classical CAH, as it lacks the features of electrolyte and cardiovascular instability associated with the salt-wasting variant.

| WHAT IS CAH?
Following birth, female infants with CCAH will have phenotypically male-appearing genitalia due to shifts in the adrenal hormone production that occur in either presentation; these physical findings can range from isolated clitoromegaly to the fusion of the labia majora. 19Males, however, will have normal-appearing genitalia with disease features occurring later in the form of precocious puberty. 21nclassical CAH represents a less severe form of the disorder as it lacks features more commonly associated with classical CAH, such as variable sexual development VSD and cardiovascular instability. 22This is due to a mild loss in the 21-OHD function, allowing for less funneling to the formation of other adrenal hormones.As such, this is diagnosed much later in life, either during puberty or beyond, due to hyperandrogenic symptoms such as precocious puberty, cystic acne, and tall stature. 21Reproductive capabilities, particularly in females, can also be hindered due to ovarian dysfunction associated with high androgen levels. 23

| PRENATAL DIAGNOSIS AND MANAGEMENT
Diagnosing CAH prenatally is possible, allowing for a multidisciplinary approach to fetal and neonatal management. 24This section will delve into the currently available diagnostic and treatment methods for CAH, their importance, and their necessity.

| Diagnosis
The first reported incidence of prenatal diagnosis of CAH was reported in the 1970s by Pang et al. 25 via the measurement of amniotic fluid hormone levels; however, this technique was eventually deemed inaccurate due to falsely low levels of 17-OH-P. 26With the technological advancements in the past few decades, more precise approaches to prenatal diagnosis have been developed; these methodologies can be deemed invasive or non-invasive.
Invasive techniques are achieved by directly obtaining fetal cells and tissues through amniocentesis and chorionic villous sampling (CVS). 27Fetal samples can be amplified using polymerase chain reaction (PCR), allowing for the detection of pathogenic variants and deletions leading to adrenal enzyme dysfunction and loss. 27Although CVS and amniocentesis are diagnostic of this condition, they are associated with the risk of infections, bleeding, and even miscarriage. 28w et al. 29  administered before this timeframe to prevent this complication. 29In a majority of at-risk pregnancies, DEX is given to a fetus unaffected by CAH until the diagnosis can be confirmed. 30Thus, an ethical dilemma arises as we want to avoid unwarranted DEX exposure to unaffected fetuses.
Non-invasive screening methods for CAH can be done using fetal cell-free DNA (cff-DNA) that can be traced in the maternal circulation and potentially supersede invasive protocols, as a diagnosis can be made before the completion of organogenesis. 31,32While this method is currently primarily used for screening trisomy and sex chromosome aneuploidies, there is potential for its use in the diagnosis of CAH. 33is approach was made possible in 1997 when Lo et al. 34 discovered that fetal cff-DNA was present in maternal plasma and could be readily detected.In their study, plasma was collected from 43 pregnant women attending John Radcliffe Hospital in the United Kingdom, and this was compared with a control population of nonpregnant women.The samples then underwent PCR testing to determine regions coding for fetal-derived Y sequences circulation in the maternal circulation; this allowed for the determination of fetal sex as male in 70% of samples collected from pregnant women, while the samples for nonpregnant women were negative. 34The use of cell-free fetal DNA may eventually replace invasive diagnostic procedures and direct interaction with developing fetal structures to be bypassed. 35Cff-DNA extraction and analysis can help diagnose various inherited genetic disorders, including X-linked genetic disorders, CAH, and fetal gender.This technique is advantageous in male fetuses, as sex can be detected as early as the fifth gestational week by using PCR to target Y Chromosome sequences. 36,37Once determination of the fetal gender and analysis of the CYP21A2 gene has been made, prenatal intervention should be considered to prevent virilization in affected female fetuses; however, if fetal sex is determined to be 46 XY, prenatal treatment is not indicated as complications of virilization would not apply. 36,38study by Khattab et al., 30 in which plasma was taken from 14 expectant mothers carrying fetuses at risk of CAH, showed that the fetus's allelic composition could determine abnormalities in the CYP21A2 gene.Their results showed that the outcomes from noninvasive procedures were comparable to invasive CAH diagnostic procedures 100% of the time.
Similar results were seen in a study by Ma et al., 39 where plasma samples from pregnant women whose gestations ranged from the first to the second trimester were collected.These authors performed next-generation sequencing on maternal and paternal DNA to identify if the fetus inherited wild-type or pathological CYP21A2 alleles.Their study showed that CAH could be identified in all 14 participants, with early diagnosis possible at 8 weeks of gestation, proving that testing maternal plasma can be an alternative to invasive procedures.
New et al. 32  determined using a PCR assay with an SRY probe.In 14 families affected by CAH due to CYP21A2 pathogenic variants, seven affected fetuses, five carriers, and two unaffected fetuses were identified as early as 5 weeks and 6 days of gestation.
As previously discussed, noninvasive methods using cff-DNA can be helpful for screening; however, more work must be done before this can replace the gold standard of invasive diagnosis that CVS and amniocentesis allow.
Simpson et al. 31 demonstrated the feasibility of using preimplantation genetic diagnosis for CAH in embryos, as these techniques can be used to locate single gene mutations when the chromosomal location is known.
This technique was further implemented by Fiorentino et al., 40 who attempted to diagnose single gene disorders prenatally in 250 cycles using multiplex PCR.They could detect the presence of CAH in embryos, although pregnancies did not occur following implantation. 40is prophylactic evaluation of genetic defects is particularly an issue of paramount importance for women undergoing assisted reproductive technology. 31,41Another method known as the preimplantation genetic-testing monogenic (PGT-M) requires a biopsy from an embryo in the blastocyst stage of development.While this approach has the advantage of an extremely early diagnosis, it could be at the cost of risk of injuring the developing fetus. 31Reihani-Sabet et al. 42 discussed a case where an Iranian couple decided to undergo PGT-M following the miscarriage of a child with CAH and having a female child with the same condition.Out of the six embryos harvested and tested one embryo was found to be mutant homozygous, two were healthy, two were heterozygous carriers, and the last embryo's status could not be determined as cells could not be tested. 42

| Management
The effectiveness of in utero intervention can be determined by the precise prenatal diagnosis of the CAH and the fetal gender determination via cff-DNA and the timing of management.
Since 1984, the mainstay prenatal treatment for CAH has been DEX; this corticosteroid is utilized as it does not readily bound to maternal cortisol-binding hormones and is not inactivated by placental 11ß-hydroxysteroid dehydrogenase, allowing it to reach the fetus and suppress excessive ACTH activity. 4One of the first studies highlighting the use of DEX therapy in CAH was described by David et al. 43 in the 1980s.Their research showed that DEX administration prevented virilization in one female infant and partially in another. 43X is typically given before the 8-ninth week's gestation as the adrenal glands have not started to secrete androgen.It is important to highlight that DEX cannot prevent a child from being born with CAH, but it may prevent virilization of the developing female genitalia. 4,44This timeframe is preferred as we would like to ensure that the fetal labioscrotal folds have yet to close and the female fetuses do not start to develop genitalia that physically fit a male phenotype; initiation following this timeframe may prove to be more ineffective compared to counterparts that did not receive DEX as promptly. 45X dosing is continued throughout the pregnancy until fetal sex is determined to be male or if the fetus is deemed not to have CAH 46 (Figure 2).The concern arises regarding the continued use of DEX in unaffected females and affected male fetuses, as animal studies suggest that long duration treatment with DEX can potentially lead to adverse effects on the developing fetal brain, highlighting the need to stop the DEX therapy as soon as it is deemed to be clinically unnecessary. 47The dose regimen given to mothers at risk of possible virilization is 20 μg/kg/day, based on the maternal pre-pregnancy weight administered in three divided doses to receive a total value of about 1.5 mg/day. 4,48In 2021, Stachanow et al. 49 suggested that rather than a dosage of 20 μg/kg/day, an appreciably lower dose of 7.5 μg/kg/day could be started at the sixth gestational week until the 16th gestational week could be as effective for preventing virilization.
Following the seminal work by David et al., 43 multiple studies have been produced to confirm the value of in utero treatment.A study by Rivkees et al. 50showed that DEX therapy could effectively suppress adrenal androgen production, preventing that affected female fetuses progress to virilization allowing for normal skeletal development and linear growth, which proves that when steroids are given in utero, it can effectively overcome the complications associated with untreated CAH.Nimkarn et al. 4  results gained from these sites showed that while each clinic adhered to the recommended prenatal dosage of DEX 20 μg/kg/day, variance in the daily dose distribution was a common finding. 51While the European medical standards do support the conventional steroid regimen, a consensus across the participating European countries has shown that the duration of treatment and postnatal follow-ups needs to be determined as significant side effects, including intellectual disability and endocrine abnormalities, are particularly common. 51e to this, prenatal treatment is performed only as a part of approved Institutional Review Board protocols, where the benefits and risks of fetal steroid exposure to be charted will allow for the finetuning of the treatment regimen and determining long-term complications. 52

| Pitfalls of DEX treatment
As with any treatment, treating the developing fetus with DEX is not without risk, as studies have demonstrated that there can be possible maladaptive effects on the fetus's metabolism and intellect. 53This prompts the following question: "Do the benefits of using steroid OKPAISE ET AL. therapy to prevent fetal virilization in utero outweigh the risks?"This question is critical to consider as while steroid intervention can defer virilization and prevent the need for genital feminization surgery, complications related to the therapy can cause long-standing physical and mental complications to both mother and baby, considered to be substantially worse off than genital masculinization. 48rvikoski et al. 54 highlighted that several animal studies have shown long-term side effects in animals exposed to in utero glucocorticoid therapy, raising concerns about the long-term ramifications in infants.In the murine model, fetuses treated with 20 μg/kg/day during the entire gestational period had lower birth weights and a higher chance of hypertension, type 2 diabetes mellitus, hyperlipidemia, and ischemic heart disease later in life. 55Monkeys were receiving this dosage of DEX were also prone to neurotoxicity, leading to an increased risk of behavioral and emotional issues. 55n't Westeinde et al. 45 described a wide range of fetal effects that DEX therapy can have, from aberrant molecular programming to epigenetics.Molecular signaling associated with neurogenesis is thought to be impacted by exogenous steroids, which can lead to potential behavioral and cognitive complications.Genetic modification can be a subsequent complication of steroid use, leading to disarrayed action in organs such as the HPA and the placenta; these changes allow for increased fetal exposure to cortisol. 45inwell et al. 56 demonstrated that fetuses receiving antenatal DEX had a higher incidence of cerebral palsy than controls (placebo).
Glucocorticoid receptors are located throughout the brain, particularly in areas that control executive functioning, memory, and emotions, such as the prefrontal cortex, amygdala, and hippocampus. 46Because of the high concentration of glucocorticoid receptors, these areas can be damaged when prenatal treatment is administered.
The association between behavioral issues and prenatal DEX exposure has been reported for nearly three decades.In 1995, Trautman et al. 57 were pioneers in linking the use of corticosteroids in pregnancy with developmental delays and postpartum abnormalities.While their findings were inconclusive, the studies that followed showed that DEX-treated children tended to be diagnosed with mental disorders 1.4 years younger compared to their non-steroidtreated counterparts. 58In addition to an increase in mental disorders, Swedish authors have reported alterations in social and cognitive functioning, particularly in females treated prenatally with DEX compared to cohorts that were not exposed. 48ternal consequences of antenatal treatment for CAH were weight gain, mood disturbances, and the development of acne; in severe cases, preeclampsia and even spontaneous miscarriage occurred. 59Van't Westeinde et al. 45 described features of hypertension, gastrointestinal issues, and gestational diabetes occurring particularly in women with full-term exposure to DEX.Some women experienced such severe symptoms that they opted not to use DEX in future pregnancies. 59,60This was highlighted in a review by Lajic et al. 46 showing that one-third of Swedish mothers reported being highly unlikely to use DEX prenatally to treat CAH due to the adverse effects they experienced during their use.

| FUTURE RESEARCH
Emerging therapies for more effective diagnosis of CAH have recently been proposed as the concerns of long duration therapy with DEX in pregnancy have been raised. 61These potential new therapies are designed to reduce glucocorticoid and mineralocorticoid therapy complications, including decreased bone density, infertility, and cardiometabolic mortality. 62cently, HPA suppression via the inhibition of CRF1 (corticotrophin-releasing factor 1) receptor antagonists have been theorized.CRF1 acts as a monoclonal antibody that can regulate the release of ACTH; therefore, CFR1 inhibitors allow for dosedependent reductions in ACTH levels.While potential has been seen in animal trials, implementation of human therapy has yet to occur. 62Surgical adrenalectomy reduces the risk of virilization in women and the need for high doses of glucocorticoids 62,63 and would effectively diminish the symptoms of CAH.However, loss of adrenal function, particularly in utero, can disrupt normal fetal development as glucocorticoids play an essential role in the development of organs such as lungs, kidneys, heart, and vasculature. 64iki et al. 65 have determined that gene therapy via stem cell implantation can allow for the differentiation and subsequent replenishment of defective adrenal enzymes.In another study, Naiki et al. 66 have also documented that transduced fibroblasts could produce 21-OHD in mice, and this technique could be applied to enhance human therapy, although the function of 21-OHD is not entirely compatible with the human enzyme system.

| CONCLUSION
The adrenal glands are essential organs in maintaining the homeostasis of the cardiovascular system.Related metabolic disorders of the adrenal glands can affect the developing fetal genitalia in utero.
As such, it is equally important to have an early diagnosis and therapeutic intervention to prevent long-standing abnormalities correlated with CAH, particularly in female fetuses with simple virilizing, as the virilization effects can be jarring to parents.Notwithstanding, early treatment can avert surgical gender interventions later in life.
DEX therapy remains the recommended treatment in female fetuses diagnosed with CAH; however, long-term complications in mothers and fetuses from this treatment should be carefully monitored and evaluated.While treatment options are available, in certain countries, parents do have the right to consider the termination of pregnancy, particularly if significant genetic anomalies have already occurred as a consequence of the disease progression. 67Further studies with new potential genetic therapies for CAH are still necessary.

3. 1 |
Etiology and geneticsCAH leads to adrenal cortical dysfunction, which impairs the feedback mechanisms of the HPA axis, causing an increase in the levels of 636 -OKPAISE ET AL.

3. 2 |
Clinical presentation of CAHFollowing delivery, the presentation of CAH can be classified as classical (CCAH) or nonclassical (NCAH).The classical form of CAH is predominately seen in the neonatal period, and although it affects

F
I G U R E 1 A flowchart that highlights adrenal steroidogenesis.[Colour figure can be viewed at wileyonlinelibrary.com]OKPAISE ET AL.
examined invasive diagnostic procedures in 532 pregnancies between 1978 and 2001.Fetal cells were collected for DNA analysis of the Human Leukocyte Antigen-DR locus and/or direct DNA analysis of the CYP21A2 gene.The acquirement of fetal cells was done by two arms, amniocentesis and CVS; in 365 pregnancies, the diagnosis was attempted via amniocentesis, while the remaining 165 used CVS.After testing the cells obtained from invasive prenatal tests, a total of 116 fetuses were diagnosed with CAH, 105 being classical and 11 nonclassical.As fetal genital organogenesis is fulfilled around the ninth gestational week, affected female infants will progress to form genital ambiguity secondary to the androgenesis of CAH; thus, low-dose dexamethasone (DEX) was used massively parallel sequencing to demonstrate the utility of cff-DNA testing and the strategy for prenatal diagnosis of CAH in affected families.Sequence analysis was performed using SNPs linked to the CYP212A2 gene in the parents.Gender was 638 -OKPAISE ET AL.
reported that DEX administration around the ninth week's gestation could significantly improve Prader scores in affected female infants.Maternal and infantile complications later in life following their study were also negligible.A European study by Nowotny et al. 51 assessed the management of CAH secondary to 21-OHD deficiency by sending questionnaires to 45 European medical centers.Out of the 45 centers contacted, 36 centers from 14 different European countries chose to participate;

F I G U R E 2
Flowchart showing the process of antenatal congenital adrenal hyperplasia (CAH) management.[Colour figure can be viewed at wileyonlinelibrary.com] 640 -OKPAISE ET AL.