Screening for Macroprolactinemia Is Indispensable for Proper Treatment of Hyperprolactinemia Microadenoma

BACKGROUND: Macroprolactin microadenoma is a combination of non-functional microadenomas with macroprolactinemia. This study aimed to explore the signicance of macroprolactinemia screening for the antidiastole of prolactin microadenoma and macroprolactin microadenoma. METHODS: Retrospective analysis of patients with pituitary microadenoma and screening for macroprolactinemia in patients with hyperprolactinemia was conducted. Based on the prolactin content and screening results, patients were divided into non-functional microadenoma, prolactin microadenoma, and macroprolactin microadenoma groups. The existing forms of prolactin in serum samples were analyzed by gel ltration chromatography and a luminescence immunoassay analyzer. The clinical course of patients and treatment were retrospectively reviewed. RESULTS: The results of gel ltration chromatography conrmed that prolactin in patients with macroprolactin microadenoma was mainly in the form of macromolecules, and the small molecular prolactin was within the normal range. Among 84 patients with hyperprolactin microadenomas, 9 (10.7 %) were diagnosed with macroprolactin microadenoma, and 5 (55.6 %) were treated with bromocriptine. The prolactin content (55.9 ~ 81.0 ng/mL) in the macroprolactin microadenoma group was elevated before the screening and signicantly decreased (11.2 ~ 21.2 ng/mL) after screening. The incidence of clinical manifestations was the same as that of the non-functional microadenoma group, but the rate of drug use was higher, which was similar to that of the prolactin microadenoma group. CONCLUSION: Macroprolactinemia screening effectively identify prolactin microadenomas and macroprolactin microadenomas, prevent misdiagnosis and mistreatment of macroprolactin microadenomas. we macroprolactin microadenoma macromolecules, there no small molecule was a microadenoma macroprolactinemia. The of macroprolactin microadenomas we not diagnosis of prolactin microadenomas for patients with clear imaging diagnosis of microadenoma and hyperprolactinemia unless the macroprolactinemia screening is accomplished. The macroprolactinemia screening could prevent non-functional microadenomas from being misdiagnosed as prolactin microadenomas. In addition, several immunoassay analyzers were used to detect the value of prolactin in the chromatographic elution from the sample of macroprolactin microadenomas. These results showed that various commercial prolactin detection reagents, Roche's second-generation prolactin reagent, high cross-reactivity with macroprolactin. Therefore, screening for macroprolactinemia in hyperprolactinemia cases is indispensable and universal, regardless of the detection reagents and instruments used.


Background
Based on clinical manifestations and endocrine hormone levels, pituitary adenomas are classi ed as non-functional adenomas, prolactinomas, growth hormone-secreting adenomas, adrenocorticotropic hormone-secreting adenomas, etc. According to the size of the tumor, pituitary adenomas < 10 mm in diameter are named microadenomas. The two main forms of microadenomas are prolactin microadenoma and non-functional microadenoma; both have similar imaging features, but different hormone (prolactin) levels. Excessive secretion of prolactin in patients with prolactin microadenoma leads to hyperprolactinemia, which often causes symptoms, such as menstrual disorders, amenorrhea, galactorrhea, and erectile dysfunction. Dopamine agonists are the rst choice for treatment [1,2].
Although there is no excessive secretion of prolactin in patients with non-functional microadenoma, they may have the symptoms mentioned above caused by compression of the pituitary [3]. If so, follow-up monitoring of prolactin may provide critical information for treatment [4,5]. Therefore, emerging hyperprolactinemia is the key to distinguish prolactin microadenoma and non-functional microadenoma.
However, macroprolactinemia, reported in recent years, is a pseudohyperprolactinemia caused by the accumulation of macromolecule prolactin, which is formed in the blood circulation when prolactin is combined with autoantibodies, resulting in an accumulation in the circulation [6,7]. If a non-functional microadenoma is accompanied by macroprolactinemia (hereinafter referred to as macroprolactin microadenoma), it could easily be misdiagnosed as prolactin microadenoma. In this study, we conducted macroprolactinemia screening in patients with hyperprolactinemia microadenoma and evaluated the value of macroprolactinemia screening in the differential diagnosis of macroprolactin microadenoma and prolactin microadenoma, thus avoiding misdiagnosis and mistreatment of macroprolactin microadenoma.

Patients
A total of 108 patients were included in this retrospective study according to the following inclusion criteria: female sex, age ranging from 18 to 50 years, visit to the Zhejiang Provincial People's Hospital between January 2014 and March 2019, diagnosis of pituitary microadenoma (diameter of adenoma < 10 mm) during the rst magnetic resonance dynamic enhancement examination, simultaneously diagnosis of hyperprolactinemia (prolactin > 50 ng/mL) before macroprolactinemia screening (84 patients), and microadenoma with prolactin content within the reference range (prolactin < 25 ng/mL) in non-treated conditions (24 patients). Exclusion criteria included pregnancy, menopause, thyroid disease, growth hormone adenoma, renal adrenal adenoma, and depression [5]. The medical history and related symptoms of the included cases were recorded. This study was approved by the Ethics Committee of Zhejiang Provincial People's Hospital.

Prolactin content assay
The I2000SR chemiluminescence immunoassay analyzer (Abbott Laboratories, Chicago, IL, USA) and its supporting kit (Lot: 91424UI00, Abbott Ireland Diagnostics Division, Lisnamuck, Longford, Ireland) were used to test the prolactin levels in serum samples. For gel ltration chromatography elution, the prolactin level was analyzed using several other luminescence immunoassay analyzers, such as Immulite2000

Macroprolactinemia screening and diagnosis
Polyethylene glycol (PEG) precipitation was used for the screening of macroprolactinemia in hyperprolactinemia samples [8,9]. Brie y, 200 µL of serum samples were mixed with an equal amount of 25% PEG6000 (Sigma, St. Louis, MO, USA), set aside at room temperature for 10 min, centrifuged at 1,800 g for 10 min, and the supernatant was assayed on an I2000SR chemiluminescence immunoassay analyzer. The recovery rate of prolactin was calculated as follows: recovery rate = (prolactin content in the supernatant after PEG precipitation × 2) × 100% / prolactin content before PEG precipitation. A recovery rate of prolactin < 50%, means that the prolactin in serum is mainly macromolecular prolactin, and this is judged as macroprolactinemia. Conversely, if the recovery rate is ≥ 50%, the prolactin in serum is primarily the type of small-molecule prolactin and it is true hyperprolactinemia.

Case grouping
All included cases were divided into three groups according to the results of prolactin testing and macroprolactinemia screening. In total, 75 patients with microadenomas were screened for true hyperprolactinemia and classi ed as prolactin microadenomas, with an average age of 35.0 ± 9.3 years. Nine patients with a mean age of 29.0 ± 9.6 years were screened for macroprolactinemia and classi ed as macroprolactin microadenomas. Twenty-four patients with an average age of 36.3 ± 10.3 years were classi ed as non-functional microadenomas because the concentration of prolactin was within the reference range in the non-treated state. There was no signi cant difference in age distribution among the three groups.

Gel ltration chromatography analysis of prolactin
Serum samples from patients with prolactin microadenomas and macroprolactin microadenomas were separated by gel ltration chromatography (HiLoad 16/600 Superdex 75 pg, GE Life Sciences, USA). The chromatographic elution was collected at 1.0 mL/tube, and the forms of prolactin in the chromatographic solution were analyzed using a luminescence immunoassay analyzer.

Follow-up
The patients diagnosed with macroprolactin microadenomas were followed up by telephone, and their clinical situations or symptoms of menstrual disorders, amenorrhea, galactorrhea, and medication were recorded.

Statistics
The date of prolactin concentration was non-normally distributed and was shown as median (quartile). Statistical analyses were performed using SPSS 17.0. The statistical signi cance between groups was determined by the Mann-Whitney test. The incidence of symptoms, such as galactorrhea, between groups was compared using the chi-square (χ2) test. P < 0.05 was considered statistically signi cant.

Detection of macroprolactin microadenomas
The magnetic resonance images of pituitary microadenomas were indistinguishable between prolactin microadenoma (sample A) and macroprolactin microadenoma (sample B), as shown in Fig. 1. Gel ltration chromatography and an I2000SR luminescence analyzer were combined to determine the form of prolactin in the serum of patients with hyperprolactinemia microadenomas. The results showed that the prominent peak representing small-molecule prolactin was present in the prolactin microadenoma group, whereas the main peak representing macromolecular prolactin was in the macroprolactin microadenoma group (Fig. 2A). The concentrations of macromolecular or small-molecule prolactin were calculated by the area under the curve, which was 58.9 ng/mL and 19.1 ng/mL, respectively. The samples from the macroprolactin microadenoma group were also detected by three other illuminating analyzers (immulite2000, E 602, and Access2), and the detection results were identical to those of I2000SR (Fig. 2B). After gel ltration chromatography screening for macroprolactinemia, 9 of 84 (10.7%) hyperprolactinemia microadenoma patients were diagnosed with macroprolactin microadenomas.
Prolactin concentration is within the normal range in patients with macroprolactin microadenomas.
The concentration of prolactin in the serum was detected before and after macroprolactinemia screening. The results in Fig. 3 show that prolactin content were 14.1 (9.9 ~ 20.6) ng/mL, 60.0 (55.9 ~ 81.0) ng/mL, and 86.1 (65.5 ~ 128.0) ng/mL, in non-functional microadenomas, macroprolactin microadenomas and prolactin microadenomas, respectively, before the screening. There was a statistical difference in these three groups, especially between the two groups of macroprolactin microadenomas and non-functional microadenomas (P < 0.001). After macroprolactinemia screening, the prolactin content in the macroprolactin microadenoma group decreased to 15.0 (11.2 ~ 21.2) ng/mL, which was similar to that of the non-functional microadenoma group, and the difference was not signi cant (P > 0.05).

Follow-up of the clinical information
We retrospectively reviewed the clinical data of the included patients. The details of symptoms and medication of the cases are shown in Table 1. Analytically, the incidence of menstrual disorders, amenorrhea, and galactorrhea were not statistically signi cantly different between the non-functional microadenomas and the macroprolactin microadenomas (P > 0.05). The incidence of menstrual disorders and galactorrhea in the prolactin microadenoma group were 38.7% and 28.0%, respectively, which were higher than those in the non-functional microadenomas (16.7% and 0.0%, respectively). The difference was statistically signi cant (P < 0.05). The usage rate of bromocriptine in macroprolactin microadenomas and prolactin microadenomas was 55.6% and 38.7%, respectively. There was no signi cant difference between the two groups (P > 0.05), but they were both higher than that in nonfunctional microadenoma group (P < 0.01).

Manifestations of patients with macroprolactin microadenomas
The details were reviewed in patients with macroprolactin microadenomas. Before macroprolactinemia screening, 5 out of 9 (55.6%) patients with macroprolactin microadenomas received bromointine treatment for 1-4 years, respectively, with no signi cant difference in tumor size, which was unchanged in 3 patients and even slightly larger in 2. The other 4 patients were not treated with bromocriptine. During the follow-up in March 2019, all 9 patients had no symptoms of galactorrhea, menstrual disorders, or other relevant symptoms.

Discussion
Prolactinoma is characterized by excessive secretion of prolactin from the pituitary gland, accounting for one-half of pituitary adenomas, 80% of which are microadenomas [10]. Non-functional adenomas account for one-third of pituitary adenomas, and the vast majority of them do not cause clinical manifestations due to the lack of excessive hormone secretion and their small size. Non-functional microadenomas often exhibit no clinical symptoms, and their diagnosis needs to entail the evaluation of multiple hormone levels and the making of an exclusion diagnosis [11,5]. For patients with a speci c imaging diagnosis of microadenomas, clinical manifestations combined with laboratory tests, such as growth hormone, insulin-like growth factor 1, adrenocorticotropic hormone, cortisol, and thyroidstimulating hormone, can effectively exclude growth hormone adenoma, adrenal hormone adenoma, and thyroid-stimulating hormone adenoma. The remaining prolactin microadenomas and non-functional microadenomas are distinguished by examining the excessive secretion of prolactin in the pituitary gland. Excluding some physiological or pathological conditions, such as pregnancy, thyroid disorders, and antidepressants, hyperprolactinemia can effectively manifest as excessive secretion of pituitary prolactin [2]. However, macroprolactinemia is a pseudohyperprolactinemia caused by the accumulation of macromolecular prolactin, mainly the complex of prolactin and its autoantibody, in the circulation, which is not an excessive secretion of pituitary prolactin [12,7]. Therefore, neglecting the existence of macroprolactinemia can lead to the misdiagnosis of non-functional microadenomas as prolactin microadenomas and even result in the wrong treatments.
Through gel ltration chromatography and prolactin luminescence detection, we con rmed that prolactin in macroprolactin microadenoma samples was mainly in the form of macromolecules, and there was no accumulation of small molecule prolactin, which con rmed that this was a non-functional microadenoma combined with the presence of macroprolactinemia. The presence of macroprolactin microadenomas indicated that we should not make a diagnosis of prolactin microadenomas for patients with clear imaging diagnosis of microadenoma and hyperprolactinemia unless the macroprolactinemia screening is accomplished. The macroprolactinemia screening could prevent non-functional microadenomas from being misdiagnosed as prolactin microadenomas. In addition, several chemiluminescence immunoassay analyzers were used to detect the value of prolactin in the chromatographic elution from the sample of macroprolactin microadenomas. These results showed that various commercial prolactin detection reagents, including Roche's second-generation prolactin testing reagent, have high cross-reactivity with macroprolactin. Therefore, screening for macroprolactinemia in hyperprolactinemia cases is indispensable and universal, regardless of the detection reagents and instruments used.
In this study, we identi ed 9 patients (10.7%) with macroprolactin microadenomas among 84 patients with hyperprolactinemia microadenomas, which is lower than the prevalence rate (15% -46%) of macroprolactinemia in hyperprolactinemia [13,14] and higher than the incidence (3.5%) of macroprolactinemia in prolactinomas [15]. During the follow-up of patients with macroprolactin microadenoma: (1) the 5 patients receiving bromocriptine treatment showed no change in tumor size and had no corresponding clinical symptoms as the 4 patients without bromocriptine treatment, indicating that bromocriptine therapy was not only ineffective, but also unnecessary for patients with macroprolactin microadenomas. (2) The results in Table 1 suggest that there was no statistically signi cant difference in the related symptoms between the two groups of macroprolactin microadenomas and non-functional microadenomas. (3) After screening for macroprolactinemia, the concentration of prolactin in patients with macroprolactin microadenomas was within the normal range and similar to that in patients with non-functional microadenomas. These data fully demonstrate that the nature of macroprolactin microadenoma is actually non-functional microadenoma [5]. Moreover, there are few or no associated symptoms in macroprolactin microadenomas in the absence of biological activity of macroprolactin in the body. Menstrual disorders, in some cases, may be related to other factors besides the pituitary, which is consistent with related reports [16].
Although the macroprolactin microadenomas are actually non-functional microadenomas, the prolactin concentration within them is moderately elevated (50 ~ 150 ng/mL) before macroprolactin screening ( Figure. 3C). Due to the absence of screening for macroprolactinemia, some patients with macroprolactin microadenomas were mistakenly treated with bromocriptine. The rate of drug use reached 55.6%, which was signi cantly higher than that of non-functional microadenomas (0.0%, P < 0.05). However, there was no signi cant difference in the rate of drug use between the macroprolactin microadenomas and prolactin microadenomas (38.7%, P > 0.05).

Conclusion
Macroprolactin microadenomas are non-functional microadenomas accompanied by macroprolactinemia. In addition, there is a high proportion of macroprolactin microadenomas in patients with hyperprolactinemia microadenomas. Without screening for macroprolactinemia, more patients with macroprolactin microadenomas could be misdiagnosed as prolactin microadenomas, and receive unnecessary treatment.

Declarations
Ethics approval and consent to participate This study was approved by the Ethics Committee of Zhejiang Provincial People's Hospital. Informed consent was not required owing to the retrospective design. Consent for publication Table  Table 1 The incidence of symptoms and drug use rates in patients with non-functional microadenomas, macroprolactin microadenomas and prolactin microadenomas