Adrenal Gland Pathology

#### **Chapter 3**

## Combination of High Prevalence Sign/Symptom Pairs: An Approach to the Diagnosis of Cushing' s Syndrome

*Aída Verónica Araya, Claudio Liberman, Claudia Munizaga, Pedro Pineda, Marcela Barberán, Francisco Cordero, Alejandra Lanas and Luís Toro*

#### **Abstract**

Most of the clinical features of Cushing's syndrome (CS) are nonspecific and could be present in obesity, particularly when this condition is associated with metabolic syndrome. Our objective was to evaluate the frequency of clinical manifestations and changes in general laboratory tests, in patients with confirmed endogenous CS, to identify diagnostic dyads. We evaluated in each patient the rate of coexistence of 2 elements either: symptoms, clinical signs, or laboratory alteration. The prevalence of a combined pair of clinical features or dyad in over 30% of the cases, was considered clinically significant. Fourteen dyads were identified as clinically relevant. Facies + buffalo hump; facies + eosinopenia; buffalo hump + supraclavicular fat pads and facies + supraclavicular fat pads, were present in over 50% of cases. Facies + muscular atrophy; centripetal fat distribution + muscular atrophy and facies + striae were present in 42–49%. Hirsutism/acne + eosinopenia; buffalo hump + eosinopenia; muscular atrophy+ eosinopenia; eosinopenia + accelerated weight gain; buffalo hump + muscular atrophy; hirsutism/acne + muscular atrophy and hirsutism/acne + supraclavicular fat pads, were observed in 33–38% of patients. Its application will facilitate the correct diagnosis of CS.

**Keywords:** Cushing, Cushing's syndrome, Cushing's syndrome/diagnosis, dyads, eosinopenia, muscle weakness, muscle atrophy

#### **1. Introduction**

Classical manifestations of Cushing's syndrome (CS) are related to cortisol excess. Frequent physical signs are moon face and plethora, buffalo hump, supraclavicular fat pads, central obesity, width and dark red striae, thin limbs, thin skin, hirsutism, and acne. Frequent symptoms are accelerated weight gain, muscular weakness, amenorrhea, pathological fractures/osteoporosis. In general laboratory tests,

alterations secondary to hypercortisolism can be found (examples: hyperglycemia, hypertriglyceridemia, hypokalemia, low eosinophils, and lymphocytes count.

Nevertheless, most of the clinical features of CS are nonspecific and could be found in obesity, particularly, in association with metabolic syndrome. For this reason, diagnosis is often overlooked, and treatment is delayed unless the evaluation is done by an expert endocrinologist.

A meta-analysis published in 2020 (which included 44 studies) showed that mean time delay to diagnosis CS was 34 months. This was shorter in ectopic CS (14 months) compared with adrenal CS (30 months) and pituitary Cushing's (38 months), probably due to the more aggressive behavior of these neoplasms [1].

For this reason, it seems necessary to establish clinical features of suspicion of this disorder that can be used by non-specialists.

More than fifty years ago, it was reported that in about 50% of patients with suspected CS, the diagnosis could be confirmed or excluded using clinical or general laboratory tests with greater accuracy than steroid screening [2].

Nevertheless, with the increasing prevalence of obesity and its associated metabolic disorders, experts questioned the use of the classical clinical parameters and validated only specific signs related to hypercortisolism (osteopenia, thin skin, and proximal muscular weakness). When they are used to confirm or discard CS diagnosis, the probability is 95% [3, 4].

Over decades, different authors have proposed methods to approach clinical diagnosis of this syndrome (**Table 1**). In the sixties, Nugent et al. studied the incidence of


*\*Index calculated by dividing the prevalence of each feature in CS by its prevalence in reference [2] of 159 mostly obese patients in whom the diagnosis of Cushing's syndrome was suspected but not biochemically substantiated. \*\*Index calculated by dividing the prevalence in authors series with the prevalence reported in reference [4]. \*\*\*Obtained using a probability of 0.2% and a likelihood ratio of 116 for thin skin, 18 for osteopenia, and 4 for ecchymoses*

#### **Table 1.**

*Diagnostic models of Cushing syndrome based on prevalent signs and symptoms.*

*Combination of High Prevalence Sign/Symptom Pairs: An Approach to the Diagnosis… DOI: http://dx.doi.org/10.5772/intechopen.106521*

different signs in patients with and without the syndrome. They reported that the more significant findings were osteoporosis, central or generalize obesity, weakness, plethora, leukocytosis (≥11,000/μL), acne, striae (red or purple), diastolic hypertension (≥105 mmHg), edema, hirsutism, ecchymoses, and low serum potassium (≤3.6 mEq/L). These signs were used to calculate the probability of CS. The results suggested that diagnosis could be confirmed or excluded with a high degree of confidence in at least half of patients with suspected CS [2].

In the early 80s, another paper showed that bruising, muscle weakness, and hypertension were the most discriminating features of CS (discriminant index over 4) [5].

Other authors evaluated the prevalence of signs and symptoms in a series of 73 cases of CS and calculated a discriminant index. The signs and symptoms, with a higher index (2 or greater) were recurrent infections, red striae, amenorrhea, abdominal fat distribution, plethora, muscular weakness, and hirsutism [6].

Another group developed a risk score to predict CS in a population of over 300 subjects at-risk. They propose two models, one based on the assessment of clinical symptoms and signs, and in the other, late-night salivary cortisol (LNSC) determination was added to the clinical features. The multivariate logistic regression analysis showed that muscular atrophy, osteoporosis, and dorsal cervical fat pad remained independent variables associated with CS. The ROC analysis showed that a score of 4 resulted in sensitivity and specificity of 96.2% and 82.9%, respectively. With this cut-off value, 83% of subjects without CS were correctly identified and only one of 26 CS was missed. However, they reported several false positives, mainly related with the LNSC level [7]*.*

Applying technology, in a study of 20 patients with CS, a facial appearance classification software was tested to discriminate patients with Cushing from healthy controls. The software correctly classified 85% of patients and 95% of controls, with a total classification accuracy of 91.7%. Nevertheless, this study only evaluated women, which limited its utility [8].

On the other hand, within general laboratory tests, impaired blood glucose and abnormal lipid levels may be present in a high percentage of the obese population, however, low potassium levels in absence of diuretic use and low eosinophil count may be typical manifestations of hypercortisolism.

Eosinopenia is defined as a reduction of circulating eosinophils < 10/μL or < 0.1% of total leukocyte count. In physiological conditions, during acute stress, eosinopenia is mediated by adrenal glucocorticoids and epinephrine. In patients treated with corticosteroids, eosinopenia would result from an impairment in the release of these cells from the bone marrow and its sequestration of the blood pool [9]. Chronic hypercortisolemia in CS can explain this typical finding which should be taken into account when evaluating a suspected case.

Because laboratory tests performed to confirm the diagnosis of CS are not routinary done and are not widely available, the selection of cases that requires evaluation should be done by a specialist but given a large number of obese patients, this is impracticable. For this reason, combinations of signs, symptoms, and general laboratory tests could be a useful tool for clinicians when CS is suspected.

Our objective was to evaluate the frequency of clinical signs, symptoms, and biochemical alterations in patients treated in a single center with a confirmed endogenous CS and identify dyads of specific symptoms and/or signs.

#### **2. Methodology**

This was a retrospective review of clinical records, laboratory tests, radiological exams, and biopsies from patients treated in one university center in the period 1980– 2017. We excluded patients under corticosteroid therapy, those whose physical examination was not performed by an endocrinologist and with incomplete records. At least two of the following tests confirmed the diagnosis of CS: free urinary cortisol (FUC), overnight 1 mg Dexamethasone suppression test (Nugent's test), and LNSC. The etiology of CS was confirmed by the following: plasma ACTH, hormonal functional tests, and radiological exams. Operated patients had a confirmatory biopsy. This protocol was approved by the local scientific Ethics Committee.

We determined patient characteristics, including age, gender, reason for consultation, features at physical examination, and general laboratory tests, establishing the frequency for each parameter.

We evaluated the rate of coexistence of two features either: symptoms, clinical signs, or laboratory alterations in each patient. A prevalence of a combined pair or dyad in over 30% of the cases was considered clinically relevant. Finally, according to expert endocrinologist opinion and based on scientific literature on the subject, we selected diagnostic dyads with the more specific features of hypercortisolism. Statistical analysis was performed using STATA SE v 15.0 software (StataCorp LLC).

#### **2.1 Definitions of some specific signs of hypercortisolism**


#### **3. Results**

In the analyzed period, 102 patients, 89 women (87.2%), 36.9 13 years old (15–72 yo), had confirmed CS.

*Combination of High Prevalence Sign/Symptom Pairs: An Approach to the Diagnosis… DOI: http://dx.doi.org/10.5772/intechopen.106521*

The etiology of CS was pituitary (68%), adrenal (23%), and ectopic ACTH (9%).

Clinical characteristics are shown in **Table 2**. The more frequent clinical symptoms were accelerated weight increase (57.3%), hirsutism and/or acne (23%), and muscular weakness (22%). At physical examination, the most frequent signs were moon face (90%), centripetal fat depot (78.4%), buffalo hump (65.7%), supraclavicular fat pads (53.9%), hirsutism and/or acne (mainly in women) (65%), systolic and/or diastolic hypertension (60%), muscular atrophy (52%) and striae (42%). At the laboratory tests, we found eosinophils count < 0.1% of total leukocytes in 84.2%.

From 520 combinations of data pairs, we obtained 18 dyads present in over 30% of cases (**Table 3**). From these, we selected 14 dyads which feature combinations were considered more specific to hypercortisolism. Facies + buffalo hump; facies + eosinopenia; buffalo hump + supraclavicular fat pads and facies + supraclavicular fat pads, were present in over 50% of cases. Facies + muscular atrophy; centripetal fat distribution + muscular atrophy and facies + striae were present in 42–49%. Hirsutism/acne + eosinopenia; buffalo hump + eosinopenia; muscular atrophy+ eosinopenia; eosinopenia + accelerated weight gain; buffalo hump + muscular atrophy; hirsutism/ acne + muscular atrophy and hirsutism/acne + supraclavicular fat pads, were observed in 33–38% of patients.

#### **4. Discussion**

Cushing's Syndrome represents a clinical challenge even for specialists. As we mentioned before, many features related to hypercortisolism could be found in other clinical conditions. Some of them correspond to pseudo-Cushing's states. This condition shares clinical signs of CS and equivocal evidence of hypercortisolism in laboratory tests [11]. One study disclosed that obesity (BMI>30 kg/m<sup>2</sup> ) was significantly more frequent in pseudo-Cushing than in CS and only ecchymoses and osteoporosis were more frequent in CS [12]. Another study showed that obesity, moon face, and buffalo hump were present in over 50% of pseudo-Cushing's patients [13].

Nonetheless, even in this condition, a meticulous clinical evaluation could improve diagnostic accuracy and the finding of combination of frequent signs, symptoms, and biochemical tests could be the best approach to continue with more specific evaluations.

Interestingly, classical signs of hypercortisolism were present in a high proportion of our cases, as is shown in **Table 2**. Accelerated weight gain and change in body fat distribution were frequent causes of consultation and feature at physical examination (58% and 78% respectively).

*In vitro* and *in vivo* studies have shown that glucocorticoids (GCs) increase hypothalamic endocannabinoids. GCs and cannabinoids have been shown to increase hypothalamic adenine monophosphate protein kinase-A (AMPK) activity, resulting in increased appetite. Moreover, GCs have been shown to upregulate gene expression of orexigenic peptides such as Y neuropeptide (NPY) and agouti-related peptides. This could explain cravings for fat and carbohydrates observed in endogenous GCs excess states as Cushing's disease [14].

Some studies in human healthy controls or subjects with inflammatory conditions evaluated the effect of short-term use of oral GCs on energy intake, body weight, body composition, or appetite but, results have not been conclusive [15].

Still, more studies are necessary to establish the effects of long-term use of oral GCs and endogenous hypercortisolism, on appetite and dietary intake.

#### *Adrenal Glands – The Current Stage and New Perspectives of Diseases and Treatment*


*Combination of High Prevalence Sign/Symptom Pairs: An Approach to the Diagnosis… DOI: http://dx.doi.org/10.5772/intechopen.106521*


#### **Table 2.**

*Clinical features in patients with Cushing's syndrome.*


#### **Table 3.**

*Diagnostic dyads.*

On the other hand, changes in fat redistribution are determined by chronic hypercortisolism, leading to an increase in centripetal adiposity and visceral abdominal fat. Regulation of adipose tissue mass is complex and involved mechanisms are multiple and still not completely understood. In general, the effects of GCs on adipose tissue depend on the duration of the exposure to GCs and on the type of adipose tissue considered (subcutaneous or visceral) [16]. Visceral fat would be differentially responsive to GCs than subcutaneous fat. In experimental models, GCs induce the differentiation of preadipocytes, specific to visceral fat, but not for subcutaneous fat. Glucocorticoid receptor expression is higher in visceral than in peripheral subcutaneous fat. Chronic corticoid exposure increases lipogenesis in visceral adipose compartment and increases lipolysis in subcutaneous adipose tissue [17]. Also, there is a higher cortisol production in visceral adipose tissue, due to an increased expression of GC receptors and 11B-hydroxysteroid dehydrogenase type 1 (11BHSD1) enzymatic activity. The 11BHSD1 is widely expressed throughout the body, including liver, visceral and subcutaneous fat, and promotes the conversion of inactive cortisone to

cortisol. These mechanisms among others, explain the characteristic phenotype of CS [18].

Another relevant clinical feature is muscle involvement. In our case series, muscle atrophy was present in 52% of cases. In previous reports, the prevalence of muscle weakness or atrophy was 20–45% [6, 7, 12]. Chronic hypercortisolism generally has a more prominent effect on the proximal muscles. GC-induced muscle atrophy affects mainly fast-twitch or type II fibers with less or no impact on type I or slow-twitch fibers. This explains the characteristic findings at physical examination of thin limbs with loss of muscle mass.

GCs reduce skeletal muscle mass both by inhibiting protein synthesis and by increasing the rate of protein degradation. GCs stimulate myostatin, an inhibitory growth factor that downregulates protein synthesis and also, proliferation and differentiation of muscle satellite cells, precursors of skeletal muscle cells. GCs inhibit the transport of amino acids into the muscle and interfere with the stimulatory action of insulin and Insulin-like Growth Factor 1 (IGF1) on the protein synthesis pathways. Moreover, GCs inhibit the production of IGF1 in muscle, which contributes to muscle proteolysis and apoptosis [18]. On the other side, GCs can indirectly affect skeletal muscle by downregulating gonadal function and reducing the expression of the androgen receptor. This would explain the authors' finding of high frequency of myopathy in males compared with females (65% *vs* 45%) [19].

Recently, some researchers compared muscle mass and muscle function in patients with CS and matched obese controls. The CS group showed a significant decrease in muscle function tests (chair rising time and hand grip strength) versus the obese non-CS group. Interestingly, both groups did not show differences in muscle mass, fat mass and waist-to-hip ratio, suggesting that CS could be associated with impaired muscle quality and functional alterations more than in trophic effects [20]. Functional tests could be other clinical tool to objectify the diagnosis of CS.

It is interesting that skin manifestations were not observed with high frequency in our cases (only 29%), as has been reported previously [21]. In that report, they used a caliper to measure the skinfold over the proximal phalanx of the middle finger of the nondominant hand. Our diagnosis was based on observation only. Also, we must consider that our patients have different ethnic backgrounds.

Another issue observed in CS patients is metabolic complications, as hyperglycemia, dyslipidemia and higher cardiovascular risk. These are due to several GCs related actions on the liver, skeletal muscle, pancreas, and adipose tissue [see Ref. 22]. In omental, but not in subcutaneous adipocytes, GCs induce insulin resistance by increasing free fatty acid, inducing deregulation in adipokines secretion, and increasing leptin and resistin levels. IL6 and TNFα receptor 1 are also increased in CS patients compared to BMI-matched controls. In some series, a prevalence of diabetes between 15–45% was reported [6, 7, 12, 19] and 46% of dyslipidemia [7]. In our series, 50% had hyperglycemia and hypertriglyceridemia. It should be mentioned that not all cases had a glucose tolerance test, so this probably underestimated the frequency of hyperglycemic disorders.

A remarkable finding observed, is a decrease in eosinophil count. Eosinopenia/ aneosinophilia determined by excess of GCs. has been described for more than 6 decades. Patients with CS may present leukocytosis and neutrophilia and a low lymphocyte and eosinophil count or eosinopenia [9, 23, 24]. In the absence of exogenous corticosteroid use, eosinopenia would be the most specific laboratory finding. Patients with pseudo-Cushing, present various clinical signs and even altered laboratory tests such as FUC or Nugent's test, but do not have eosinopenia [11]. We found an

*Combination of High Prevalence Sign/Symptom Pairs: An Approach to the Diagnosis… DOI: http://dx.doi.org/10.5772/intechopen.106521*

eosinophil count < 0.1% of total leukocyte count in over 80% of cases, unlike lymphopenia, observed only in 18% of patients. Then, we consider eosinopenia a significant diagnostic element.

The association of eosinopenia with signs such as hypertension or hirsutism, which are observed commonly in other pathologies with similar characteristics to CS, like polycystic ovary syndrome (PCOS), leads toward the diagnosis of Cushing's. The combination of hirsutism/acne + eosinopenia and systolic hypertension + eosinopenia were prevalent in CS (38% and 35%).

Decreased bone mass density was frequent in other series [7, 12]. But, in our patients we found osteoporosis only in 5%. However, it should be noted that this was due to the fact that bone densitometry was performed in a minority of cases. Then, this sign was underestimated in our cases and always should be considered if it is present together with any of the diagnostic dyads.

#### **5. Conclusion**

We present a casuistry with a large number of patients with a confirmed Cushing's syndrome which makes our findings very significant. The combinations of high-risk clinical pairs or dyads associated with determined disease have been used for the identification of patients at risk of other pathology such as acromegaly [25].

The presence of dyad combinations of clinical features may be a useful tool in a practical clinical setting, to assist physicians in identifying patients at risk of CS, without requiring complex or expensive tests.

With training, that allows recognizing the specific signs of hypercortisolism and general laboratory tests, the use of these sign/symptom dyads (shown in **Table 3**), will facilitate that the correct diagnosis of CS can be reached with high certainty.

An early diagnosis and treatment of Cushing syndrome, most probably will reduce the severe comorbidities associated with this condition and will improve the prognosis of these patients.

#### **Acknowledgements**

To the neurosurgery team of the University of Chile Clinical Hospital, who referred many patients for evaluation.

#### **Conflict of interest**

The authors declare no conflict of interest.

#### **Author details**

Aída Verónica Araya1,2\*, Claudio Liberman1 , Claudia Munizaga2 , Pedro Pineda<sup>1</sup> , Marcela Barberán<sup>2</sup> , Francisco Cordero<sup>1</sup> , Alejandra Lanas<sup>1</sup> and Luís Toro<sup>3</sup>

1 Endocrinology Section, University of Chile Clinical Hospital, Chile


\*Address all correspondence to: draveronicaaraya@gmail.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Combination of High Prevalence Sign/Symptom Pairs: An Approach to the Diagnosis… DOI: http://dx.doi.org/10.5772/intechopen.106521*

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#### **Chapter 4**

## Primary Hyperaldosteronism: The Role of the General Surgeons in Diagnosis and Treatment

*Thawatchai Tullavardhana*

#### **Abstract**

Primary hyperaldosteronism (PA) is the cause of endocrine hypertension, which commonly occurs in young patients with uncontrolled hypertension that leads to worsening cardiovascular-related mortality. Patients suspected of developing PA should have their plasma aldosterone concentration and plasma renin activity (PRA) assessed for screening purposes. After verifying the diagnosis of PA, adrenal venous sample (AVS) is the gold standard diagnostic technique for differentiating unilateral from bilateral disease. Since adrenalectomy may benefit patients with unilateral disease, laparoscopic adrenalectomy, a minimally invasive surgical approach that provides better postoperative outcomes than open surgery, has become the standard treatment for unilateral PA. Laparoscopic adrenalectomy resulted in a 53% cure rate of hypertension after surgery, as well as all patients had improved hypertension control, including the remission of hypokalemia. The conventional laparoscopic adrenalectomy approaches are transperitoneal and retroperitoneal, with similar postoperative outcomes. However, for general surgeons with limited laparoscopic adrenalectomy experience, the transperitoneal technique may offer an advantage over the retroperitoneal approach in terms of faster learning curve time, better surgical anatomy view, and the ability to resect adrenal tumors larger than 5 cm. This chapter focuses on the diagnosis and treatment of PA from the general surgeon's perspective.

**Keywords:** primary hyperaldosteronism, hypertension, adrenalectomy, minimally invasive surgery, transabdominal approach

#### **1. Introduction**

Primary hyperaldosteronism (PA) is a leading cause of endocrine hypertension attributable to the autonomous secretion of aldosterone by the adrenal gland, which inhibits the renin-angiotensin system, resulting in sodium retention, potassium diuresis, and volume overload. PA was reported as 5–11% patients with hypertension are related to 20% of drug-resistant hypertension [1–3]. In addition, patients may experience hypokalemia symptoms, such as proximal muscular weakness, muscle cramps, and palpitations. This chapter focused on the diagnosis process and the proper minimally invasive surgical treatment of PA from the general surgeon's perspective.

#### **2. Prevalence**

The PA was classified into two subtypes, which are as follows: 1) unilateral adrenal gland involvement (adrenal adenoma) in 33–95% of patients, and 2) bilateral adrenal gland involvement (adrenal gland hyperplasia) in 5–60% of patients, according to certain studies. Thus, in patients with unilateral gland involvement, the goal of normalizing aldosterone levels by surgical management may be advantageous in contrast to the patients having bilateral adrenal involvement, these were more susceptible to medical treatment [4, 5].

Excessive aldosterone secretion may damage the cardiovascular system, increasing the risk of myocardial infarction, heart failure, cardiac arrhythmias (atrial fibrillation), stroke, renal function impairment, and consequences to increase cardiovascular-related mortality [6, 7].

The following were the indications for PA screening in hypertensive patients: 1) hypertension in young adults (age < 40 years), 2) severe/treatment-resistant hypertension (blood pressure > 160/100 mmHg), 3) sleep apnea, 4) hypokalemia, 5) atrial fibrillation without structural cardiac diseases, 6) first degree relative with PA, and 6) incidentaloma [8].

#### **3. Screening test**

#### **3.1 Plasma aldosterone concentration and plasma renin activity**

The plasma aldosterone concentration (PAC) is greater than 15 ng/dL, and the plasma renin activity (PRA) is less than 1 ng/mL/hour. This is the cut-off result for screening tests and was used to diagnose PA. Nonetheless, there are various methods for evaluating aldosterone levels in clinical settings, such as immunological assays and liquid chromatography-tandem MS/MS (LC–MS/MS), which may influence the cut-off values of each test.

As a result, aldosterone to renin ratio (ARR) greater than 30 ng/dL per ng/mL/h is more accepted in determining positive screening tests for PA with a sensitivity of 68–94%. To prevent a false negative result, antihypertensive medications, particularly angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARB), should be discontinued between 4 and 6 weeks prior to the screening is recommended [9, 10].

#### **4. Confirmatory test**

Almost all patients who had positive screening test results needed a confirmation test to access dynamic aldosterone hypersecretion that affects the imbalance of the renin-angiotensin-aldosterone system to confirm the diagnosis of PA. However, the confirmation test may not be required in patients with the following conditions: 1) PAC levels larger than 20 ng/dL and 2) spontaneous hypokalemia.

#### **4.1 Oral sodium loading test**

After achieving good blood pressure control and collecting hypokalemia, the patients were required to intake 5000 milligrams (mg) of sodium (equal to 12.8 grams *Primary Hyperaldosteronism: The Role of the General Surgeons in Diagnosis and Treatment DOI: http://dx.doi.org/10.5772/intechopen.107460*

of sodium chloride) for 3 days. After 3 days of the high sodium diet, 24-hour urine samples were collected for sodium and aldosterone measurements. The diagnosis of PA was confirmed by: 1) 24-hour urinary sodium excretion greater than 200 mEq, and 2) 24-hour urinary aldosterone excretion greater than 12 micrograms. The sensitivity and specificity of the oral sodium loading test for the diagnosis of PA were reported as 96% and 93%, respectively [11, 12].

#### **4.2 Intravenous saline infusion test**

In order to reduce aldosterone production, this approach involves intravenous delivery of normal saline solution (0.9% sodium chloride) at a rate of 2 liters every 4 hours following an overnight fast. For PAC assessment, a blood sample was obtained after a complete saline infusion. In normal patients, the PAC level might be decreased to less than 5 ng/dL, however, a PAC level greater than 10 ng/dL is consistent with the diagnosis of PA with high sensitivity and specificity of 87% and 94%, respectively [13].

#### **4.3 Fludrocortisone suppression test (FST)**

Patients are given 0.1 mg of fludrocortisone orally every 6 hours for 4 days, along with potassium chloride and sodium chloride (6 gram/day) supplements to maintain serum potassium level greater than 4 mmol/L and 24-hour urine sodium excretion greater than 200 mEq. Blood samples were obtained at 7 AM to assess plasma cortisol, and at 10 AM to assess PAC, PRA, and cortisol levels. The diagnosis of PA was confirmed by: 1) a PAC level greater than 6 ng/dL, 2) a PRA level less than 1 ng/mL/h, and 3) a cortisol level at 10:00 AM that was lower than at 7:00 AM. In comparison with the saline infusion test, both tests were reliable, but FST seems to be more expensive and complex in the diagnosis of PA after a positive screening test [14].

#### **4.4 Captopril challenge test**

After 1 hour of the patients remaining in a sitting or standing position, captopril 25–50 mg was administered orally. Blood samples were taken to access PAC and PRA at 0, 1, and 2 hours after the medication was administered. The diagnosis of PA was confirmed by: 1) a decrease in PAC of more than 30% at 2 hours, 2) inability to suppress PAC to less than 11 ng/dL, and 3) an ARR of 20 ng/dL per ng/mL per hour. Furthermore, an ARR greater than 30 ng/dL per ng/mL per hour after ingesting captopril is strongly indicated for the diagnosis of PA [15, 16].

#### **5. Radiologic imaging**

#### **5.1 Computed tomography scan (CT scan)**

Contrast-enhanced CT scan is the first step in radiologic imaging in a PA patient for subtype evaluation, which is useful in distinguishing unilateral adrenal adenoma from bilateral adrenal hyperplasia, particularly in tumor sizes greater than 1–2 centimeters (cm). The CT scan detected unilateral adrenal involvement with 100% accuracy in a patient under the age of 35 with hypokalemia and a PAC greater than 30 ng/dL. The accuracy rate of CT scan declined in the patients over the age of 35, which is attributed to the high prevalence of bilateral disease and the limitations of

#### **Figure 1.**

*Computed tomography (CT) shows a well-defined left adrenal nodule (arrow) of size 1.3 cm. (18 HF on noncontrast CT) compatible with adrenal adenoma: (A) Sagittal view, and (B) coronal view.*

the CT scan in differentiating the normal appearing adrenal gland from microadenomas (size less than 1 cm).

There are no CT criteria for differentiating adenoma from bilateral adrenal hyperplasia in terms of size and Hounsfield units. Nevertheless, the study by Lingam et al., demonstrates that an adrenal limb greater than 3 millimeters is beneficial in the diagnosis of adrenal hyperplasia with 100% sensitivity and specificity [17, 18]. **Figure 1** illustrates CT scan-detected left aldosterone-producing adenoma.

#### **5.2 Magnetic resonance imaging (MRI)**

High-resolution MRI, including T1 and T2 weight images, has the same detection rate for unilateral adrenal adenoma as CT and should be used as a diagnostic adjunct in patients with strong clinical indications of PA and negative or equivocal CT findings. Furthermore, the diagnostic value of MRI for bilateral adrenal hyperplasia is limited in patients older than 40 years, with 68% sensitivity and 57% specificity [19, 20].

#### **6. Role of lateralization with adrenal venous sampling (AVS)**

Adrenal venous sampling is the gold standard diagnostic tool for differentiating unilateral from bilateral disease in PA patients. Aldosterone hypersecretion was discovered in the absence of an enlarged adrenal gland or a nodule. Thus, AVS improves the accuracy of the diagnosis of PA by 20–38% when compared to CT or MRI imaging, which can guide proper management and minimize unnecessary adrenalectomy in 20% of patients using the imaged-base technique.

The current guidelines for PA care are determined based on AVS findings. An interventional radiologist used percutaneous femoral vein access to perform this procedure. Blood samples were taken from the veins in the adrenal glands and the inferior vena cava (IVC).

The unilateral lateral disease was diagnosed when the ratio of normalized aldosterone to cortisol between the dominant and nondominant adrenal glands (lateralization index) was at least 4.0 and the ratio of aldosterone normalized to cortisol between the nondominant adrenal gland and peripheral blood (suppression ratio) was equal or less than 1.0.

*Primary Hyperaldosteronism: The Role of the General Surgeons in Diagnosis and Treatment DOI: http://dx.doi.org/10.5772/intechopen.107460*

#### **Figure 2.**

*Angiographic imaging of adrenal venous sampling via the left adrenal vein.*

Furthermore, AVS-based decision-making was found to be superior to imagebased decision-making in unilateral pathology, resulting in hypokalemia correction and improved blood pressure control with 35–60% requiring no further antihypertensive drugs [21–23]. **Figure 2** illustrates adrenal venous sampling in patients with left primary hyperaldosteronism.

#### **7. Role of surgical management in primary hyperaldosteronism**

Patients with bilateral disease were considered for medical treatment with mineralocorticoid receptor antagonists, such as spironolactone or eplerenone, in conjunction with potassium chloride supplementation. In contrast, surgical adrenalectomy may reduce aldosterone levels to normal in unilateral hyper-aldosterone-producing adenoma. Adrenalectomy can cure hypertension among 30–60% of patients and completely correct hypokalemia, resulting in a decreased incidence of atrial fibrillation and cardiovascular-related mortality in PA patients [24, 25].

#### **7.1 Open surgery versus minimally invasive surgery**

Historically, open adrenalectomy was the treatment of choice for functioning adrenal adenoma. After the development of minimally invasive surgical techniques, Gagner et al. first described laparoscopic adrenalectomy in 1992 for the management of Cushing's syndrome and pheochromocytoma [26].

Laparoscopic adrenalectomy provides an advantage over open surgery in terms of decreased intraoperative blood loss, less postoperative complications, shorter return to diet time, shorter length of hospital stays, and faster return to normal activity [27].

Laparoscopic adrenalectomy is beneficial for long-term blood pressure control and correction of hypokalemia in patients with PA. The cure rate for hypertension was 51%, with conversion to open surgery and postoperative morbidity rates of 3.2% and 8.0%, respectively [28]. Currently, laparoscopic adrenalectomy is considered the standard treatment for unilateral aldosterone-producing adenoma.

#### **7.2 Transperitoneal versus retroperitoneal approach**

The transperitoneal and retroperitoneal approaches are used for standard laparoscopic adrenalectomy techniques. The retroperitoneal technique is advantageous in cases of previous extensive abdominal surgery to avoid visceral organ injury, but it should be avoided in adrenal tumors greater than 7 cm in size, since this may limit anatomical landmarks in the retroperitoneal space. Previous research indicates that both procedures are comparable in terms of operating time, intraoperative blood loss, hospital stay, and postoperative complications [29, 30].

Attributed to the reason that the author's institution is a medium-sized university hospital, general surgeons must play an important role in the minimally invasive surgical management of patients with functional adrenal tumors, particularly pheochromocytoma and primary hyperaldosteronism.

Transperitoneal approach appears to be an appropriate surgical technique for general surgeons with limited experience in laparoscopic adrenalectomy because of 1) shorter learning curve time, 2) superior surgical view through the use of laparoscopy, 3) comfort with intraperitoneal anatomical landmarks over retroperitoneal space, and 4) ability to resect adrenal tumors larger than 5 cm. The retroperitoneal technique is appropriate for experienced surgeons who have conducted at least 20 procedures and also have a tumor size of less than 5 cm [31–32].

#### **8. Operative techniques for laparoscopic transperitoneal adrenalectomy**

#### **8.1 Right adrenalectomy**

#### *8.1.1 Patient position*

The operation was performed under general anesthesia and the patients were placed in a left lateral decubitus position, and the surgical table was flexed between 100 and 120 degrees at an umbilical level to enhance the distance between the costal margin and the iliac crest [33–36].

#### *8.1.2 Trocars site placement*

A 12 mm trocar was introduced into the peritoneal cavity using an open method at 3 cm below the right costal margin at the anterior axillary. Under laparoscopic guidance, two 5 mm working trocars were placed in the posterior axillary line and

*Primary Hyperaldosteronism: The Role of the General Surgeons in Diagnosis and Treatment DOI: http://dx.doi.org/10.5772/intechopen.107460*

mid-clavicular line. The author preferred to add a 5 mm trocar at the subxiphoid for a fan-shaped liver retractor.

#### *8.1.3 Operative technique*

After carbon dioxide insufflation, pneumoperitoneum is created with a pressure of 10–12 mmHg. The triangular ligament was divided and the right lobe of the liver was lifted upward with a fan-shaped liver retractor. The posterior peritoneum was dissected with an electrocautery hook or harmonic scalpel (Ethicon Endo-Surgery INC - Johnson & Johnson, NJ, USA) to expose the right kidney, right adrenal gland, and inferior vena cava (IVC).

The first and most crucial step in right adrenalectomy is vascular control. Thus, the superomedial aspect of the adrenal gland, which is located between the confluence of the IVC and the right renal vein, was meticulously dissected to identify the right adrenal vein.

The right adrenal vein was clipped with nonabsorbable polymer clips (hem-olock(R), Teleflex Medical, Durham, NC, USA) and divided by a laparoscopic scissor or harmonic scalpel. The author recommended the use of double clips on the IVC site to avoid clip slippage, which could result in exsanguinate bleeding.

Following control of the right adrenal vein, the adrenal gland was raised up with an atraumatic grasper, and dissection should be extended to the posterior and lateral aspects to complete adrenal gland removal.

The gland is placed in a sterile bag before being retrieved via a 12 mm trocar incision that can be extended as needed. Before closing the skin incision, complete

#### **Figure 3.**

*Laparoscopic transperitoneal right adrenalectomy: (A) Position of the patient and placement of the trocars; (B) identification of the right adrenal vein; (C) clips control of the right adrenal vein; and (D) after removal of the right adrenal tumor.*

hemostasis was confirmed, and routine drainage was not recommended. **Figure 3** illustrates the surgical technique for laparoscopic transperitoneal right adrenalectomy.

#### **8.2 Left adrenalectomy**

#### *8.2.1 Patient position*

The patients were placed in the right lateral decubitus position and the operating table was flexed, as described in right adrenalectomy.

#### *8.2.2 Trocars site placement*

An open 12 mm trocar was introduced into the peritoneal cavity 3 cm below the left costal margin at the anterior axillary line. Two 5 mm and 12 mm working trocars were placed at the subxiphoid and mid-clavicular lines, respectively. An extra 5 mm trocar can be placed in the posterior axillary line as necessary for the fan-shaped retractor.

#### *8.2.3 Operative technique*

The procedure for creating pneumoperitoneum is the same as that described in right adrenalectomy. The splenic flexure colon and spleen were mobilized initially by dissecting the splenocolic, splenorenal, and lienorenal ligaments through the diaphragm for adequate exposure of the left adrenal gland.

The upper pole of Gerota's fascia serves as a landmark for identifying the left adrenal gland during dissection. The dissection was carried out medially close to the level of the left renal vein. Meticulous dissection was required to locate the left adrenal vein, which was subsequently clipped and separated from the left renal vein using nonabsorbable polymer clips (hem-o-lock(R), Teleflex Medical, Durham, NC, USA).

The adrenal gland's medial aspect was carefully dissected, and the small arterial branches from the aorta that supply the left adrenal gland were clipped and divided. Completer gland removal was accomplished by continuing dissection to the upper portion of the gland while avoiding harm to the splenic artery and pancreatic tail. After hemostasis was confirmed, the specimen was retrieved using a sterile bag. **Figure 4** illustrates the surgical technique for laparoscopic transperitoneal left adrenalectomy.

#### **8.3 Postoperative complication**

For the right adrenalectomy, significant bleeding occurs as a result of IVC injury caused by avulsion or right adrenal vein or clip slippage, necessitating conversion to open surgery. This complication may have been avoided with proper trocar site placement and precise dissection to allow adequate exposure of the surgical landmarks via the laparoscopic view.

Left adrenalectomy complications included hollow viscus organ perforation, vascular injury, and pancreatic tail injury. These could be prevented by using the proper trocar site placement and surgical dissection technique. In the case of small adrenal tumors, the upper pole of Gerota's fascia is an essential surgical landmark for determining the appropriate dissection plane to identify the adrenal gland. This technique could prevent the dissection of the incorrect plane, thereby preventing bleeding from the renal and splenic veins.

*Primary Hyperaldosteronism: The Role of the General Surgeons in Diagnosis and Treatment DOI: http://dx.doi.org/10.5772/intechopen.107460*

#### **Figure 4.**

*Laparoscopic transperitoneal left adrenalectomy: (A) Patient positioning and trocar site placement; (B) identification of the left adrenal vein; (C) control of the left adrenal vein with clips; and (D) control of the arterial supply to the left adrenal gland with clips.*

In the circumstance that renal artery or renal vein injury occurs during adrenal gland dissection. The damage site might be controlled with an atraumatic grasper and repaired with an intracorporeal suture. However, if the hemorrhage cannot be controlled, immediate conversion to open surgery will be required.

Hollow viscous organ perforation should be repaired with an intracorporal suture under laparoscopic vision, similar to pancreatic tail injury; in this circumstance, closed-suction drainage at the surgical site should be considered.

#### **9. Postoperative disease monitoring**

For immediate postoperative disease monitoring, blood tests for plasma aldosterone, renin, and potassium levels should start on the first day. Therefore, antihypertensive medications and potassium supplements should be discontinued or tapered to prevent interfering with biochemical measurements.

Furthermore, serum aldosterone and renin levels should be measured at 1 and 6 months after surgical management to confirm that PA is curable. Patients who underwent adrenalectomy under AVS-based therapy had a high biochemical cure rate of 80%, attributable to the improvement in hypertension control and hypokalemia cure rates [37, 38]. **Video 1** (https://drive.google.com/file/ d/1J5Z03Kb7ZCXVOGZEWthVpEZOc-p3u3EM/view) shows a technique in laparoscopic left adrenalectomy via transperitoneal approach in a patient with primary hyperaldosteronism.

**Figure 5.**

*Surgical specimen: (A) Gross specimen reveals 1.5 and 0.3 cm of golden yellow adrenal adenoma, and (B) microscopic examination of tumor cells reveals an alveolar and trabecular pattern of growth without capsular or vascular invasion.*

The postoperative hypertension cure rate following adrenalectomy was found to be 53% at 6 months and 49.6% at 12 months. Age older than 55 years, a long history of hypertension, and a tumor size greater than 2 cm are indeed risk factors for postoperative persistent hypertension. All of the patients, however, had curative hypokalemia and needed fewer antihypertensive medications following the surgery [39–41]. **Figure 5** illustrates a surgical specimen from laparoscopic left adrenalectomy in PA patients.

#### **10. Conclusion**

Laparoscopic adrenalectomy is the gold standard treatment for unilateral PA, with a low postoperative morbidity rate and an excellent clinical success rate for hypertension control. General surgeons, particularly in small to medium-sized hospitals, may play an important role in the surgical management of PA. Transperitoneal laparoscopic adrenalectomy is a safe and effective technique that is recommended for general surgeons with limited laparoscopic adrenalectomy experience.

#### **Acknowledgements**

The author wishes to thank Associate Professor Prinya Akkranurakul for the comments, who contributed in the preparation of this chapter.

#### **Conflict of interest**

The author declared no conflict of interest.

*Primary Hyperaldosteronism: The Role of the General Surgeons in Diagnosis and Treatment DOI: http://dx.doi.org/10.5772/intechopen.107460*

#### **Author details**

Thawatchai Tullavardhana Department of Surgery, Faculty of Medicine, Srinakharinwirot University, Ongkharak, Nakhon Nayok, Thailand

\*Address all correspondence to: thawatchait@g.swu.ac.th

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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#### **Chapter 5**

## Congenital Adrenal Hyperplasia

*Adina Mariana Ghemigian and Nicoleta Dumitru*

#### **Abstract**

Congenital adrenal hyperplasia (CAH) is a rare pathology with an estimated incidence of 1:14,000–18,000 births. It includes a group of inherited diseases with autosomal recessive transmission. The genetic defect consists of mutations of the genes encoding the enzymes involved in adrenal and eventually gonadal steroidogenesis. The most common mutation is the gene encoding 21 hydroxylase the enzyme involved in cortisol and aldosterone synthesis. However, other enzymatic defects can be identified. The excess of steroid precursors in the adrenal cortex will be directed towards adrenal androgen synthesis. Finally, the clinical picture includes a series of manifestations specific to the enzymatic deficiency, the severity depending on the degree of the genetic defect. Thus, we can meet severe deficits with clinical expression in newborns and toddlers or partial, non-classical forms with manifestation in adolescence or adulthood. Once the diagnosis of CAH is established, patients will require specific therapy and long-term monitoring.

**Keywords:** congenital adrenal hyperplasia, adrenal steroid synthesis, 21-hydroxylase deficiency, hyperandrogenism, dexamethasone

#### **1. Introduction**

Congenital adrenal hyperplasia (CAH) includes a heterogeneous group of autosomal recessive disorders caused by total or partial impairment of steroidogenesis, which is characterized by decreased synthesis of cortisol and, or aldosterone [1]. Cortisol deficiency causes an increase in ACTH levels by losing the effect of negative feedback on the hypothalamic-pituitary region. In turns this leads to chronic overstimulation of the adrenal cortex and consecutive adrenal hyperplasia.

From the point of view of steroid synthesis, the adrenal cortex is divided into three areas: zona glomerulosa or the outer layer, the site of mineralocorticoid synthesis, the fasciculata area, the middle largest layer, the site of glucocorticoid synthesis and the inner layer or zona reticularis involved in androgen biosynthesis [1]. Steroidogenesis involves the conversion of cholesterol to active steroid hormones and is performed under the action of many enzymes, cofactors, and accessory proteins (**Figure 1**) [1]. These enzymes are specific expressed in the three areas of the adrenal cortex, depending on the type of steroids synthesized, but some are also expressed in the gonads or placenta.

The pathophysiological mechanism of CAH consists in mutations in most of the genes encoding the enzymes involved in adrenal and eventually gonadal steroidogenesis. Impaired enzyme function in a specific step of adrenal steroid biosynthesis leads to a unique combination of elevated precursors and deficient products [2]. For this

**Figure 1.**

reason, the clinical phenotype is variable and depends on the severity of the enzyme deficiency. The most common enzyme deficiency that disrupt cortisol synthesis leading to CAH is that of 21 hydroxylase [1–4].

### **2. 21-hydroxylase deficiency**

#### **2.1 Epidemiology**

CAH due to 21-hydroxylase deficiency (21OHD) represents approximately 90–95% of CAH cases encountered in clinical practice [3, 4].

Depending on the degree of enzyme deficiency, it is classified into classical and non-classical form. Classical form occurs in 1:14,000 to 1:18,000 live births based on screening programs [1, 4, 5]. Non-classical form is more common with a prevalence of 1:500 to 1:1000 in the general white population [4]. In certain population groups like the Ashkenazi Jewish the prevalence is even higher with 1 in 27 individuals affected and 1 in 3 are carriers of the allele, making this form the most frequent autosomal recessive disorder in population [2].

#### **2.2 Pathophysiology**

CYP21A2 is a microsomal enzyme belonging to the cytochrome P450 family, also known as P450c21 [1]. The major substrate of CYP21A2 is 17 hydroxyprogesterone (17OHP), which is converted to 11-deoxycortisol in zona fasciculata, an intermediate metabolite in the cortisol synthesis stage. In zona glomerulosa, CYP21A2 acts on progesterone, converting it to 11-deoxycorticosterone within the aldosterone pathway [1]. Blocking this enzymatic step will cause cortisol deficiency with a secondary increase

in ACTH secretion [2]. This in turn, will stimulate excessive synthesis of precursor molecules in both pathways blocked by the enzyme deficiency, namely 17OHP and progesterone. The precursor will be redirected to adrenal androgens synthesis via the 17,20-lyase activity of CYP17A1, the step unaffected by the enzyme deficiency [1, 2].

Intrauterine exposure to excess adrenal androgens will not significantly influence male sexual differentiation. The situation is totally different in the case of a fetus 46, XX. In this case, excess of androgens will interfere with the differentiation of the external genitalia, causing prenatal virilization. The severity of virilization is quantified using a five-point scale developed by Prader, where grade I involves minimal virilization with clitoral hypertrophy, while grade V is a typical male aspect of the external genitalia [2].

#### **2.3 Genetics**

The gene that encodes CYP21A2 is located on the short arm of the 6 chromosomes within the HLA class III region (6p21.3) arranged in tandem with its inactive pseudogene (*CYP21A1P*) [1, 3]. Both *CYP21A2* and *CYP21A1P* genes share a high nucleotide homology of about 98% and 96% in exons and introns respectively, but only *CYP21A2* gene encodes the active microsomal P450 enzyme, 21-hydroxylase (CYP21A2, P450c21) [1, 6]. The pseudogene CYP21A1P is inactive because of the presence of multiple pathogenic variants, small insertions or deletions and point pathogenic variants that prevent the synthesis of a functional protein [3, 7].

In about 70–75% of cases *CYP21A2* mutations arise from gene microconversion, namely transfer of deleterious mutations from *CYP21A1P* during meiosis, generating point mutations with consecutive reduction of the enzymatic activity of CYP21A2 [1, 6]. In 20–25% of the cases, *CYP21A2* mutations are due to gross misalignment owing to unequal crossing over during meiosis leading to gene deletions, gene duplications or the appearing of a hybrid gene or pseudogene-gene chimeras, yielding a non-functional *CYP21A2* gene [3, 6]. Less than 5% of the pathogenic variants in the CYP21A2 gene are de novo point mutations, most of these affected patients are heterozygous [3, 6].

#### **2.4 Genotype-t correlation**

Most patients with 21OHD are heterozygous compounds, with more than one mutation in one or both *CYP21A2* alleles, which is why there is a wide spectrum of phenotypes [1, 4]. In general, the phenotype reflects the residual activity of the lightest mutation [3, 7].

Scholastically, two forms of this condition are described, namely the classical and non-classical form. In turn, the classic form is subdivided into salt-wasting (SW) and simple virilizing (SV) form [1, 4].

The SW form is characterized by mutations in both alleles leading to complete loss of enzymatic activity, such as deletions, large gene conversions, nonsense mutations, frameshifts, and missense mutations [1].

The SV form occurs by mutation associated with complete loss of function on one allele and on the other allele a mutation that reduces enzymatic activity by 1–5%, like a nonconservative amino acid substitution, p.Ile172Asn (I172N) or splicing mutation on intron 2 [1, 4]. This mutation that alters splicing of intron 2 is particularly common in SV patients, but also in SW ones.

In the nonclassical (NC) form, most of the affected individuals are compound heterozygotes, with various mutations on each of the two alleles, of which at least one can allow enzymatic activity of 20–50% [6]. The genetic defect may be a point mutation in exon 7 (p.Val281Leu), or a missense mutation as p.Pro30Leu (P30L) [1, 4, 6]. However the clinical observations suggest that patients carrying the P30L allele are somewhat more symptomatic, being rather a borderline form between SV and NC [1].

Although we still use this classification of CAH forms, in practice we must consider the *CYP21A2* allelic variants with their phenotypic manifestations as a continuum clinical spectrum.

#### **2.5 Clinical picture**

The classic form of CAH due to 21OHD is identified in 1 of 14,000–18,000 newborns and includes the two variants: salt-wasting and simple virilizing form [1, 4].

#### *2.5.1 Classic salt-wasting 21-OHD CAH*

Of the two variants, the SW form is the most severe, but also the most common, representing about 75% of all cases [6]. It is characterized by null or minimal enzymatic activity (<1%) in both *CYP21A2* alleles, resulting in severe cortisol and aldosterone deficiency and elevated androgen levels [1, 3]. Clinically it is characterized by prenatal virilization of female girls by exposure to potent androgens (testosterone and Δ4-androstenedione) at critical stages of sexual development [6]. Aldosterone deficiency will be manifested soon after birth by the appearance of hypovolemia with hyperreninemia, hyponatremia and hyperkalemia, which associated with hypoglycemia due to severe cortisol deficiency, will generate an increased risk of seizures in these infants [2]. Also, these children are at high risk for adrenal crisis, that can occur at 1–6 weeks after birth, with azotemia, vascular collapse, shock, and finally death [6]. Affected males are more prone for a salt-wasting adrenal crisis at home because their normal male genitalia do not raise the suspicion for this condition, being discharged from the hospital without diagnosis [4, 6]. The episodes of salt loss in the infant period are similar in symptoms to pyloric stenosis or gastroenteritis with poor feeding, weight loss, failure to thrive, vomiting, dehydration [6]. The differentiation from the latter is achieved by the fact that children with CAH maintain diuresis.

#### *2.5.2 Classic simple virilizing 21-OHD CAH*

This second variant of classic CAH represents about 25 of the cases and is characterized by a reduced enzymatic activity of only 1–5%, which generates a clinical picture in which the exposure to excess androgens predominates. These children do not have episodes of salt loss due to the relatively normal production of aldosterone, but they do associate cortisol deficiency. In utero exposure of females to adrenal androgen excess results in external genital virilization at birth with varying degrees of clitoris enlargement, fusion of the labioscrotal folds, and formation of a urogenital sinus [6]. Excess androgens are imperceptible at birth in males. It should be noted that it is difficult to make a difference between SV and SW form based on the degree of virilization of an affected female at birth [6]. This is why all infants detected with classic CAH will be treated with glucocorticoid and mineralocorticoid treatment at least within the first year of life, because infants have a relative renal tubular resistance to the salt-retaining effects of aldosterone and a low sodium content diet [1].

#### *Congenital Adrenal Hyperplasia DOI: http://dx.doi.org/10.5772/intechopen.106520*

Postnatal, both females and male's infants with SV CAH develop signs of androgen excess including precocious development of pubic and axillary hair, acne, seborrhea, comedones, acceleration of linear growth, advanced bone maturation leading to short final stature, with a consecutive catecholamine deficient secretion [6]. The pubertal development of untreated children consists:


Persistence of glucocorticoid deficiency in adulthood may lead to impaired vascular tone and cardiac function, as a result of impaired development and function of adrenal medulla [6].

#### *2.5.3 Non-classic 21-OHD CAH*

As mentioned above, the genetic defect associated with NC form of CAH retains an enzymatic activity of 20–50% for CYP21A2. Thus, cortisol production is sufficient for a normal lifestyle, but insufficient to adequately suppress ACTH synthesis, and so the excessive androgen secretion is maintained. External genitalia are normal at birth, as are 17-hydroxyprogesterone levels, which is why these cases are not detected in neonatal screening [2, 7]. This form is usually diagnosed later during childhood or adolescence or even in adulthood, therefore in the past it was called the "late-onset" form [6, 7].

The clinical features of androgen excess are related to age at diagnosis [7]. In the case of prepubertal children, both sexes show accelerated growth, with advanced skeletal maturation compared to chronological age, premature development of pubic hair with a prevalence up to 30%, described even at the age of 6 months [2, 7]. Girls may have clitoromegaly, and boys may have enlarged penis with prepubertal testicles, similar to classic SV form [6]. In 30–60% of cases the ACTH 1–24 test reveals a slight cortisol deficiency, requiring replacement therapy during periods of stress.

In adolescents and adult women, the signs of hyperandrogenism predominate with treatment-resistant nodulo-cystic acne, seborrhea, androgenic alopecia, hirsutism, risk of infertility, increased rate of miscarriages [2, 6, 7]. Clinically they may associate a profile like polycystic ovary syndrome (PCOS) [7]. Up to 30% of women with PCOS have undiagnosed NCCAH [6].

Affected men are often asymptomatic. In their case, the diagnosis is made either from an affected female member or in the context of evaluation for peripubertal gynecomastia, short stature, infertility, oligospermia, or adrenal incidentalomas [2]. Up to 3% of adult men with bilateral adrenal events may have undiagnosed NCCAH [6].

#### **2.6 Diagnosis**

Diagnosis of CAH must be suspected in infants born with ambiguous genitalia [2]. CAH is a life-threatening disorder for this reason it is important to establish the diagnosis as early as possible.

#### *2.6.1 Hormonal evaluation*

Diagnosis of 21-OHD CAH can be confirmed biochemically by identifying high values of 17-OHP in a blood sample. Such hormonal evaluation is the basis of the

newborn-screening program developed to identify affected patients with classical form of CAH [2]. This neonatal program screening is currently being used in more than 30 countries around the world to prevent morbidity and mortality from adrenal crisis [1, 4]. The screening procedure implies collection of blood samples by heel puncture at more than 24 h after birth, but not later than 1 week [8]. Clinical guidelines recommend as first-tier screens the use of immunoassays to measure 17OHP in dried blood spots, on the same filter paper cards used for other newborn screening tests [4].

A value of 17OHP on the 3rd day of life less than 30 nmol/L or 10 ng/mL is considered normal. However, there are no universally accepted standards for stratifying infants, most laboratories use a series of birth weight-adjusted cut-offs [4]. It should be noted that neonatal screening:


For infants with positive newborn screening for CAH the guidelines recommend referral to pediatric endocrinologists and evaluation by cosyntropin stimulation test [4].

When CAH is suspected later in childhood or in an adult, diagnosis confirmation is based on an early morning 17OHP [7]. After the newborn period, a morning 17OHP value below 2.5 nmol/L (or 0.8 ng/mL) in children, respectively below 6 nmol/L (or 2 ng/mL) in adults excludes the diagnosis of CAH (**Table 1**) [7]. It should be noted


#### **Table 1.**

*The basal and ACTH stimulated 17OHP levels.*

that 17OHP has a pulsating secretion so, the values obtained mid-morning or in the afternoon may be normal. For this reason, it is recommended that blood be collected in the morning, 7.30–8.30 AM. In addition, in women it should be measured in the follicular phase of the menstrual cycle or in amenorrhea [7]. It should be noted that a simple determination of 17OHP may be accompanied by a false negative result, estimated at 2–11%, especially in adults with NC form [7].

The same as in the case of classic form, improving screening for NC CAH form can be achieved through 17OHP measurement by LC-MS/MS. This method provides increased analytical specificity, the ability for multiplex analysis (simultaneously measure multiple analytes like androstenedione, testosterone, 21-deoxycortisol), and the advantage of using minute specimen volumes [4].

#### *2.6.2 Additional tests*

If the clinical suspicion remains despite the use of LC-MS/MS, the next diagnostic step is the ACTH stimulation test, which is the golden standard for the diagnosis [2, 6, 7]. The ACTH stimulation test is performed in the morning, 8.00–9.00 AM. It involves administration of an intravenous injection of 250 μg of synthetic ACTH (Cosyntropinum, Cortrosyn), or 36 μg/kg in children under 1 year, with measuring the levels of 17OHP, androstenedione and cortisol at baseline and 60 min after the injection (**Table 1**).

Given the fact that in girls and women with non-classical form of CAH, the features of hyperandrogenism predominate in the clinical picture, it is important to be able to differentiate the etiology of this virilization syndrome. Thus, we can use the long dexamethasone test, with administration of dexamethasone for 4 days at a dose of:


In CAH, in contrast to PCOS or Cushing syndrome there will be a suppression of androgen values (DHEAS, 17 (OH) P, testosterone) with minimum of 50 (75%) of the basic values, and also for cortisol level.

#### *2.6.3 Molecular genetic testing*

Molecular testing can detect CYP21A2 mutations and should be carried out in equivocal cases to support the diagnosis, for genetic counseling and for better prognostic and treatment guidance [2, 4, 7]. There are commercial kits that detect the most common 10–12 mutations. Testing should be performed in parents to confirm the parental origin of each mutation, to rule out the coexistence of two mutations on the same allele (cis), to determine compound heterozygosity, distinguish hemizygosity from homozygosity in the index case, and estimate the recurrence risk [1]. Once the *CYP21A2* pathogenic variants have been identified in an affected family member, molecular genetic prenatal testing for 21-OHD CAH can be performed [6]. Most often prenatal testing is considered when the parents have a previous child with 21OHD [1].

There are two methods used: chorionic villus biopsy and amniocentesis, implying invasive sampling [1]. By chorionic villus biopsy is obtained fetal DNA at gestational week 9–11. Amniocentesis was the initial method available for prenatal diagnosis [1]. It allows analysis of fetal hormones in amniotic fluid and can be performed at gestational week 12–14 [1]. Both methods imply an increased risk of fetal loss and do not totally allow avoiding prenatal treatment in male fetuses as they cannot be performed earlier than week 9 of gestation [1, 3].

However, noninvasive prenatal diagnostic method has been developed, which prevent prenatal treatment of males and unaffected females [5]. This method is based on PCR amplification of cell-free fetal DNA that can be isolated from maternal plasma as early as week 6–9 [1]. Protocols must include screening for Y chromosomal DNA in maternal blood, by detecting SRY gene [3]. Sequencing of cell-free fetal DNA can ascertain *CYP21A2* mutations, but not suitable in clinical settings [1, 3]. Instead, it can identify single nucleotide polymorphisms flanking CYP21A2 that are specific for the mother, father, and proband, determining the maternal and paternal alleles inherited by the fetus [1].

#### **2.7 Management of 21OHD CAH forms**

Treatment goals [1, 4]:


#### *2.7.1 Prenatal treatment*

The prenatal treatment involves the use of dexamethasone in dose of 20 μg/kg/ day, to maximum 1.5 mg/day given in three doses to pregnant women with a fetus at risk for classic CAH [1]. Dexamethasone has increased half-life, crosses the barrier placenta and is not degraded by placental 11βhydroxysteroid dehydrogenase type 2 (HSD11B2). It can reduce the fetal ACTH and subsequently the androgen levels, with the aim of preventing prenatal virilization of external genitalia in affected girls. The effectiveness of prenatal therapy with dexamethasone depends on when treatment is started, optimum by gestational week 6–7. The benefit is reduced to 15% in case of late initiation, early discontinuation, or reduced compliance with therapy, thus

#### *Congenital Adrenal Hyperplasia DOI: http://dx.doi.org/10.5772/intechopen.106520*

allowing slight virilization of the fetus. The treatment is controversial due to safety concerns related to exposure to dexamethasone in this stage of embryonic development that may impact much more than the hypothalamic-pituitary-adrenal axis [1].

#### *2.7.2 Treatment of classic 21OHD CAH forms*

#### In SW form:


Due to relative mineralocorticoid resistance at this age and secondary to the antimineralocorticoid effects of elevated 17OHP, neonates and young infants require higher fludrocortisone doses [1]. However, the need for fludrocortisone diminishes with time. The monitoring will be done by electrolytes, plasma renin, and blood pressure measurement [2].

• Glucocorticoid replacement with hydrocortisone is the preferred option, because of its shorter half-life that minimizes the adverse side effects [1].

The hydrocortisone tablets are crushed and mixed into food or suspended and given in a dose of 10–15 mg/sqm daily, in 3–4 divided doses [1]. Treatment will be monitored every 3 months in infant younger than 18 months and every 4–6 months in older children, or more frequently after a change in dosing [1, 4]. Serum levels of 17OHP and androstenedione will be monitored. The suggested target for 17OHP level is 2–3 times the upper normal level, but not higher than 10 ng/mL (or 30 nmol/L) when measured in the early morning before medication [1]. It is recommended to avoid the normalization of the 17OHP level because of the risk of glucocorticoid overdosing causing iatrogenic Cushing syndrome [1, 2, 4].

The hydrocortisone dose may need to be dubled in case of minor intercurrences with fever below 39 degrees or tripled in case of fever above 39 degrees. Raising the dose is not recommended for everyday mental and emotional stress, minor illness, or before routine exercise [1, 4].

As growth cartilage closes, teens and later adults may receive slow-release corticosteroids, such as prednisone 5–7.5 mg/day or dexamethasone 0.25–0.5 mg/ day at bedtime. However, in adults hydrocortisone remains the preferred option for glucocorticoid treatment due to its better metabolic, cardiovascular and bone mineral density outcome compare to dexamethasone or prednisone [1, 4].

Adolescents and women with clinical and ultrasound criteria for PCOS may benefit from treatment with oral contraceptives that may be associated, or not with antiandrogens (spironolactone) [4].

In SV form:

Treatment includes hydrocortisone at a dose of 8–15 mg/sqm, doses being increased in conditions of overload, except for intense physical effort [1, 4].

Replacement with mineralocorticoids is done only during infancy to allow the use of minimum doses of glucocorticoids. After this period the child will receive mineralocorticoids only if the renin values are increased [1].

A multidisciplinary team is needed in approaching the individual with genital ambiguity consisting of specialists in pediatric endocrinology, pediatric urology/ surgery, clinical genetics, and psychology is essential for the best diagnosis and management [6].

Genitoplasty surgery in girls involves clitoroplasty, opening of the vaginal introitus and labioplasty [1]. Surgical techniques are intended for erectile tissue removal, keeping the clitoris with sensitive innervation, ensuring a proper vaginal opening, preventing recurrent urinary tract infections [1].

#### *2.7.3 Treatment of non-classic 21OHD CAH form*

Decisions about starting treatment should be individualized and based on clinical symptoms [2]. The guidelines do not recommend routine treatment with glucocorticoid in asymptomatic individuals [1, 4]. The main goal in children with NC CAH is to maintain normal growth and pubertal development. This childrens will be regularly monitored clinically for height, weight, signs of androgen excess, puberty, and bone age advancement [7].

Hydrocortisone treatment will be recommended in case of:


#### *2.7.4 Management of adrenal emergency in CAH*

Adrenal crisis is estimated to be responsible for up to 42% of deaths in patients with CAH [1, 5]. Patients with SW are the most exposed to this risk, but not only them. The most common precipitants of this medical emergency are infectious illnesses, gastrointestinal and upper respiratory tract infections, for all ages [1, 5].

Prevention of adrenal crisis is accomplished through patient education on stress dosing (2–3 times usual doses) of glucocorticoid [1, 5]. But there may be situations when oral stress doses will not prevent the progression to adrenal crisis [1]. Each family should have a glucocorticoid injection kit for emergency use at home if oral medication is not tolerated during episodes of major stress [1, 2, 4]. All family members should be trained for its intramuscular administration, especially for patients living

*Congenital Adrenal Hyperplasia DOI: http://dx.doi.org/10.5772/intechopen.106520*

far from medical facilities. The injectable dose of hydrocortisone in an emergency is 25 mg for infants, 50 mg for children under 40 kg, and 100 mg for children over 40 kg and for adults [2].

Management of adrenal crisis involves giving an immediate bolus of hydrocortisone 50–100 mg/sqm intravenously or intramuscularly, followed by hydrocortisone 50–100 mg/sqm/day as either continuous infusion or divided at every 6 h [2]. Glucocorticoid doses will be adjusted according to clinical status (state of consciousness, pulse, blood pressure), blood glucose level and serum ionogram. Next day, parenteral administration will be reduced by 1/3 of the dose starting on the day the digestive tolerance reappears and, in parallel with the reduction of parenteral therapy, oral therapy is gradually reintroduced, returning to predecompensation doses.

In combination with glucocorticoid therapy, the patient is rehydrated by rapid infusion of intravenous fluids: 1000 mL of 0.9% sodium chloride during the first 60 minutes, or 20 mL/kg up to 60 mL/kg normal saline in children, continuation of therapy will be guided by level of dehydration [1, 2].

If hypoglycemia is present may require dextrose bolus of 0.5–1 g/kg can be given intravenously at 2–3 ml per minute [2].

If the patient has also hyperkalemia, it requires cardiac monitoring for EKG changes and should be treated using insulin with glucose infusion [2].

All patients with CAH should wear medical alert identification tags (Medical Alert bracelet or medallion) indicating the diagnosis of adrenal insufficiency, for prompt therapy initiation in case of emergencies [1, 2, 4].

#### **2.8 Comorbidities asociated with 210HD**

#### *2.8.1 Effect on growth and final adult height*

As mentioned earlier, exposure to excessive androgens determines advanced somatic and epiphyseal development accompanied by premature epiphyseal maturation and closure, resulting in a lower final adult height that expected from parental heights. An early diagnosis with the initiation of appropriate treatment could improve the final adult height. However, it is necessary to use an optimal dose of glucocorticoid, avoiding overdose, which could have a negative impact on growth [2, 5].

#### *2.8.2 Impaired bone mineral density (BMD)*

Is another consequence of long-term use of supraphysiological doses of glucocorticoid, dexamethasone having the most deleterious effects on BMD compared to hydrocortisone or other intermediate-acting glucocorticoids [1, 5]. In terms of bone metabolism, adrenal androgens including DHEAS can increase BMD, mainly cortical bone [1, 7]. Thus, regarding bone mass late diagnosis and or a poor hormonal control may improve BMD by exposure to higher androgen levels [1]. Similar to the general population, these patients may also benefit from adequate vitamin D intake, a highcalcium diet, and physical activity to prevent bone loss.

#### *2.8.3 Tumor risk*

It is estimated that 20–30% of adult patients with CAH have adrenal masses: benign adrenal tumors (29%) or myelolipomas (8.6%). They generally occur in

patients with a poor hormonal control, suggesting a role of ACTH stimulation in pathogenesis [5]. There is no evidence that adrenocortical carcinoma is more prevalent in CAH patients [1].

A common and important complication in individuals male with CAH is development of testicular tumors of adrenal-like tissue or testicular adrenal rest tumors (TARTs) [5]. TARTs are bilateral benign testicular tumors, mostly painless, centrally located in the rete testis and easily identified by ultrasound [1, 5]. The central location of TARTs can compress the seminiferous tubules, leading to irreversible damage to the surrounding testicular tissue with gonadal dysfunction and infertility [5]. According to the guidelines, boys with classic CAH should have a testicular ultrasound upon completion of puberty and regular examination for TARTs every 2–5 years [1, 4]. TARTs are rare in NC form, so routine ultrasound is not recommended in NC CAH males [4].

It should be noted that TARTs tumors may also occur in other adrenal enzyme deficiencies like 11β-hydroxylase and 3β-hydroxysteroid dehydrogenase type 2 deficiencies [1].

Due to its strong adrenal-suppressive effect, dexamethasone is preferred in the treatment of TARTs. Testis sparing surgery may be an option, but usually does not improve gonadal function, so this patients may be recommended cryopreservation of sperm before surgery [1].

#### *2.8.4 Fertility*

Is affected in both men and women with CAH, especially those with a classic form.

In males fertility is impaired due to hyper- or hypogonadotrophic hypogonadism or through TARTs [2, 7].

In women, elevated androgen and 17OHP levels result in menstrual irregularities and anovulatory cycles [7]. Adverse effects of elevated progesterone on the uterine lining, combined with secondary development of PCOS with oligo-amenorrhea, increases the risk of sub-fertility or infertility [5]. In general, there is an association between the severity of the CAH phenotype and the level of gonadal dysfunction and fertility, mostly reported in women with the salt-wasting subtype [1, 5]. A major cause for low child rates in women with CAH is considered to be a lowest interest in motherhood, especially with the SW phenotype [1]. This may be caused by prenatal androgen exposure that influence gender role behavior, associated with a lack of a partner, dissatisfaction with genital appearance, decreased sexual satisfaction and sexual dysfunction as a result of corrective surgery [1].

#### *2.8.5 Cardiovascular and metabolic disease*

Patients with CAH have increased cardiometabolic morbidity [1]. A Swedish population-based study found an increased prevalence of obesity, type 2 diabetes mellitus, obstructive sleep apnoea, hypertension, elevated lipids, atrial fibrillation and venous thromboembolism in CAH individuals compared with control [1, 5]. Regular follow-up with lifestyle interventions to prevent obesity, and screening for diabetes, especially gestational diabetes, and dyslipidemia may improve cardiometabolic outcome [1, 5].

### **3. 11β-hydroxylase deficiency**

CAH owing to 11β-hydroxylase deficiency (11β-OHD) is the second cause of CAH, accounting for 5–8% of all cases [2].

Steroid 11-hydroxylase (CYP11B1, P450c11β) and aldosterone synthase (CYP11B2, P450c11AS, P450aldo) are closely related enzymes, encoded by duplicated genes, and catalyze the final steps in the synthesis of glucocorticoids and mineralocorticoids [1]. CYP11B1 is expressed abundantly in the zona fasciculata, where it converts 11-deoxycortisol to cortisol and deoxycorticosterone (DOC) to corticosterone (**Figure 1**), and also in the zona reticularis, where it initiates the 11-oxo-pathway [1]. CYP11B2 expression is less abundant and confined to the zona glomerulosa where it catalyzes the 11 β-hydroxylase, 18-hydroxylase, and 18-methyloxidase activities which lead to conversion of DOC to aldosterone [1]. This steroid 11-hydroxylase are [1].

11β-OHD occurs as a result of mutations in *CYP11B1*, the gene encoding 11β-hydroxylase, with occurrence of deficient cortisol, and increased DOC and adrenal sex steroids [1]. *CYP11B1* gene is located on the long arm of chromosome 8 (8q24), about 40 kb from the homologous *CYP11B2* gene encoding aldosterone synthase [2]. It consists of 9 exons and 8 introns. Mutations in this gene tend to be grouped into exons 2, 6, 7 and 8. Several types of mutations affecting *CYP11B1* have been described, including missense mutations, splicing, small deletions, small insertions, regulatory deletions, large deletions, or complex rearrangements [9].

The general incidence of this form is estimated to be 1 in 100,000 individuals, and in contrast to 21 hydroxylase deficiency CAH, the disorder is more prevalent in the Middle East and North Africa (1:5000–1:7000 individuals). A mild non-classical form of 11β-OHD CAH has been reported, with an unknown frequency [9].

#### **3.1 Clinical picture**

The prominent clinical features of 11β-OHD are female virilization, similar to classical 21OHD and low renin hypertension [9]. Clinically we will encounter virilization in females, childhood gynecomastia, early isosexual/contrasexual pseudopuberty, accelerated bone maturation with reduced adult final height.

Elevated blood pressure is secondary to overproduction of DOC which causes salt retention and hypertension despite the fact that it is a less potent mineralocorticoid than aldosterone [9]. High blood pressure may or may not be associated with hypokalemic metabolic alkalosis and occurs in approximately 2/3 of patients, usually later in childhood or in adolescence [9]. Although the excess of DOC is incriminated, the etiology of hypertension is not fully elucidated. There are other factors involved considering that the decrease in the level of DOC after the administration of dexamethasone is not always associated with BP normalization. Likewise, clinical signs of mineralocorticoid excess and the degree of virilization are not well correlated. Some severely virilized females are normotensive, whereas mildly virilized patients may experience severe hypertension [9].

There have been reported cases of salt loss in neonates with 11β-OHD, incompletely explained pathophysiological, probably due to the natriuretic effect of pregnenolone and progesterone [9]. These episodes could be precipitated by the initiation of glucocorticoid therapy due to a sudden decrease in DOC or by conditions of infectious intercurrences.

Along with the classic form of 11β-OHD there has been described a rare form of non-classical 11β-OH. It presents with excess androgens but without hypertension and has been diagnosed in normotensive children with mild virilization or precocious pubarche and in adults with signs of hyperandrogenemia [9].

#### **3.2 Diagnosis**

It is established based on the hormonal profile, that is characterized by:


### **4. 3-β hydroxysteroid dehydrogenase deficiency type II**

There are 2 human 3β-hydroxysteroid dehydrogenase (HSD3B) genes: type I and type II [1, 9].

HSD3B1 encodes an isozyme found in the placenta, brain, liver, skin. HSD3B2 encodes an isoenzyme found mainly in the adrenals and gonads [1, 9]. Both of these isozymes can convert delta 5 (Δ5) to delta 4 (Δ4) steroids (**Figure 2**) [1, 9]. Mutations in HSD3B2 cause a rare form of CAH, characterized by elevated ratios of Δ5/Δ4 steroids, notably 17OHPreg/17OHP, that are >8 SD above normal [1]. About 45 mutations in HSD3B2 have been described, of which 37 are missense, nonsense, major deletion, or complex rearrangements.

Classical form of HSD3B2 deficiency causes genital ambiguity in both sexes: genetic females are mildly virilized because some fetal adrenal DHEA is converted to testosterone by HSD3B1, that can act on low concentrations of steroids in the circulation; genetic males synthesize some androgens by peripheral conversion of DHEA, but these are insufficient for complete male genital development [1].

Also, hepatic HSD3B1 permits conversion of some of the elevated 17OHPreg to 17OHP, engendering false positives in newborn screening for 21OHD [1].

Diagnosis of HSD3B2 deficiency is based on identification of elevated serum levels of Δ5 steroids and their urinary metabolites (pregnentriol and 16-pregnenetriol) and by elevated ratios of Δ5/Δ4 steroids.

It is worth mentioning that newborns show a physiological increase in steroid levels Δ5. In this case, the diagnosis requires evaluation by the ACTH stimulation

**Figure 2.** *Enzymatic conversion mediated by HSD3B.* test. Increases of more than 2 standard deviations of 17 OH pregnenolone, DHEA, Δ5 17OHPregnenolone/17 OH progesterone ratio and Δ5 17OHPregnenolone/cortisol ratio are considered diagnostic for HSD3B2 deficiency.

Nonclasic form of HSD3B2 deficiency manifest itself in form of precocious development of pubic and axillary hair at both sexes and virilization signs in females.

#### **5. 17α-hydroxylase/17,20 lyase deficiency**

17α-hydroxylase (CYP17A1, P450c17) is a double-acting enzyme, both 17α-hydroxylase and 17.20-lyase. The 17α-hydroxylase activity mediate conversion of pregnenolone to 17OHPreg and progesterone to 17OHP, and the 17,20-lyase activity convert 17OH-Preg to DHEA, and to a lesser extent 17OHP to androstenedione [1].

CYP17A1 is absent in zona glomerulosa, has only 17α-hydroxylase activity in zona fasciculata, and is fully expressed, with both activities in the zona reticularis [1].

This enzyme is encoded by the *CYP17A1* gene, located on the long arm of chromosome 10 (10q24.3). Mutations in the CYP17A1 gene with 17-hydroxylase deficiency (17OHD) are rare, account for approximately 1% of all CAH cases, more common in Brazil or China [1, 9]. At least 100 mutations affecting CYP17A1 have been described so far, such as missense/nonsense, splicing, small/large deletions, small insertions, but also complex rearrangements. 17OHD affects steroid synthesis in both the adrenals and gonads [9].

Lack of CYP17A1 blocks sex steroid biosynthesis, thus we will encounter undervirilized genitalia, hypertension, absence of pubertal sexualization and gynecomastia in males, respectively normal aspect of the genitals, hypertension, absence of pubertal sexualization (absence of telarche, primary amenorrhea), minimal body hair in females [1, 9].

Hypertension is caused by overproduction of DOC in the zona fasciculata. 17OHD is accompanied by a lack of cortisol synthesis, with low serum levels of potassium, renine and aldosterone, but very high values of corticosterone substitutes for glucocorticoid requirements [1].

Clinically, the suspicion of 17OHD should be considered in hypertensive patients with hypokalemia and low renin and aldosterone levels, in patients with delayed puberty or stopped developing puberty, in children with karyotype 46, XY with inguinal hernia or female external genitalia. It can also be considered in women with unexplained infertility, who may associate a partial deficit with normal sexualization.

Hormone evaluation will identify elevated values of DOC, corticosterone and 18 OH-corticosterone and gonadotropins, associated with low values of 17 OH pregnenolone and 17 OH-progesterone, renin, and aldosterone [9].

Cases of isolated 17.20 lyase deficiency have been reported [1, 9]. In this situation the conversion of pregnenolone to sex steroid precursors with 19 carbon atoms is blocked, without affecting cortisol synthesis. Clinically we will meet external female genitals at birth, regardless of genetic sex.

#### **6. P450 oxidoreductase deficiency**

P450 oxidoreductase (POR) is a flavoprotein that transfers electrons from NADPH to all microsomal cytochrome P450 (CYP) enzymes, including CYP17A1, CYP21A2, CYP19A1 (aromatase, P450aro) [1].

POR mutations will be associated in varying degrees with concomitant decrease in the activity of 17α-hydroxylase/17.20 lyase, 21 α-hydroxylase and aromatase. It is a very rare disease [1]. Splicing, missense, or nonsense mutations, small or large deletions, and small gene insertions affecting the gene located on the long arm of chromosome 7 (7q11.23) cause POR deficiency [9].

Clinical findings are various:


Patients with POR deficiency typically has normal electrolytes and mineralocorticoid function, nearly normal cortisol levels that respond poorly to ACTH stimulation, increased levels of 17OHP, but lower than in the isolated 21OHD, and low levels of sex steroids [1].

#### **7. Congenital lipoid adrenal hyperplasia**

To initiate steroidogenesis, cholesterol from cytoplasmic storage depots is transported to the inner mitochondrial membrane by steroidogenic acute regulatory protein (StAR), a transporter protein. At this level CYP11A1 (P450scc) will converted it to pregnenolone [1].

Mutations in StAR cause a rare form of CAH called congenital lipoid adrenal hyperplasia. In this disorder we have hormone deficiency on all 3 lines: mineralocorticoid, glucocorticoid, and sex steroids. Furthermore, lipoid CAH is typically associated with very large adrenals secondary to the accumulation of cholesterol esters in the adrenal glands [1].

From a clinical point of view, children have normal length and weight at birth; male newborns have a completely female phenotype but lack the mullerian derivatives; adrenal insufficiency is manifest from birth, with severe salt loss and dehydration; in 2/3 of cases, we find skin hyperpigmentation and in 1/4 cases hypoglycemia during infancy [9].

Attenuated forms are also described, in which the salt loss becomes manifest later, after the age of 1 month. Female patients with a minor deficiency may have a normal phenotype in childhood, but with the absence of puberty or spontaneous puberty, but stopped in evolution.

These patients will have electrolyte imbalances with severe hyponatremia and hyperkalemia, very low levels of cortisol, aldosterone, DHEA, but also their precursors with much higher levels of ACTH, plasma renin and gonadotropins, the latter even in middle childhood.

The treatment of this condition is common with the classic forms of CAH with adequate glucocorticoid and mineralocorticoid substitution.

#### **8. Conclusions**

Under the name of congenital adrenal hyperplasia, there is a heterogeneous spectrum of diseases whose phenotype depends on the type and degree of enzyme deficiency. The diagnosis has an important impact on both the patient and his family. Given that 21 hydroxylase deficiency is the most common etiopathogenesis of the disease, it often becomes synonymous with congenital adrenal hyperplasia. However, we must also consider the possibility of the existence of other enzymatic defects. In conclusion, we are talking about a group of complex diseases whose diagnosis and management require a multidisciplinary approach with the formation of a team of experts in neonatology, endocrinology, genetics, surgery, psychology, these patients requiring regular long-term evaluation.

### **Acknowledgements**

The authors would like to thank to their teachers, professor doctor Constantin Dumitrache and professor doctor Mihail Coculescu, from which they inherited the passion for the secrets of endocrinology.

### **Conflict of interest**

The authors declare no conflict of interest.

### **Author details**

Adina Mariana Ghemigian\* and Nicoleta Dumitru C.I. Parhon National Institute of Endocrinology, "Carol Davila" University of Medicine and Pharmacy, Bucharest, Romania

\*Address all correspondence to: adinaghemi@yahoo.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

#### **References**

[1] Claahsen-van der Grinten HL, Speiser PW, Ahmed SF, Arlt W, Auchus RJ, Falhammar H, et al. Congenital adrenal hyperplasia-current insights in pathophysiology, diagnostics, and management. Endocrine Reviews. 2022;**43**(1):91-159. DOI: 10.1210/endrev/ bnab016

[2] Yau M, Gujral J, New MI. Congenital adrenal hyperplasia: Diagnosis and emergency treatment. 2019 Apr 16. In: Feingold KR, Anawalt B, Boyce A, Chrousos G, de Herder WW, Dhatariya K, et al., editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000 [Accessed: May 16, 2022]

[3] Arriba M, Ezquieta B. Molecular diagnosis of steroid 21-hydroxylase deficiency: A practical approach. Frontiers in Endocrinology. 2022;**29**(13):834549. DOI: 10.3389/ fendo.2022.834549

[4] Speiser PW, Arlt W, Auchus RJ, Baskin LS, Conway GS, Merke DP, et al. Congenital adrenal hyperplasia due to steroid 21-hydroxylase deficiency: An endocrine society clinical practice guideline. Journal of Endocrinology and Metabolism. 2018;**103**(11):4043-4088. DOI: 10.1210/jc.2018-01865. [Erratum in: The Journal of Clinical Endocrinology and Metabolism 2019;**104**(1):39-40]

[5] Mallappa A, Merke DP. Management challenges and therapeutic advances in congenital adrenal hyperplasia. Nature Reviews Endocrinology. 2022;**18**(6): 337-352. DOI: 10.1038/s41574-022- 00655-w. Epub 2022 Apr 11

[6] Nimkarn S, Gangishetti PK, Yau M, et al. 21-Hydroxylase-deficient congenital adrenal hyperplasia. 2002

Feb 26 [Updated 2016 Feb 4]. In: Adam MP, Mirzaa GM, Pagon RA, et al., editors. GeneReviews® [Internet]. Seattle (WA): University of Washington, Seattle; 1993-2022. Available from: https://www.ncbi.nlm.nih.gov/books/ NBK1171/. [Accessed: June 02, 2022]

[7] Nordenström A, Falhammar H. Management of endocrine disease: Diagnosis and management of the patient with non-classic CAH due to 21-hydroxylase deficiency. European Journal of Endocrinology. 2019;**180**(3):R127-R145. DOI: 10.1530/ EJE-18-0712

[8] van der Kamp HJ, Wit JM. Neonatal screening for congenital adrenal hyperplasia. European Journal of Endocrinology. 2004;**151**(Suppl. 3): U71-U75. DOI: 10.1530/eje.0.151u071

[9] New M, Yau M, Lekarev O, et al. Congenital adrenal hyperplasia. [Updated 2017 Mar 15]. In: Feingold KR, Anawalt B, Boyce A, et al., editors. Endotext [Internet]. South Dartmouth (MA): MDText.com, Inc.; 2000. Available from: https://www.ncbi.nlm. nih.gov/books/NBK278953/. [Accessed: June 03, 2022]

#### **Chapter 6**

## Congenital Adrenal Hyperplasia – The Main Effect of 21-Hydroxylase Deficiency

*Berta Carvalho, Filipa Carvalho and Davide Carvalho*

#### **Abstract**

Congenital adrenal hyperplasia (CAH) consists of a group of autosomal recessive disorders resulting from enzymatic defects in steroidogenesis. More than 95% of CAH cases result from a deficiency of the 21-hydroxylase enzyme, which leads to cortisol deficiency, with or without aldosterone insufficiency, and also an excess of androgen. The clinical spectrum varies from milder symptoms to severe cases settled by the functional impairment of the corresponding pathogenic variant in the *CYP21A2* gene. The two major forms of CAH caused by 21-hydroxylase deficiency are the classical form and the non-classic, or late onset form. There are two subtypes of the classic form: salt wasting and simple virilized. Diagnosis is clinically confirmed by 17OH-progesterone measurements, although genotyping is now progressively assuming an essential role for characterising patients. Genotyping is sometimes challenging, due to the existence of the highly homologous *CYP21A1P* pseudogene. The 21-hydroxylase enzyme is encoded by the *CYP21A2* gene, where most of the pathogenic variants defects are due to meiotic recombination phenomena events between the *CYP21A2* and *CYP21A1P*. Complete gene analysis is recommended to obtain a correct diagnosis and a better understanding of the underlying mechanisms of the disease in patients with CAH, and is relevant for prognosis and for prescribing the appropriate type of genetic counselling.

**Keywords:** CAH, cortisol, aldosterone, androgens, 21-hydroxylase, *CYP21A2*

#### **1. Introduction**

Congenital adrenal hyperplasia (CAH) consists of a group of autosomal recessive disorders caused by a defective activity of one of the enzymes involved in the steroidogenic pathway on the zona fasciculata of adrenal cortex, leading to an impairment in cortisol synthesis by the adrenal gland [1–5]. From the several enzymes involved in the adrenal steroids pathway, the 21-hydroxylase (21-OH) enzyme is responsible for the majority of CAH cases [5, 6]. Less frequently, other rare deficits in other enzymes may occur, namely: 11β-hydroxylase (11β-OH), 17α-hydroxylase (17α-OH), 3β-hydroxisteroid dehydrogenase (3β-HSD), and StaR protein cholesterol side chain and cholesterol 20–22-desmolase. 21-OH, which is an enzyme belonging to

the cytochrome P450 enzyme group, is responsible for the conversion of progesterone into deoxycorticosterone and for the conversion of 17-hydroxyprogesterone (17-OHP) into 11-deoxycortisol [7, 8]. The impairment of this enzyme leads to the accumulation of precursor substrates for 21-hydroxylation, particularly 17-hydroxyprogesterone (17-OHP), which in turn causes elevated levels of androstenedione, testosterone, dihydrotestosterone, and other peripherally estrogens [4, 7–9]. Deficits in cortisol or aldosterone and the consequent excess of androgens caused by the accumulation of precursors may be responsible for distinct levels of enzyme activity, leading to different forms of the disease, ranging from severe, moderate, to mild. This monogenic autosomal disorder is transmitted in a recessive pattern, requiring two mutated alleles for the disease to occur [8]. The clinical manifestations are distinct and, in general, correspond to the enzymatic activity of the less-affected allele [6, 10, 11]. Approximately 95% of CAH cases are the result of an impairment of the 21-hydroxylase enzyme, which is one of the several enzymes involved in the androgens pathway. Pathogenic variants of the encoding *CYP21A2* gene may lead to a partial or complete loss of function and an impairment of cortisol and aldosterone synthesis. In turn, the depletion of cortisol concentration may inhibit the negative feedback loop, leading to a compensatory release of the corticotrophin release hormone (CRH) and the adrenocorticotropic hormone (ACTH) by the hypothalamus and the pituitary gland, respectively, and also an adrenal cortex hyperplasia.

The phenotypic spectrum of CAH is widely variable, and the major clinical manifestations of adrenal insufficiency caused by aldosterone and cortisol deficits and androgen excess constitute the main clinical concern, with morbidity and mortality becoming less common. Therapy with hormone replacement constitutes a considerable benefit for patients since early diagnosis, although precise and individualised treatment is required for better outcomes.

Genetic counselling is essential, especially because this disease affects many individuals of reproductive age [1–3], and for the elucidation of the consequences and complications for offspring. Molecular analysis of the gene responsible for 21-OH deficiency is a necessary tool for the confirmation of clinical diagnosis of the affected individual, which can also provide added valuable information for relatives or offspring at risk.

#### **2. Clinical manifestations of CAH due to 21-OH deficiency**

Based on distinct phenotypes, the disease was classified into two forms: the classic form, subdivided into the salt-wasting (SW) and simple virilising (SV) forms, and the non-classical or late-onset form (**Table 1**) [12]. In both forms, decreased cortisol synthesis causes increased secretion of ACTH, which in turn stimulates the adrenal gland to produce cortisol precursors, including androgens and their intermediates (DHEAS, androstenedione and testosterone). In this way, hyperplasia of the adrenal cortex is caused, as mentioned above [13].

#### **2.1 Salt-wasting (SW) form**

The SW form of CAH is the most severe form of this disease, accounting for 75% of cases of the classic form of the disease [13]. This form is characterised by a complete absence of enzymatic activity, with consequent cortisol and aldosterone deficiency [14]. SW form patients present alterations in the body electrolyte balance


#### **Table 1.**

*Clinical characteristics, hormonal levels and genotype–phenotype correlation for the most common pathogenic variants, according to the percentage of enzyme activity.*

due to the deficient production of mineralocorticoids. Appropriate therapy should be administered in a timely manner, as this form can range from severe forms to more discrete cases, with increased plasma renin activity [15]. In all cases, the decreased production of cortisol and aldosterone poses a threat to survival, with acute adrenal insufficiency occurring between the 1st and 3rd weeks of life [16]. Approximately 50% of salt wasting crisis occur between the age of 6 to 14 years old [15]. In the SV forms, external genitalia may also be affected, caused by a deficiency in cortisol and the consequent increase in adrenal androgens [9]. Affected males typically presented severe electrolyte imbalances within the first 2 weeks of life, however, in general, they do not show signs of excess androgen production, with only hyperpigmentation of the genital area occurring (**Table 1**) [17]. The excess of androgens produced during pregnancy in individuals with 21-OH cause the virilization of female foetuses, resulting in pseudohermaphroditism [4]. Prior to the onset of newborn screening programmess, affected females were more rapidly identified, on account of the simultaneous presence of genital ambiguity.

#### **2.2 Simple Virilizing (SV) form**

In the SV form of CAH there is a partial enzymatic deficit, which is manifested by a decrease in cortisol [18]. SV CAH patients present hypocortisolism and simple **Classic Salt-Wasting—severe form (0% enzymatic activity)** Failure to thrive Cortisol deficiency Mineralocorticoid deficiency Hyponatremia Hyperkalemia High Plasma Renin Activity (or renin) Hypovolemic shock Excess androgen production, early in life Virilization of external genitalia in females **Classic Simple Virilizing—intermediate severity form (1–2% enzymatic activity)** Virilization of external genitalia in females Progressive premature pubarche Progressive virilization with clitoromegaly (female) or increased penile size (male) Elevated androgen levels cause accelerated growth velocity and advanced bone age but premature fusion of the epiphyses is also observed causing final short stature. **Non-Classic Adrenal Hyperplasia—mild form (20–50% enzymatic activity)** Between asymptomatic or with signs of androgen excess appearing later in life (acne; hirsutism; menstrual irregularities; anovulation; infertility)

#### **Table 2.**

*Phenotypes of 21-hydroxylase deficiency.*

virilisation. Furthermore, in the perinatal period, newborn female foetuses may present ambiguous genitalia without overt salt loss in affected females (**Table 2**) [19]. Virilization can be classified according to the Prader Stages scale, which ranges from 1 to 5, with Stage 5 being the most severe. Despite these external features, the female internal organs are not affected [4]. Male children may also present signs of early virilization, due to excess androgens, however, the undervaluation of signs of early virilization can delay the diagnosis of this disease in male individuals [4, 16].

#### **2.3 Non-classical (NC) form**

In the NC form of CAH due to 21-OH deficiency, the enzymatic impairment is partial and usually there is no cortisol deficiency [20]. This form is less severe and of late onset and manifests itself by signs of precocious puberty and gynaecological anomalies, which may occur either in late childhood or in early adulthood [14] (**Table 2**). Some affected women have no symptoms at all, and a considerable high number of men remain asymptomatic and only have a genetic diagnosis during segregation family analysis [20].

#### **3. Prevalence in different ethnic populations**

The overall incidence of classical CAH due 21-OH deficiency is 1:10,000–15,000 births, with a carriers' frequency of approximately 1:60–100 [3, 4, 10, 21]. Heterozygosity for 21-OH deficiency in the classical form is estimated to be 1:60 in non-Jewish Caucasians and 1:3 in Ashkenazy Jews [7, 8, 22]. The incidence is much lower in Afro-Americans, ranging from 1:25,000 to 1:42,000 in different studies [10, 11]. The most common form of CAH is the NC form, or the late onset form,

#### *Congenital Adrenal Hyperplasia – The Main Effect of 21-Hydroxylase Deficiency DOI: http://dx.doi.org/10.5772/intechopen.107092*

which affects between 0.1% and 1% of the overall population. The NC-CAH form presents non-specific features, oligomenorrhea, hirsutism and infertility, which causes an ascertainment bias which in turn leads to a diagnosis of a higher number of affected females. Affected males are mostly identified during family screening studies, since androgen excess manifestations are not commonly recognised. NC-CAH patients do not often oversecrete CRH and ACTH, although some patients might demonstrate an increase in glucocorticoids response to ACTH stimulation, reflecting a subtle adrenal hyperplasia [9, 14]. The increase of the secretion of androgens by adrenal glands without an overstimulation of ACTH can be justified by the alteration in kinetic enzymatic activity of CYP21A2 [22]. In addition, an increased level of androgens in NC-CAH may be the result of the peripheral conversion of precursors, or can be due to ovarian hypersecretion, as NC-CAH women frequently have polycystic ovaries.

Among hirsute women, the prevalence of CAH attains between 1% and 10% [17, 22, 23]. A clinical study based on ACTH-stimulated 17-OHP concentrations reported the incidence to be highest among Ashkenazi Jewish populations [24]. In addition, Ashkenazi Jews are reported to have the pathogenic variant p.Val282Leu with a high allelic frequency of approximately 63%, which is mostly associated with non-classic CAH. On the contrary, p.Val282Leu was not detected in Yupikspeaking, Eskimos of Western Alaska, Native Americans, East Indians and Asians. Interestingly, the Yupik Eskimos, represent an isolated geographic population and the splice site intronic variation c.293-13A/C > G in intron 2 is found to be represent a founder effect for this population [25].

Several studies regarding the genotyping of 21-OH deficiency have been presented over recent years. A large study conducted on the French CAH population showed a frequency of the most common pathogenic variants, for the classic form, of 30% of c.293-13A/C > G in intron 2, 25% for large rearrangements, 17% for p.Ile173Asn (I172N) in exon 4, and 7% of p.Gln319\* (Q318X) in exon 8. For the NC form, the same study found a frequency of 55% for p.Val282Leu (V281L), 9% for c.293-13A/C > G, 8% for large rearrangements, 4% for p.Ile173Asn, and 3% for p.Gln319\* [25]. In Brazil, three novel pathogenic variants were described, namely: an insertion of adenine 1003∧1004 insA in exon 4, a transition C > T in codon 408 (p.Arg408Cys), and a transition A > G in intron 2, IVS2 − 2A > G, which it is suggested is due a founder effect, as it had previously been found in the G424S pathogenic variant in the same population [26–28].

In Finland, multiple pathogenic variants have been analysed and found to have an independent founder effect, with each one being associated with a distinct haplotype, where some are identical to other European populations. This is probably due to remote immigration phenomena originating from the Scandinavian or the Baltic countries, as well as other specificities from that population, albeit with a more recent origin.

In Tunisia, the pathogenic variant p.Gln319\* (Q318X) was found to be the most prevalent in that population (35.8%) [29].

A study of the population of Iran showed the contrary, demonstrating that the common deletion of 8 nt in exon 3 is the most frequent pathogenic variant of the *CYP21A2* gene (10%), followed by the c.293-13A/C > G, p.Ile173Asn, and the promotor deletion in the gene. Unlike other populations, p.Arg357Trp (R356W) in exon 8, was not found in Iran [30].

Gene rearrangements, such as the large 3 kb deletion, are frequent among the Anglo-Saxon population (28%). Furthermore, the frameshift pathogenic variant p.Gln319\* in exon 8 of *CYP21A2* gene was found in 16% of East Indians and among Croatians, the missense p.Arg356Trp variant is one of the most frequent pathogenic variants (14%) [29].

The pathogenic variant in exon 7, p.Val282Leu is often found in NC-CAH forms and represented 86% of all alleles studied [31, 32]. *De novo* rearrangements and deletions/conversions of *CYP21A2* gene represent 1% of the affected alleles. Several forms of the chimeric *CYP21A2/CYP21A1P* gene have been described in different populations [33, 34].

#### **4. Differential diagnosis**

#### **4.1 Biochemical**

The biochemical diagnosis of CAH is made using hormone assays. 17-hydroxyprogesterone (17-OHP) is the gold standard screening marker for this disease [16], however other markers can also be used, such as DHEA, cortisol, testosterone, aldosterone, and renin (or plasma renin activity). Individuals with the classic form of CAH present a marked increase in serum 17-OHP, with values greater than 100 ng/ mL, without an increase in 11-deoxycortisol and with a less marked increase in basal dehydroepiandrosterone and testosterone [14]. In patients presenting a SW form, there is also an increase in plasma renin activity, and manifestations such as hyponatremia and hyperkalemia [19]. CAH carriers frequently present normal basal 17-OHP levels and diagnostic strategy involves stimulation with synthetic ACTH with subsequent hormone measurement after 60 minutes. A concentration of 17-OHP greater than 15 ng/mL is indicative of an impairment in 21-OH activity, however many carriers' individuals have only slightly increase hormone concentration after stimulation [7].

#### **4.2 Molecular genetics**

Molecular genetic testing of the *CYP21A2* gene is considered to be essential for the establishment of correlations between genotype and phenotype, as well as the confirmation of clinical and biochemical diagnosis, measuring the status of severity of the patients, distinguishing between severe and milder cases, and, very importantly, making the decision regarding the appropriate genetic counselling for at risk family members and their couples. The molecular diagnosis of CAH is based on the identification of point pathogenic variants and small deletions or insertions, which are mostly transferred from the pseudogene to the active gene [35]. The molecular analysis of the *CYP21A2* gene involves amplification with specific primers just for the *CYP21A2* gene, in order to ensure the targeting of the functional gene, instead of the inactive pseudogene [12, 36]. As *CYP21A2* is constituted by 10 exons and intronic regions of reduced size, it is possible to amplify the entire gene, enabling the possibility to analyse all exons and their intron-exon flanking regions. The most common strategy employed to achieve this specificity is to select a primer that is located at the site with a specific and unique gene sequence [8]. The presence of sequence variations can cause alterations in the sequence reading and interpretation and different forward or reverse primers have to be used to achieve a complete analysis, emphasising the complexity of CAH genomics.

#### *4.2.1 Structure of the CYP21A2 gene*

21-OH CAH deficiency is mainly caused by pathogenic variants in the *CYP21A2* gene, which is the gene that encodes the 21-OH enzyme [21]. *CYP21A2* is located in the Major Histocompatibility Complex (MHC) class III region, on the short arm of chromosome 6 (6p21.3), which is a region that displays a complex organisation of genes with high variability in gene size and copy numbers [37, 38] (**Figure 1**). Approximately 30 kb apart from the *CYP21A2* gene is a pseudogene – *CYP21A1P*. Both the *CYP21A2* gene and the *CYP21A1P* pseudogene share 98% homology between exons and 96% in introns, and both are constituted by 10 exons, spanning 3.1 kb [21, 39, 40].

Throughout evolution, the inactive *CYP21A1P* pseudogene has acquired multiple pathogenic variants, as well as small insertions or deletions and point pathogenic variants that prevent the synthesis of a normal functional protein. The high degree of homology between the gene and the pseudogene enables the occurrence of unequal pairing during meiosis between homologous chromosomes and sister chromatids [41]. Approximately 95% of pathogenic variants in the *CYP21A2* gene result from meiotic recombination events between the gene and the pseudogene [8], with approximately 75% of pathogenic variants resulting from conversion events of large sequences from the pseudogene to the gene or punctual conversion of single alterations (microconversion). Approximately 20% of the pathogenic variants found in this gene are the result of an unequal crossing-over during meiosis, leading to the occurrence of duplications and/or deletions. Large gene conversions and deletions may result in the *CYP21A1P/CYP21A2* chimeric genes, comprising around 20% of the pathogenic variants. The remaining 5% correspond to new pathogenic variants which are not related with recombination events and *de novo* pathogenic variants account for approximately 1–2% of cases [41]. Three other genes are placed adjacent and alternately to *CYP21A2* and *CYP21A1P,* namely the *RP1*, *C4*, *TNXB* genes, as well as two pseudogene – *RP2* and *TNXA* [42]. This configuration is designated by the RCCX module (RP-C4-CYP21-TNX) and it extends to approximately 30 kb, where the orientation from telomere to centromere is: *RP1-C4A-CYP21A1P-TNXA-RP2- C4B-CYP21A2-TNXB* (**Figure 1**) [42]. The *C4B* and *C4A* genes encode for the fourth component of serum complement [41] and the *TNXB* gene encodes for an extracellular matrix protein termed tenascin-X23. In turn, *RP1* encodes a DNA helicase nuclear protein [42]. The RCCX module organisation is usually bimodular, one with the active *CYP21A2* gene, and the other with the inactive pseudogene *CYP21A1P*, which is present in around 69% of the Caucasian population. Monomodular presentation is present in around 17% of the population, whereas trimodular haplotype occurs in almost 14% of cases [33, 42].

**Figure 1.**

*RCCX modules: Bimodular haplotype (upper) and a three modular haplotype with two modules harbouring the CYP21A1P pseudogene and one the CYP21A2 gene.*

*Adrenal Glands – The Current Stage and New Perspectives of Diseases and Treatment*

#### **Figure 2.**

*CYP21A2 and CYP21A1P interconversion events responsible for the conversion of the most common pathogenic variants from the pseudogene to CYP21A2 gene.*

The monomodular organisation is characterised by a deletion of 26 or 32 kb, depending on whether the *C4B* is a short or a long gene, without or with the HERV-K sequence. Several deletions have been described, giving rise to different chimera *CYP21A1P*/*CYP21A2* genes [34]. The most common deletion involves the 3′ end of *CYP21A1P*, the entire *C4B* gene, and the 5′ end of the *CYP21A2* gene, which produces a single non-functional chimeric gene with 5′ and 3′ ends of *CYP21A1P* and *CYP21A2*, respectively (**Figure 2**).

The trimodular haplotype may exhibit two copies of the *CYP21A1P* gene and one copy of the *CYP21A2* gene, or two copies of the *CYP21A2* gene and one copy of the *CYP21A1P* pseudogene, the latter being described in patients who present both the p.Gln319\* pathogenic variant and chimeric *CYP21A1P/CYP21A2* genes together [35, 36, 43]. The existence of different haplotype with simultaneous distinct copy number variation with a large number of sequence variants presents a challenge for the characterisation of *CYP21A2* alleles. Pathogenic variants have been described along the entire gene, in the coding region, as well as in the intron-exon boundaries and beyond, and also in the 5′ and 3′ untranslated region, which demands a careful full gene analysis.

CAH can also be caused by uniparental isodisomy events, although this phenomenon is less frequent [44]. To date, more than 1000 genetic variants have been reported, but only a quarter of them affect human health, with most of these resulting in classic CAH cases [45]. Several sequence variants have been described in the non-translated regions of the gene and even in the promoter sequence, many of them associated with NC-CAH [46]. 153 of the 230 genetic variants were demonstrated to be missense pathogenic variants [45] and these can result in all forms of the disease, whereas nonsense and frameshift pathogenic variants are prone to result in classical forms.

#### *4.2.2 CYP21A2 gene pathogenic variants*

The use of the screening approach for the detection of the ten most common pathogenic variants was the most-used practice among molecular genetic laboratories for several years, providing a molecular confirmation for the majority of cases. The most common pathogenic variants evaluated in classic and non-classic forms were responsible for almost 80% of the cases of CAH caused by 21-OH deficiency:

• **p.Pro31Leu (P30L or Pro30Leu):** This pathogenic variant has an enzyme activity of around 20–60% in cultured cells [45], however this enzyme activity is quickly lost when cells are lysed, pointing to a relative instability of this enzyme. p.Pro31Leu is associated with the mild form of disease and is found in one sixth of NC-alleles, although it can be present with a higher prevalence in specific ethnicities, such as the Japanese [47]. NC-CAH patients who carry this pathogenic variant may present more serious manifestations of androgen excess *Congenital Adrenal Hyperplasia – The Main Effect of 21-Hydroxylase Deficiency DOI: http://dx.doi.org/10.5772/intechopen.107092*

than those patients who carry the most common pathogenic variants associated with NC forms, with p.Val282Leu being associated as having a dominant effect on patients [48].


Many other different pathogenic variants have been described, some of which are from wide families, while many others are private from particular families. Most of the pathogenic variants associated with CAH due to 21-OH deficiency are missense pathogenic variants. Their study requires functional studies to be correlated with a clinical form, although nonsense, frameshift, and rearrangement pathogenic variants are promptly deduced as being severe. Genotype–phenotype correlation data demonstrated that some variants were associated with the severe SW form, some with the SV form, and others with the NC form. It was also observed that some of these pathogenic variants confer different phenotypes, depending on whether they are isolated or are associated with another pathogenic variant, resulting in a synergistic effect [57]. Some sequence variants do not affect the protein production and are considered to be benign variants [57]. Some of these sequence variants are also present in *CYP21A1P* gene and can also be transferred by conversion events, although they do not affect the enzyme activity and have no effect on the clinical phenotype.

#### *4.2.3 Targeted screening of the ten most common pathogenic variants*

For several years, the study of *CYP21A2* gene was based on the evaluation of the ten most frequent pathogenic variants. Several different molecular methods and strategies have been described to cover just a limited number of pathogenic variants. Polymerase chain reaction (PCR), specific primer sequence (SSCP), restriction fragment length polymorphism (RFLP), and direct DNA Sanger sequencing were among the methods available for screening. The initial screening for the most frequent pathogenic variants included the search for large deletions or conversions events involving the promoter region of the *CYP21A2* gene (del/conv of promoter region), as well as pointing to the following pathogenic variants along the *CYP21A2* gene: exon 1, p.Pro31Leu; intron 2, which is a splicing pathogenic variant c.293–13A/C > G; exon 3, which is a 8 bp deletion (p.Gly111Valfs21); exon 4, p.Ile173Asn; exon 6, which is a cluster of three point pathogenic variants (p.Ile237Asn, p.Val238Glu, p.Met240Lys); exon 7, p.Val282Leu, and a thymine insertion (p.Leu307PhefsX5); exon 8, p.Gln319\*, and p.Arg357Trp; and p.Pro454Ser in exon 10 [8].

#### *Congenital Adrenal Hyperplasia – The Main Effect of 21-Hydroxylase Deficiency DOI: http://dx.doi.org/10.5772/intechopen.107092*

The study of the large deletions/conversions involving the promoter region of the *CYP21A2* gene was achieved by a PCR-based experiment, followed by digestion with the *Taq I* restriction enzyme [8]. This method covers a large number of pathogenic variants found in CAH patients who are affected by 21-OH deficiency owing to alterations in *CYP21A2*, as the screening strategy fails to identify other pathogenic variants outside the target regions.

#### *4.2.4 Complete gene analysis by sanger sequencing*

Whole gene sequencing is nowadays the gold standard for genotyping the *CYP21A2* gene. The full gene analysis of *CYP21A2* by Sanger sequencing allows the detection of the above-described pathogenic variants, as well as the identification of new sequence variations. Specific gene amplification by PCR has dramatically improved the sensitivity of the various techniques available to detect *CYP21A2* pathogenic variants. However, the existence of a highly homologous *CYP21A1P* pseudogene which carries most of the pathogenic variants of interest has revealed the major difficulty in finding a strategy for the selective amplification of the *CYP21A2* gene, owing to the lack of specific primers. Indeed, locus-specific mutation primers and PCR conditions were revised and now enable the gene-specific amplification of *CYP21A2.* Gene sequencing usually analyses both the coding regions and the flanking intron-exon regions of the gene. *CYP21A2* whole genomic sequence may be performed by selecting the *CYP21A2* functional gene and amplification by PCR into two partially-overlapping fragments, with 1517 and 2214 base-pairs (bp), avoiding the coamplification of the pseudogene *CYP21A1P* [58]. After the selective amplification of the targeted genes and their subsequent purification, the PCR products are sequenced with internal primers that cover the entire *CYP21A2* gene [59].

#### *4.2.5 Large rearrangements*

The molecular approach for the identification of large rearrangements is distinct from that used for the analysis of sequence pathogenic variants.

A diversity of methods is available for the detection of large rearrangements in the exonic and/or intronic regions of *CYP21A2* gene and also in the promoter and contiguous regions, such as for neighbours' genes (e.g., *C4B* gene). The southern blot technique has been used as a gold standard for many years in the study of large deletions in the *CYP21A2* gene, however this technique has now been substituted by MLPA (Multiplex Ligation-dependent Probe Amplification). The MLPA experimental technique enables the detection of large rearrangements, where several gene sequences can be simultaneously analysed. The detection of known deletions and duplications in the *CYP21A2* gene and in the *CYP21A1P* pseudogene is currently achieved by using this method, which analyses the variations in the copy number of several exons of the gene, and also identifies sequence variations, as well as contiguous genes and control sequences.

This semi-quantitative method is a simple, reliable, and highly sensitive approach for detecting copy number variations in genomic sequences based on the hybridisation of probes to locus-specific targets and the amplification of the effectivelymatched targets. The products of amplification are then separated by capillary electrophoresis and comparison is made between a peak-area pattern obtained from reference and the tested samples enable the determination of which probes/locus have aberrant copy numbers [60, 61].

Another eligible method for detecting copy number variations (CNV) is the quantitative real time PCR, which evaluates the progression of a PCR reaction in real time and simultaneously quantifies the amount of product amplified. This method is based on the detection of the fluorescence produced by a reporter molecule, which increases as the reaction proceeds, and is characterised by a quencher of fluorescence at the opposite end. The proximity of the reporter to the quencher prevents the detection of its fluorescence. During a real-time reaction, in each cycle the probe hybridises and elongates and the reporter that produces fluorescence that is captured is then released which is subsequently measured for each cycle. The increment of fluorescence is proportional to the increase of product, which is quantified to evaluate gene copy number variations through the co-amplification of a control gene [38].

#### **5. CAH genotype: Phenotype correlation**

Several studies have reported high levels of concordance between genotype and phenotype in patients with CAH, however genotype–phenotype correlation is weaker in less severe forms of the disease [42]. As described above, these pathogenic variants are almost entirely correlated with the clinical severity and are well characterised and are associated with distinct clinical forms of the disease. Accordingly, both the classic and non-classical forms of the disease are associated with different genotypes [21]. *In vitro* studies of the expression of the CYP21A2 protein enable the determination of the rate of enzymatic activity that is associated with each pathogenic variant. The majority of CAH patients due to 21-OH deficiency are characterised by compound heterozygotes and there are severe phenotypes of classic forms, which must present two severe pathogenic variants and have no mild pathogenic variants [10, 16, 21]. Alongside there is a genotype of a NC form which has either moderate pathogenic variants in both alleles or one severe mutation and one moderate mutation [38]. The mild pathogenic variant allows for the synthesis of 21-hydroxylase enzyme up to 50% of normal activity, although the severe pathogenic variant does not contribute to any synthesis (**Table 3**).


#### **Table 3.**

*Genotype–phenotype correlation for the most common pathogenic variants, according to the percentage of enzyme activity.*

#### *Congenital Adrenal Hyperplasia – The Main Effect of 21-Hydroxylase Deficiency DOI: http://dx.doi.org/10.5772/intechopen.107092*

Patients with the classic form of CAH usually present severe pathogenic variants in both alleles, presenting nil or less than 5% of 21-OH enzymatic activity [23]. The well-known intronic splice site pathogenic variant in intron 2, which consists of the substitution of an A/C nucleotides by a guanine, constitutes a serious alteration, since it causes the occurrence of alternative splicing and an abnormal protein [21]. Many patients present this pathogenic variant in homozygosity with low levels, or even a complete absence of aldosterone, which is typical of the SV form. In turn, patients with the SV form often present only 1–2% of enzyme normal activity, which, despite being low, is sufficient for the synthesis of aldosterone (**Table 3**) [38].

The clinical spectrum of CAH varies widely between the different forms of the disease, however clinical presentation can be particularly diverse in less severe forms. The phenotype is usually predicted by the less severely-affected allele, although the existence of a more severe pathogenic variant in a second allele, rather an intermediate pathogenic variant, can develop into a more severe phenotype [25]. Compound heterozygous for a mild and a severe pathogenic variant has been reported in NC-CAH patients, showing higher basal and stimulated levels of 17-OHP and hyperandrogenic signs when compared with both mild pathogenic variants [6, 62]. Furthermore, CAH carriers are also characterised by higher than normal 17-OHP levels after ACTH stimulation, albeit not as high as the levels registered in CAH patients.

Although there is a relatively high concordance between genotype and phenotype, there are several examples of variability, particularly for moderately affected patients [63]. Both the pathogenic variants designated as c.293-13A/C > G (IVS2–13) and p.Ile173Asn (I172N) result in variable degrees of 21-hydroxylase activity. Those patients who would generally be expected to be SV cases can sometimes be SW, while others can have a NC-CAH-like phenotype [52, 58]. Another example is the p.Pro31Leu (P30L) in exon 1 of the *CYP21A2* gene, which is usually associated with the NC-CAH form, but is also often present in the SV-CAH phenotypes [63]. The lack of correlation between genotype and phenotype is probably the result of an incomplete gene sequencing, particularly in cases where the whole gene sequencing was not performed. The impairment of 21-OH deficiency is responsible for three major types of the disease, representing a continuum spectrum of clinical severity [22].

Familial segregation studies are recommended for a correct analysis of the parental segregation of the pathogenic variants in order to obtain a better understanding of the disease. Pathogenic variants may be placed in opposite alleles (*trans* configuration) or in the same allele (*cis* configuration). In the case of the latter, the individual is not clinically affected. A possible pitfall of PCR-based strategies is that the interpretation of the diagnosis can be complicated, due to the failure to amplify one haplotype, which results in misdiagnosis. The evaluation of flanking microsatellite markers in all family members can minimise this issue and the establishment of a familial segregation pattern is recommended for individuals with homozygous or hemizygous genotypes (i.e., those who have a pathogenic variant in one chromosome and a deletion in the other one).

#### **6. Prenatal diagnosis and treatment**

The objective of prenatal diagnosis and treatment of CAH is to avoid genital ambiguity in females and prevent precocious puberty in males, as well as to minimise the psychological and physical aspects related with manifestations of an excess of

androgen [39]. Prenatal diagnosis is also of major importance for families at risk of having children with the classic form of CAH, due to the determination of previous carrier status, or due to previous CAH-affected children [39]. Different methods of prenatal detection have been used for several years, such as measurements of 17-OHP in amniotic fluid, or chorionic villus sampling and the molecular genetic analysis of pathogenic variants through DNA that is extracted from chorionic villus cells or amniocytes [27]. These procedures can be informative for fetal gender and carrier/ affected status and they can be carried out late on during the first trimester through to the second trimester by the 10th week of gestation for chorionic villus sampling, or around the 16th–17th week in the cases of amniocentesis [39]. The prenatal treatment of this disease involves the administration of dexamethasone to the pregnant woman, starting from 6th to the 7th week of gestation, in order to minimise the effects of genital virilization in affected female foetuses [64]. Dexamethasone is a very powerful glucocorticoid with weak mineralocorticoid activity and is still considered to be a controversial therapy in the prenatal context, as the effectiveness of this treatment is approximately 75%, and the adverse effects for both pregnant woman and the fetus in the long term are not yet fully known [21, 64]. Postnatal treatment can be administered chronically or acutely during a salt wasting crisis and in both cases high amounts of sodium chloride (NaCl) need to be infused, as well as hydrocortisone in an acute case [15]. A careful assessment of the efficiency and suitability of glucocorticoid therapy is carried out by the monitorisation of the urinary excretion of 17-ketosteroids, pregnanetriol, and also plasma levels of 17-OHP [15].

#### **7. New clues at genotyping**

A promising aspect that can result from genotyping is prevention of CAH. *CYP21A2* genotyping is strongly recommended in couples with a personal or familial history of CAH who are trying to conceive, in order to obtain a genetic diagnosis of clinically-confirmed cases and prescribe suitable genetic counselling. Although there is a strong correlation between genotype and phenotype, occasionally the genotype interpretation is rather difficult to make due to genetic complexity. In the case of a couple where one of the partners has CAH, the risk of giving birth to an offspring who is affected is dependent on the partner's genotype. If a CAH-homozygous patient with p.Val282Leu has a partner who is a carrier of a CAH pathogenic variant, then the risk of having a child with NC-CAH is 50%. The probability of an individual from the general population being a carrier of a severe pathogenic variant is 1:60 (approximately 1.7%) [22, 24] and the probability of a NC-CAH patient being a carrier of a mild pathogenic variant is approximately 60%, which occurs in about two thirds of cases. In such a case, the risk of having a child with a classical form of CAH is expected to be just 1:600 [62]. Nevertheless, it is understood that the real frequency would be higher, due to the higher carrier frequency in particular ethnic groups.

Therefore, the genotyping of both parents should be a mandatory procedure as part of a prenatal study protocol for couples in which one has CAH [65]. Prenatal diagnosis using nucleic acids circulating in maternal blood (cff-DNA: cell free-fetal DNA) enables gender determination during early pregnancy. By being able to identify the *SRY* gene sequences in maternal circulation, male foetuses do not need to continue to be exposed to prenatal treatment with glucocorticoids, contrary to female foetuses, in which the prevention of genital ambiguity is a concern. Besides determining

#### *Congenital Adrenal Hyperplasia – The Main Effect of 21-Hydroxylase Deficiency DOI: http://dx.doi.org/10.5772/intechopen.107092*

gender, an analysis of the *CYP21A2* gene by using the most recent massive sequencing technologies for fetal fraction present in maternal blood should be used, however this procedure is very complex and it is subject to a relatively large rate of false positive and negative results.

Traditional invasive methods still provide more confident results, albeit these are usually only performed rather late during pregnancy, which thus compromises decision making regarding the administration of dexamethasone-suppressive treatment during pregnancy when genetic severe forms are identified [66]. Furthermore, Preimplantation Genetic Testing (PGT) in conjunction with *in vitro* fertilisation (IVF) techniques is already a reality, which is designed to avoid the transmission of various diseases. Embryos are genetically analysed by using molecular genotyping and haplotyping techniques and only genetically transferable embryos (i.e., without the disease) are transferred to the woman's uterus, which accordingly stops the transmission of a disease within a family [67].

#### **8. Conclusions**

As congenital adrenal hyperplasia is a common autosomal recessive disorder, its incidence in a carrier is higher, particularly in specific populations. The typical symptoms are manifested during the neonatal period, childhood, adolescence, and adulthood, and may lead to an ascertainment bias in favour of the identification of affected females. This is particularly the cases in NC-CAH patients, where the use of 17-OHP measurements tests are not so precise and it is essential to carry out molecular diagnostic studies at *CYP21A2* locus in addition. Genotype–phenotype correlations are of major importance for contributing to an integrated analysis, where specific treatment and genetic counselling also needs to be personalised.

#### **Nomenclature**

Nomenclature of *CYP21A2* variants is in accordance with the CYP21A2–002 ensembl transcript, namelly ENST00000418967.6 [NM\_000500 from National Center for Biotechnology Information].

### **Author details**

Berta Carvalho1 \*, Filipa Carvalho1,2 and Davide Carvalho2,3,4

1 Faculty of Medicine of the University of Porto, Genetics Unit, Department of Pathology, Porto, Portugal

2 Instituto de Investigação e Inovação em Saúde (i3S), University of Porto, Porto, Portugal

3 Department of Endocrinology, Diabetes and Metabolism, Universitary Hospital Center of S. João, Portugal

4 Faculty of Medicine of the University of Porto, Department of Medicine, Porto, Portugal

\*Address all correspondence to: bertalc@med.up.pt

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*Congenital Adrenal Hyperplasia – The Main Effect of 21-Hydroxylase Deficiency DOI: http://dx.doi.org/10.5772/intechopen.107092*

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[63] Barbaro M, Soardi FC, Ostberg LJ, Persson B, de Mello MP, Wedell A, et al. In vitro functional studies of rare CYP21A2 mutations and establishment of an activity gradient for nonclassic mutations improve phenotype predictions in congenital adrenal hyperplasia. Clinical Endocrinology. 2015;**82**:37-44. DOI: 10.1111/cen.12526

[64] Dörr HG, Sippell WG. Prenatal dexamethasone treatment in pregnancies at risk for congenital adrenal hyperplasia due to 21-hydroxylase deficiency: Effect on midgestational amniotic fluid steroid levels. The Journal of Clinical Endocrinology and Metabolism. 1993;**76**(1):117-120. DOI: 10.1210/ jcem.76.1.8421074

[65] Moran C, Azziz R, Weintrob N, Witchel SF, Rohmer V, Dewailly D, et al. Reproductive outcome of women with 21-hydroxylase-deficient nonclassic adrenal hyperplasia. The Journal of Clinical Endocrinology and Metabolism. 2006;**91**:3451-3456. DOI: 10.1210/ jc.2006-0062

[66] Witchel SF. Congenital adrenal hyperplasia. Journal of Pediatric and Adolescent Gynecology. 2017;**30**:520-534. DOI: 10.1016/j.jpag.2017.04.001

[67] Simpson JL, Rechitsky S. Preimplantation diagnosis and other modern methods for prenatal diagnosis. The Journal of Steroid Biochemistry and Molecular Biology. 2017;**165**(Pt A):124-130

#### **Chapter 7**

## The Surgical Approach in Adrenal Gland Pathology

*Radu Mirica and Sorin Paun*

#### **Abstract**

The pathology of the adrenal gland and the clinical management of the adrenal clinical entities are particularly complex. The surgical approach to adrenal disorders, both in the classic way and especially in the minimally invasive way, is reserved for highly addressable centers and experienced surgeons. The surgical treatment is dedicated to both functional and nonfunctional adrenal tumors, closely following specific criteria. Regarding adrenal pathology, the surgical treatment is indicated for adrenal tumors that secret mineralocorticoid hormones (Conn syndrome), adrenal tumors secreting glucocorticoids (Cushing syndrome), pheochromocytomas, paragangliomas, neuroblastomas, adrenal carcinomas, and metastases. At the same time, non-secreting tumors should be removed as soon as imaging details are recorded an increasing dimensions of these tumors during a short time interval (up to one year). Although laparoscopic adrenal removal became a gold standard procedure in the late 90s, the classic open surgical adrenalectomy is reserved for bulky adrenal tumors and adrenal cancers, but it is overshadowed by possible multiple complications such as lung damage, wound infections, thrombosis, bleeding, etc. The minimally invasive approach, either laparoscopically or robotically, is dedicated to small tumors, with the advantage of rapid patient recovery, rapid socio-professional reintegration, and reduction of complications. Laparoscopic adrenalectomy is indicated in a wide range of pathologies, ranging from Conn adenoma, Cushing syndrome, and pheochromocytoma, to hormonal inactive tumors or other pathologies. The surgical sparing of the adrenal cortex is advised in cases of hereditary disorders affecting the adrenal gland (such as the MEN2 syndrome) in order to avoid primary adrenal insufficiency after the surgical excision. The postoperative evolution must be closely monitored by the anesthetic-surgical team, and the subsequent follow-up must not be neglected. We will discuss the primary surgical indications and contraindications of adrenal gland pathology in this chapter, as well as the perioperative management of specific tumors, surgical approach types, pluses and minuses of various adrenal surgery procedures, surgical technique and tactics, potential complications, and postoperative management.

**Keywords:** adrenal gland, adrenalectomy, sparing, minimal invasive surgery, surgical approach

#### **1. Introduction**

The pathology of the adrenal gland and the clinical management of the adrenal clinical entities are particularly complex. Surgery for adrenal diseases should only be performed by skilled surgeons in highly addressable centers. The surgical treatment is dedicated to both functional and nonfunctional adrenal tumors.

Regarding adrenal pathology, the surgical treatment is indicated for adrenal tumors that secret mineralocorticoid hormones (Conn syndrome), adrenal tumors secreting glucocorticoids (Cushing syndrome), pheochromocytomas, adrenal metastases, paragangliomas, neuroblastomas, and adrenal carcinomas [1].

In adrenal gland surgery, a multidisciplinary approach involving surgeons, anesthesiologists, endocrinologists, and oncologists plays a key role in patient management. Adrenalectomy is a very frequent surgical intervention with minimal invasive approach in endocrinology surgery [2, 3].

Dr. Lamar Snow performed the first laparoscopic adrenalectomy in 1991, and Dr. Joseph Petelin presented the first description of the procedure in a video at the international symposium of laparoscopic surgery, Saskatoon, Saskatchewan, Canada in August 1992 [4]. In literature, Go H. performed his first laparoscopic adrenalectomy on January 17, 1992, and published it in 1993, after Gagner published his first laparoscopic adrenalectomy in March 1992. Laparoscopic adrenal surgery advanced significantly when Michel Gagner published the lateral trans peritoneal procedure in 1992 [2, 5]. This procedure went on to become the most popular laparoscopic adrenalectomy procedure. The video-assisted approach recognizes three forms, depending on the patient's location and access: first, anterior (trans-peritoneal), second lateral (transperitoneal or retroperitoneal), and third posterior (transperitoneal or retroperitoneal) (retroperitoneal). Soon after, laparoscopic adrenalectomy (after the cholecystectomy) was recognized as the second-best surgical treatment [1].

Transabdominal, transthoracic, and retroperitoneal surgery are the most common approaches to adrenal pathology. To provide a fair control of vascular pedicles' maneuvers therapy, these comprise huge incisions and thorough plan dissections. Postoperative morbidity may vary depending on the surgical technique used to remove the adrenal gland. The development of minimally invasive surgery (MIS) techniques has resulted in significant changes to the majority of surgical operations. The procedure of adrenalectomy is a good illustration of this. The advantages of laparoscopy are completely utilized in this form of approach [2–4].

#### **1.1 Anatomy landmarks**

The kidneys and the adrenal glands are in close proximity. The right adrenal is predominantly suprarenal, while the left adrenal is mostly prerenal and medial to the left upper pole of the kidney. An adrenal gland, which is crescent-shaped on the left and triangular on the right, sits above the superior medial pole of each kidney. While the right adrenal gland is frequently supra-renally positioned, the left adrenal gland is typically pre-renally positioned. On the left, between the 11th and 12th ribs, and on the right, between the 12th rib, the adrenal glands are situated in front of the ribs in both regions [2]. The adrenal glands are encased in a capsule that creates septa that convey capillaries into the gland's core [4]. The renal fascia surrounds the adrenal glands, which are surrounded by a significant amount of renal fat (Gerota fascia). The renal fascia's ventral and dorsal layers stretch upward, covering the adrenal glands and attaching to the diaphragm. The renal fascia, which attaches to the diaphragm,

#### *The Surgical Approach in Adrenal Gland Pathology DOI: http://dx.doi.org/10.5772/intechopen.106522*

as well as big renal veins and arteries, keep them in place. Despite their mobility, the adrenal glands are anchored to the abdominal wall due to their attachment to the diaphragm. That is why hemostasis is challenging because they move in concert with the diaphragm during breathing [5].

For radiological anatomy and also for adrenalectomy, topographic anatomy is crucial. The adrenal glands have a close architecture with several significant anatomical structures, in addition to their intimate association with the kidney. The anterior, posterior, and medial edges of the adrenals are inspected topographically [5].

The renal fat and renal fascia are in contact with the superior portion of the dorsal abdominal wall, which includes the adrenal glands. The 11th and 12th ribs, subcostals muscles, latissimus dorsi muscles, and sacrospinalis muscles are all located adjacent to each adrenal gland, as well as the diaphragmatic crus and lateral arcuate ligament that divide the adrenal glands from the pleura [6].

The right adrenal gland is located 3 mm behind the inferior vena cava and near the celiac ganglion. The gland is not proximal to the renal arteries because of its suprarenal position. It is separated from the pancreas head and duodenal loop on the inferior side by the vena cava, and by the Winslow foramen on the superior side. In contact with the lateral upper region of the anterior face is the inferior medial bare portion of the liver [6, 7].

The inferolateral portion of the anterior side is covered by the peritoneum, which is situated between the liver, kidney, and hepatic flexure. In either a laparoscopic or open adrenalectomy, the peritoneum is open after the movement of the organs, and the right lobes of the liver and gallbladder are brought up while the colon is taken down [8].

The peritoneum of the omental bursa covers the anterior portion of the left adrenal gland superiorly. The peritoneum does not cover the inferior part of the anterior face, despite being close to the medial border of the spleen, the pancreatic body, and the splenic vein and artery behind it. It is connected to the transverse mesocolon on the pancreatic inferior side. The omental bursa and stomach are separated from the adrenal by the pancreatic body. About 7 mm from the aorta, the medial side of the adrenal gland is situated in front of the celiac truncus. The left adrenal gland is near the left renal vein and renal sinus because of its prerenal location [9].

The retroperitoneal region can be reached by separating the splenic flexure and splenic lateral connections, migrating the left adrenal laterally and separating the splenic lateral connections. During the avascular dissection, the veins in front of the adrenal gland, the pancreatic body, and the spleen are mobilized and dragged to the right side. The pancreatic body can also be shifted by opening the peritoneum at the inferior and superior sides of the organ in the bursa omentalis region, especially when the adrenal glands are small. After releasing the gastrocolic ligament, this step is done [8, 9].

The middle and superior adrenal arteries are derived from the aorta, the renal artery, and the inferior phrenic artery, respectively. The lower adrenal arteries come from the renal artery. In addition to other blood vessels, the intercostal and gonadal vessels may also feed the adrenals. These arteries divide into around 50 arterioles under the glandular capsule, creating a rich plexus that needs to be carefully dissected and managed for homeostasis during the adrenalectomy [7–9].

Each adrenal is normally emptied by a single adrenal vein, as opposed to the arterial supply. Controlling this main vein is crucial for physiopathology, especially in malignancies that release excessive hormones. Normally, the left adrenal vein is longer (30mm) and connects the inferior phrenic vein, highlighting the left renal vein after emptying into it. Small (5mm), the right adrenal vein usually drains into the inferior vena cava. Approximately 5% to 10% of persons have accessory veins. These auxiliary

veins could directly drain into the left hepatic vein, the right renal vein, or the left renal vein on the left [10].

One lymphatic plexus is located deep inside the capsule, whereas the other is located in the medulla. The adrenal gland produces a vast number of lymphatic channels that run alongside bigger vessels. These lymphatic channels terminate at the lateral aortic lymph nodes surrounding the places where the renal vein drains into the vena cava, as well as the para-aortic lymph nodes close to the diaphragmatic cruris and the renal artery exit. The fact that some lymphatic veins can cross the diaphragm and leak into the posterior mediastinum or the ductus toracicus helps to explain how adrenocortical cancers can spread both locally and regionally. The sympathetic visceral nervous system gives rise to the nerves. The sympathetic celiac ganglia supply the visceral afferent fibers. Nerves and vessels are ligated concurrently during adrenalectomy [9, 10].

#### **2. Indications and contraindications**

The majority of adrenal masses are now identified as incidentalomas as a result of the extensive use of ultrasonography and cross-sectional radiological imaging.

Congruent with ESE/ENSAT guideline**s,** adrenalectomy indications are as follows: functional adrenal tumors, adrenocortical carcinoma, pheochromocytoma, and metastatic tumors. Similarly, patients with unilateral adrenal masses that seem benign on imaging or with adrenal masses that have radiological characteristics that are suggestive of malignancy and have a diameter of less than 6 cm but no imaging signs of local invasion should consider laparoscopic adrenalectomy. For unilateral adrenal tumors with radiological features indicative of cancer and imaging evidence of local invasion, open adrenalectomy is typically performed. The guidelines also suggest surgical removal if the lesion grows by more than 20% over the course of six to twelve months of monitoring of adrenal incidentalomas, combined with at least a 5-mm rise in maximum diameter. Furthermore, surgical treatment should be taken into consideration in a customized manner for individuals with autonomic cortisol secretion who also have comorbidities that may be connected to cortisol excess [11].

Moreover, several studies have reported the outcome of surgical intervention in the potential reversal of autonomic cortisol secretion. Weight reduction, reduced blood pressure, improved glucose tolerance, decreased cholesterol levels, and favorable effects on bone architecture have all been linked to improved metabolic function following adrenalectomy. Cardiovascular benefits in patients with autonomic cortisol secretion were the subject of a recent systematic analysis with adrenalectomy improving cardiovascular outcome and death [12].

An adrenocortical carcinoma poses the biggest risk to a unilateral adrenal lesion with ambiguous imaging features. Surgery is the most crucial therapeutic intervention for adrenocortical carcinoma without metastases. According to recommendations in the guidelines, only referral institutions and surgeons with experience in open and laparoscopic adrenal surgery and who execute more than 15 benign and malignant adrenalectomies annually should perform surgery on patients with this pathology [13].

Hormone-producing tumors have surgical indications regardless of their size. The following are hormone-secreting adrenal tumors for which adrenalectomy is recommended: Cushing's syndrome is caused by an overproduction of glucocorticoids in the fasciculate adrenal cortex, Conn's syndrome is caused by an overproduction of aldosterone in the glomerulosa adrenal cortex, and Pheochromocytomas are caused

#### *The Surgical Approach in Adrenal Gland Pathology DOI: http://dx.doi.org/10.5772/intechopen.106522*

by an overproduction of catecholamine in the adrenal medulla [11]. Due to pituitary or ectopic ACTH synthesis, adenomas that originate from the reticularis layer of the adrenal cortex and produce excessive levels of androgens and estrogens may infrequently require a bilateral adrenalectomy in Cushing's disease [7].

Nonfunctioning adrenocortical malignancies account for around a half of adrenocortical cancers. Thus, 50% is composed of tumors that secrete 15% cortisol, 10% androgens, 5% estrogens, and only 1% aldosterone, and the remaining 18% of tumors may secrete other hormonal varieties [7]. Malignant pheochromocytomas account for about 12% to 29% of all pheochromocytomas. Lung and breast malignancies, melanoma, renal cell carcinoma, and lymphoma frequently metastasize to the adrenal gland. Adrenalectomy may be required in these patients. The risk of malignancy associated with the size of a nonfunctional adrenal tumor is an indication for surgery. The risk of cancer is roughly 2% if the lesions are less than 4 cm in diameter. Malignancy is 6% more likely in lesions that are 4–6 cm long, and 25% more likely in lesions that are 6 cm long [11]. At the time of diagnosis, 90 percent of adrenocortical tumors have a diameter of more than 4 cm. Most adrenal masses discovered by chance are less than 4 cm in diameter and nonfunctional. Patients who have benign imaging results are not candidates for surgery, although clinical and radiological follow-up is suggested at 6–12 months. Even if that case where the imaging findings are benign, surgical therapy should be considered for each tumor separately since the risk of malignancy increases with the size of the mass [14, 15]. Some disorders, such as asymptomatic myelolipoma or a simple cyst with a diameter of more than 4 cm, may not require surgery [1, 12, 16].

#### **3. Preoperative preparation**

Surgical treatment is an option in the management of adrenal masses, respecting the indications mentioned above. In the case of adrenalectomy, the preoperative preparation is determined by the pathology for which the procedure is conducted. The preoperative preparation for an adrenalectomy for a benign nonsecretory or malignant nonsecretory disease is no different than for any other surgery. The condition in which we are dealing with pheochromocytoma, the patient necessitates specific preparation, as outlined in a previously published chapter.

Patients with pheochromocytoma are treated with alpha-blockers, intravascular volume expansion, and beta-blockers (especially if concerns regarding tachyarrhythmia are present) prior to adrenal surgery. Patients may develop substantial hypotension when the tumor is free of vascularization, particularly after dividing the adrenal vein intraoperatively. This necessitates fluid optimization and inotropic support, which may need to be continued in the immediate postoperative period. In addition, mineralocorticoid receptor antagonists, such as spironolactone or eplerenone, are frequently recommended to patients with adrenal incidentalomas and primary hyperaldosteronism. This specific medication should be stopped after adrenalectomy for primary aldosteronism in order to avoid hyperkalemia. Depending on the patient's postoperative blood pressure, other antihypertensives may be discontinued or lowered. Despite successful surgery, older patients and those with long-standing hypertension are more likely to suffer permanent vascular damage and need antihypertensive medication for a long period of time [14]. Furthermore, current endocrine guidelines support perioperative glucocorticoid medication at major surgical stress dosages for patients with autonomic cortisol secretion as part of preoperative care.

Adrenal surgery can be done either laparoscopically or openly. Transabdominal or retroperitoneal approaches can be used for both techniques. The gland can be reached transabdominal or retroperitoneal in either technique [7]. Laparoscopic surgery can be performed in a traditional or robotic manner. With a single port, both ways can be used [13]. The surgical procedure is influenced by the size and also by the kind of the lesion, the patient's general features, and the surgeon's level of experience. Laparoscopic adrenalectomy is really regarded as the best course of therapy for some people [14]. Laparoscopic transabdominal or retroperitoneoscopic surgery has benefits over open surgery in terms of shorter hospital stays, postoperative discomfort, intraoperative blood loss, total 30-day postoperative sequelae, and mortality. Over the past ten years, mortality following adrenalectomy has decreased, with a fatality rate of < 2% [1].

#### **3.1 Adrenalectomy – surgical technique**

Furthermore, the surgical strategy is determined by the surgeon's preference, expertise, and knowledge with the procedures. Aspects to take into account include the patient's unique pathological history of abdominal or retroperitoneal surgical procedures, body mass index, tumor size, and location. However, the surgical approach may be impacted by these factors [14].

Normal anatomy is included in the laparoscopic method, and if open surgery is required for special circumstances, it may be done easily [15]. Although the retroperitoneal method allows for direct access to the adrenal gland and requires less dissection and mobilization of other visceral organs, general surgeons find it difficult to master because of a lack of experience. The retroperitoneal surgical approach is also contraindicated in patients with a high body mass index and excessive retroperitoneal fat, as well as those with tumors larger than seven to eight centimeters in diameter. In addition, a tumor near the inferior vena cava on the right side or the aorta on the left may require a difficult retroperitoneal excision. In terms of operating times, blood loss volumes, intraoperative hemodynamic events, rates of morbidity and death, and rates of switching from a minimally invasive to an open surgery approach, retrospective reviews of the literature have shown that laparoscopic and robotic excision of pheochromocytomas are equivalent [9]. The advantages of robotic adrenalectomy also include three-dimensional access, increased wrist mobility for the surgeon, and a stable camera port. Robotic adrenal surgery has certain drawbacks, including rising costs, a slow learning curve, and a lack of tactile input [3].

Patients should be positioned supine on the operating table for laparoscopic transabdominal lateral adrenalectomy. A nasogastric tube and Foley catheter are normally put in after general anesthesia is administered. The patient is positioned in a lateral decubitus position with the afflicted side facing up. A raised arm board is employed, and an axillary cushion is used to support the elevated arm. The superior iliac spine should be the breakpoint for the operating table, and the bed should be bent to expand the working area. The table is bent by around 100–120 degrees in reference to the ground plane to widen the space between the iliac crest and the costal edge. The surgeon and assistant stand side by side and face the patient's front while the monitor is placed at the patient's head side [15].

CO2 insufflation is conducted after gaining peritoneal access 2 cm inferior to the right/left costal border in the midclavicular line, using either the blind (Verres Needle) or optical (Hasson) trocar. An open approach to the first trocar site can also cause pneumoperitoneum. CO2 insufflation is usually done with low/medium flow

#### *The Surgical Approach in Adrenal Gland Pathology DOI: http://dx.doi.org/10.5772/intechopen.106522*

rate of inflation until a pressure of 12–14 mmHg [15]. Between the midclavicular line medially and the anterior axillary line laterally, four 10-mm trocars are inserted 2 cm below the costal margin. Additional ports may be placed if necessary [7], often one 10-mm trocar in the upper epigastric area (to be utilized for retraction - liver for the right side or spleen for the left side).

The first port is often accessed by the midclavicular line. Adrenalectomy is frequently performed through four ports on the right and three to four ports on the left. The inferior vena cava image will remain straight and parallel throughout the procedure as long as the midclavicular line trocar is employed as a camera port on the right side of the procedure (30-degree laparoscope). The two medial ports, together with the anterior axillary line port, can all be used as functional ports. The instruments and ports, on the other hand, can be replaced as needed to give optimal exposure. A liver retractor is normally inserted through the port below the xiphoid process, parallel to inferior margin of the liver. It is strongly advisable to use only a 10-mm trocar in order to facilitate the use of different laparoscopic devices (including suction devices, seal and cut devices, and special clamps). More than 5 cm between the ports should be left to allow laparoscopic instruments to move freely [17–19].

A fourth port is typically positioned laterally on the middle axillary line or medially on the midclavicular line, although adrenalectomy can be performed with three ports on the left side, spaced 5 centimeters apart from the anterior axillary line. The abdomen is examined once the surgeon has entered the peritoneal area to look for any disease and injuries caused by the installation of the port, but because the patient is positioned on his lateral side, little information can be gathered (consequently, it is mandatory for a surgeon to investigate the patient in order not to have any intraoperative findings that can lead to a conversion to open surgery) [19, 20].

For the right adrenalectomy, two working ports are used to insert an atraumatic grasper and an L-hook cautery. If mobilization of the visceral surface of the liver is not successfully achieved by simple upward retraction of liver segments V and VI, a retractor is inserted under the liver, the liver is retracted and the right triangular ligament of the liver is first detached. Thus, the inferior vena cava is reached medially after opening the posterior peritoneum with the cautery, starting from the right triangular ligament of the liver and removing 1 cm from the edge of the liver, thus exposing and dissecting the fascia of Gerota. While the surgeon applies inferior lateral counter-traction on the superior pole of the kidney, avoiding any pressure over the adrenal tissue, the assistant must provide appropriate retraction of the liver in a superior medial way so as not to drain it and to avoid the release of any hormone product into the systemic blood circulation [9, 21, 22].

Normally, the upper medial part of the adrenal is dissected first by the surgical team, unless the adrenal tumor does not invade the lateral wall of the inferior vena cava or if the adrenal tumor is located posterior to the vein. During this dissection, we may use a Harmonic® scalpel (Ethicon Endo-Surgery INC- Johnson & Johnson Medical SPA, NJ, USA), LigaSureTM (Medtronic, Minneapolis, MN, USA), or any other medical sealing and cutting device, preferably 10 mm in diameter, so that enough connective tissue can be trapped to be sealed along with the dedicated vessels.

Using a little piece of fabric, the surgeon can gently move the adrenal gland laterally without damaging it. The adrenal vein is exposed by continuing the dissection from the medial side of the adrenal to the lateral side of the vena cava and is usually a short vein, no longer than 10–15 mm. As it approaches the liver, the right adrenal vein is often visible laterally and slightly below the inferior vena cava. Two clamps are used to transect the lateral side of the wall of the vena cava after circumferential dissection

of the adrenal vein. One clip is used to clip the adrenal side, and endoscopic scissors are used to separate it [7, 9, 21]. Sealing and cutting (without clipping) the adrenal vein is also to be performed, if the diameter of this vein is less than 7 mm, according to the technical recommendations of the dedicated laparoscopic instrument.

An auxiliary adrenal vein can be found 2–3 cm above the primary adrenal vein in around 20% of cases and should be dissected, clipped, and divided if present [6]. Alternatively, vascular staples can be used to divide the adrenal vein. The gland's inferior medial side is then dissected. Right adrenal gland's superior medial pole needs special care since there is an artery that has to be ligated or shut off because it supplies the parenchyma. Between it and the upper pole of the kidney is the periadrenal adipose layer of the adrenal gland. It is vital to locate and protect any renal arteries in the higher pole, just like on the left side [13].

The adrenal gland is then pushed up and the dissection is completed on the posterior and lateral sides of the gland as well as superiorly, and during this period of dissection, a particular concern of the surgeon should be not to accidentally injure the adrenal tissue. Otherwise bleeding will occur and it is very difficult to achieve adequate hemostasis with the electrocautery, so it is advisable to use a gauze pad for gentle compression over the adrenal tissue. The gland is removed from the 12-mm port location and placed always in a retrieval bag. If necessary, trocar incisions can be expanded. Incisional hernias should be avoided by closing the port side with a resorbable suture [6, 13, 15]. Many publications propose ligation of the adrenal vein as the first step in adrenalectomy, especially for pheochromocytoma. However, with big adrenal masses, the suitable dissection area for identifying the adrenal vein at the first stage may not be available. As a result, the adrenal vein should be dissected only when it is safe to do so (clearly visualization, good exposure, and no intense traction from the cava wall).

The mobilization of the spleen, which is performed by dissecting the splenic-parietal ligament, is the first stage in the left adrenalectomy surgery unless the division of the left colonic flexure with its proper mobilization is necessary and the first gesture after intraperitoneal video inspection. In the lateral decubitus posture, the splenicparietal ligament is plainly visible. In order to successfully retract the spleen and expose its posterior aspect, it is recommended to dissect the splenic-parietal ligament starting at the inferior and posterior edge of the spleen, leaving a margin of roughly 2 cm of peritoneum. When the splenic-parietal ligament is separated until it reaches the diaphragm, the fundus of the stomach and the left crus of the organ are visible. Thus, the splenic-parietal ligament is completely dissected, allowing the spleen to be fully mobilized [7, 10].

The splenorenal ligament is then dissected, starting from the posterior portion of the spleen and going to the tail of the pancreas, taking care not to damage this parenchyma. Between the tail of the pancreas and the fascia Gerota, a groove is delineated and an avascular plane is dissected [6]. With the help of gravity, the splenopancreatic block is moved medially out of the operative field, exposing the upper pole of the kidney and the adrenal area, which is brownish in color, not as yellow as the pancreas in the perirenal adipose tissue. The pancreatic tail and spleen are retracted if necessary, using a retractor inserted through a port positioned medially or laterally [16].

Starting on the lateral part of the gland, dissect from the upper to lower adrenal poles, remaining close to the posterior muscle plane. Energy devices are used to separate small arteries encountered during this dissection. The left renal vein is present on the inferior border of the operational field, thus care must be taken to avoid damaging it while the dissection is carried out along the inferior border of the adrenal gland at

*The Surgical Approach in Adrenal Gland Pathology DOI: http://dx.doi.org/10.5772/intechopen.106522*

the same time as the posterior plane, up to the level of the quadratus lumbaris muscle. The left adrenal vein is uncovered by continuing the dissection on the anteromedial side of the adrenal [9, 22].

On the medial side of the gland during this dissection, the left inferior diaphragmatic vein is frequently seen. The left adrenal vein should be exposed by following the left inferior diaphragmatic vein until it reaches it. The medial side of the gland is where it descends. Recognized and isolated is the left adrenal vein. It is longer than the right adrenal vein and empties into the left renal vein (at least 20–30 mm). Contrary to the side of the adrenal gland, which can either be snipped and divided with one clip or divided with an energy device, the side of the renal vein can be cut and split with two clips. Another vein that could need to be cut is the left inferior phrenic vein. If this vein's diameter is less than 7 mm, it can be sealed and severed using a sealing-cutting device, much like on the right side [6, 15].

It is typical to have a tissue tail that extends from the left adrenal gland to the hilum of the kidney. Dissecting the gland from the superior pole of the kidney, right adjacent to the kidney capsule, and all peri-adrenal fat in the specimen are required for an oncologically sound resection. At this point, attention should be paid to locating and safeguarding any superior renal arteries. The last adrenal dissection allows for the "hanging procedure." Pancreas injury should always be considered during dissection using a hook, coagulating scissors, or energy devices in order to avoid a postoperative pancreatic fistula. The adrenal is removed from the retrieval bag using a 10–12 mm trocar [13]. Installing a drain in the adrenal lodge is a solution; however, it is not frequently required. Some authors advise installing a drain there [1, 6, 7, 23, 24].

#### **3.2 Laparoscopic transabdominal anterior adrenalectomy**

For adrenalectomy, the anterior laparoscopic route is the least preferable method. The patient is positioned supine, the first trocar is implanted in the umbilicus, and three other trocars are placed in different configurations in the abdominal cavity. However, although it allows different abdominal conditions to be treated at the same time and can be performed bilaterally without changing the patient's posture, the submesocolic technique requires a longer surgical time and more port placements and therefore should be thought about beforehand. It seems to be easier to change the lateral side position of the patient for a bilateral adrenalectomy in the same surgical time [11, 24, 25].

#### **3.3 Posterior retroperitoneal laparoscopic adrenalectomy**

Supine positioning of the patient is used to provide vascular access and, if required, place a Foley catheter. The patient is placed in the jackknife position with the table bent to increase the space between the posterior rib edge and the pelvis. The surgeon is on one side of the adrenal gland, while the assistant is on the side that will not be removed. By creating a 1.5-cm incision, 2 cm inferior and parallel to the twelfth rib, cutting through the subcutaneous and posterior muscle layers, and finally reaching the retroperitoneal area, the surgeon can dissect the Gerota fascia posteriorly with his finger [11, 20, 26].

After that, a 12-mm trocar is reinserted into the area, and CO2 is insufflated at a pressure of 12 to 15 mmHg. A 45 laparoscope is used in place of the 0 laparoscope. On either side of the initial port, two additional 5- or 10-mm trocars are inserted. The adrenal gland and arteries are then located using laparoscopic ultrasonography. The

lateral and inferior portions of the adrenal gland are then dissected after the superior pole. The medial dissection is usually done last, after which the veins are identified and separated, as detailed in the previous section on the "lateral transabdominal approach." Feeling the smooth interior surface of the twelfth rib confirms that you are at the right place [7, 13, 15, 26].

Using the Hasson trocar and a direct laparoscopic view, the retroperitoneal region can be reached. Retroperitoneum can be inserted using a finger or a balloon dissector. A 12-mm trocar is then reinserted into this space and the CO2 is insufflated to 12 to 15 mmHg [7] or 20 to 25 mmHg CO2 [16]. One 5 or 10 mm working port is inserted medially from the lateral side of the para-spinal muscle to the camera port, and one port is introduced laterally from the middle axillary line to the camera port. A camera with a 30o or 45o angle is utilized [13, 15, 26].

The main markers for this procedure are the para-spinous muscle medially, the peritoneal margin laterally, the peri-renal area, which contains the kidney, adrenal gland, peri-renal fat anteriorly, and the ribs posteriorly. A strong insufflation pressure allows for clear visibility while also compressing any minor bleeding arteries. Hypercarbia and crepitus may occur, necessitating short periods of deflation or reduced insufflation pressures [13, 15, 24, 26].

By pressing down on the kidney and mobilizing the gland inferiorly, the superior attachments of the adrenal might elevate the gland superiorly. Next, the detached, free adrenal vein needs to be located. Utilizing energy-based technology, the remaining adrenal linkages are subsequently removed [15, 26].

#### **3.4 Lateral retroperitoneal laparoscopic adrenalectomy**

The patient is positioned the same way as in the lateral transabdominal approach for this type of operation. Similar port placements are used in the posterior retroperitoneal laparoscopic approach as in the posterior retroperitoneal laparoscopic technique. Instead of being on the middle axillary line, the camera port on the anterior side might be placed on the anterior axillary line. The fourth port can potentially be implanted 5–7 cm lower than the anterior port on the anterior axillary line. Important anatomical markers can be used to help with orienting once the pneumo-retroperitoneum has been formed. The fact that the psoas muscle establishes longitudinal alignment and is commonly visible is taken into account by the surgeon. Additionally, the major veins that run parallel to the psoas muscle are visible upon retraction of the kidney anteriorly and upward following medial dissection. Although complete mobilization of the renal hilar arteries is not required following adrenalectomy, the renal artery can be detected by pulse identification. The left adrenal vein needs to be dissected and located via a posterior approach, as opposed to transperitoneal laparoscopy [26, 27].

#### **3.5 Single-incision or single-port laparoscopic adrenalectomy**

The single-port approach of laparoscopic adrenalectomy can be done transabdominally or retroperitoneally. Some investigations compared the traditional laparoscopic method to single-port adrenalectomy, finding no significant changes in patient length of stay or morbidity, as well as a minor benefit in cosmetic and postoperative discomfort. There is a lack of data on this strategy, and more research is needed [11, 17, 18, 22, 23].

#### **3.6 Robot-assisted adrenalectomy**

Although laparoscopic adrenalectomy has fewer drawbacks than open adrenalectomy in terms of blood loss, pain, hospital stay, and cosmesis, it does have certain drawbacks, such as the need for dexterity, two-dimensional vision, and reliance on a camera assistant. By giving surgeons a three-dimensional magnified view, superior ergonomics, camera control, and multi-articulated instruments, robotic adrenalectomy overcomes the constraints of laparoscopic surgery. Robotic adrenalectomy has yet to achieve general acceptance because of its higher cost and lengthier operating periods. Robotic adrenalectomy can be performed successfully using either the transperitoneal or retroperitoneal technique, depending on the patient's body habitus and the surgeon's skill. It has also been reported to be a viable approach for partial adrenalectomy [19, 24].

#### **3.7 Partial adrenalectomy**

In 1983, Irvin et al. [20] suggested cortex-preserving adrenalectomy or partial (or sub-total) adrenalectomy for the treatment of hereditary, bilateral pheochromocytoma in order to retain adrenocortical function and prevent lifetime steroid replacement therapy. The results of partial adrenalectomy utilizing the retroperitoneoscopic laparoscopic approach were published in 1996 [21, 27].

In order to treat minor adrenal masses, partial adrenalectomy is increasingly being used. Therefore, the suggested standard therapy for small benign and hormonally active adrenal tumors may change to minimally invasive partial adrenalectomy [27, 28].

In addition to genetic diseases with a risk of developing numerous adrenal tumors, solitary adrenal masses are now one of the criteria for partial adrenalectomy in bilateral adrenal masses. Particularly in patients with small hormone-active tumors, partial adrenalectomy should be carried out to lower the risk of malignancy. Partial adrenalectomy should not be performed when there are nonfunctional cortical tumors. The surgical reasoning in these cases is determined by the tumor's size and rate of growth at the time of diagnosis, as well as any indication of malignancy. In these patients, a complete adrenalectomy should be necessary [27, 28].

An important consideration in adrenal-sparing surgery is the preservation of the circulatory supply to the adrenal stump. Partial adrenalectomy necessitates the least amount of tissue manipulation and dissection. Studies have shown that patients treated for adrenocortical carcinomas with minimally invasive procedures experience disease recurrence earlier and more frequently due to a greater risk of intraoperative tumor leaking and positive margins. Patients who received laparoscopic resection in Stage 1 and Stage 2 were likewise shown to have worse overall survival rates [24]. In individuals who require lymphatic dissection and adrenalectomy, open surgery should be chosen. For locally advanced malignancies that have invaded surrounding organs or major arteries, the open approach is the best option [9, 13, 24, 26, 27].

Open adrenalectomy is still an option if there are any general or specific contraindications to laparoscopy (a patient cannot undergo laparoscopy if they are unable to undergo pneumoperitoneum, for example), or if there are any specific features of the adrenal tumor that call for a safer procedure, such as the presence of large lesions (12–16 cm in diameter) or the presence of malignant tumor with invasion of nearby structures [27, 28].

The approach used will be determined by the patient's size, location, the likelihood of malignancy, and the surgeon's skill with the various approaches. In addition, the results and morbidity of open adrenalectomy differ depending on the pathophysiology of the disease and the technique used. All peritoneal techniques reduce the risk of postoperative ileus; however, extraperitoneal approaches are associated with significant rates of neuromuscular morbidities, such as persistent discomfort (14%), laxity in the flank muscles (30%), and flank numbness (30%) [2, 29, 30].

#### **4. Postoperative complications**

#### **4.1 Postoperative outcomes**

Untreated pheochromocytoma has a high rate of morbidity, which is difficult to determine. Cardiovascular reasons can be the cause of death for 71 % of patients, including myocardial infarction, hypertensive heart failure, or hemodynamic instability [18].

The goal of favorable postoperative results with cortical-sparing adrenalectomy is to avoid steroid reliance. The benefit of not using steroids must be weighed against the possibility of recurrence over time. The need for a complete adrenalectomy is determined by a genetic or familial propensity. Cortical sparing adrenalectomies are a suitable option for patients with familial illnesses including MEN 2B, VHL, and/or MEN 2A. Patients with MEN 2A or 2B syndromes who underwent cortical sparing adrenalectomy have a 51.8 percent incidence of recurrence at the age of ten. Moreover, 43 percent of patients who underwent the unilateral or bilateral cortex sparing technique became dependent on steroids. Cortical sparing adrenalectomy is another option for people with VHL syndrome [19].

To prevent recurrence or the development of metastatic illness, which can happen up to 40 years after the tumor was removed, patients with various forms of pheochromocytomas should be monitored for the rest of their lives [17].

There are some relative contraindications to laparoscopic surgery. Many patients can undergo laparoscopic surgery, despite the fact that maybe they had abdominal surgery in the past or they have obesity.

#### **4.2 Contraindication of laparoscopic surgery**

In people at high operative risk with cardiopulmonary disease and/or hematological disease, open surgery may be required. Even though previous abdominal surgery and obesity have been cited as relative contraindications for this procedure, many patients in these categories may still benefit from laparoscopic surgery. Therefore, personalized patient management by the surgical team is recommended [24].

#### **4.3 Conversion to open surgery in laparoscopic surgery**

There is a small risk when switching to open surgery from laparoscopic surgery, described in some of the studies as about 5 percent [29]. Laparoscopic adrenalectomy should be switched to classic open surgery if there is a macroscopic appearance suggestive of cancer such as invasion into surrounding structures or the

presence of suggestive regional lymphadenopathy. Also, intraoperative problems, such as uncontrolled bleeding that cannot be controlled laparoscopically, should be resolved by switching to the classic method if the surgeon fears he cannot control the situation [25].

#### **4.4 Complications of right adrenalectomy**

Bowel and vascular injuries, gas embolism, operating difficulties due to adhesions, and obesity are some of the common risks associated with the laparoscopic method. Difficulties may emerge as a result of an incorrect port placement or insufficient mobilization, as well as an incorrect surgical perspective. The right adrenalectomy has a number of particular adverse effects, including damage to the kidneys, liver, duodenum, gallbladder, common bile duct, inferior vena cava, portal vein, right adrenal vein, rupture of the adrenal capsule, and diaphragm injury [1, 6, 13].

#### **4.5 Complications of left adrenalectomy**

During port replacement, intra-abdominal organ damage is possible. When a port is inserted, there is a risk of intraabdominal organ harm. Inappropriate port placement, insufficient mobilization, and an incorrect surgical perspective can all lead to difficulties. Some possible adverse effects of left adrenalectomy include splenic artery and vein injury, spleen and pancreatic injury, and stomach injury. Bleeding may occur from poor plane dissection. It is possible to sustain injuries to the left adrenal or inferior phrenic veins. The left renal vein may be divided by mistake, assuming it to be a broad adrenal vein, especially in big tumors. There may be an injury to the left kidney's upper lateral artery, kidney parenchymal injury, gland capsular rupture, and diaphragm rupture [1, 6, 13].

#### **5. Conclusions**

In the case of adrenal gland surgery, it is critical to remember that a multidisciplinary approach involving surgeons, anesthesiologists, endocrinologists, radiologists, and oncologists plays a key role in patient management and that surgeon experience and hospital experience both have an impact on the procedure's success. Open adrenalectomy, laparoscopic adrenalectomy, robotic-assisted, and singleincision approaches are only a few of the operations on the adrenal glands that have been recorded in the literature. Surgeons should select the technique with which they are most familiar.

When compared to open surgery, several studies have shown that laparoscopic adrenalectomy is safe, effective, reduces hospital stays, operative blood loss, and wound complications, and has equal long-term outcomes; in fact, it is regarded as the gold standard for adrenal disorders that require surgery. Due to the scarcity of these cases in general practice, the learning curve for laparoscopic adrenalectomy may be difficult. For large tumors and morbidly obese patients, robotic adrenalectomy may offer advantages over laparoscopic adrenalectomy.

In the same way, single port adrenalectomy is technically viable and safe in the hands of an experienced surgeon, but it offers little, if any benefit over other typical laparoscopic adrenalectomy techniques.

### **Author details**

Radu Mirica1,2\* and Sorin Paun2,3

1 Emergency Clinical Hospital 'St. John', Bucharest, Romania

2 University of Medicine and Pharmacy "Carol Davila", Bucharest, Romania

3 Bucharest Emergency Clinical Hospital, Romania

\*Address all correspondence to: mirica\_rm@yahoo.com

© 2022 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

*The Surgical Approach in Adrenal Gland Pathology DOI: http://dx.doi.org/10.5772/intechopen.106522*

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#### **Chapter 8**
