The reliability of existing diagnostic methods for thyroid neoplasms remains questionable, which necessitates the search for alternative approaches. The use of nontarget proteomic analysis for diagnosing oncological diseases is gaining traction and represents an efficient method for multiplex analysis. This study analyzed 372 blood plasma samples collected from patients with histologically confirmed thyroid pathologies treated at the National Medical Research Center for Endocrinology in 2019-2021. The samples were obtained prior to surgical intervention. Sample preparation involved the reduction and alkylation of disulfide bonds, followed by proteolysis and purification using specialized cartridges. Proteomic analysis was performed using nanoflow HPLC coupled with high-resolution mass spectrometry in a data-dependent acquisition mode. Peptides were identified using FragPipe software, and their suitability for biomarker discovery was assessed. From this analysis, 60 candidate proteins for thyroid disease biomarkers were identified, and sequences of 2930 peptides were evaluated. Further evaluation of nine candidate proteins revealed 31 peptides with high suitability scores for quantification. These peptides can be consolidated into a single biomarker panel that will be further developed for the risk stratification of patients with thyroid diseases.

BACKGROUND:Insulinoma is a neuroendocrine tumor, the main manifestation of which is hypoglycemia. However, the symptoms of hypoglycemia can be non-specific for a long time, especially outside provocative conditions, and quite often the tumor manifests from a life-threatening condition - hypoglycemic coma. In this regard, timely laboratory diagnosis of insulinoma and determination of its aggressive course is one of the priorities in modern researches. AIM:Search for new immunohistochemical (IHC) and circulating markers (CM) of insulinoma, including its aggressive course. MATERIALS AND METHODS:The patients examined at the Endocrinology Research Centre in the period 2017-2022 and operated on for an insulin-producing tumor were included. Before surgery and 2-12 months after it, blood sampling was performed with the determination of targeted marker proteins. Some patients underwent an extended IHC examination of the tumor, surrounding tissue and islets of Langerhans with primary antibodies to target marker proteins with an assessment of the degree of their expression. To determine the aggressive course of the tumor, the degree of malignancy (Grade), the number of tumors and signs of recurrence were characterized. RESULTS:Based on the analysis of literature and pathogenetic characteristics of insulinoma, the following candidates for targeted marker proteins were selected: cocaine and amphetamine-regulated transcript (CART), chromogranin B (CrB), neuroendocrine secretory protein 55 (NESP55), glucagon-like peptide 1 (GLP1), arylalkylamine-N-acetyltransferase (AA-NAT), melatonin, and, exclusively for IHC research, protein D52 (TPD52), as well as receptors for glucagon-like peptide-1 (rGLP1) and melatonin (MTNR1b). 41 patients were included in the study, of which 10 patients underwent an extended IHC study. In patients with both aggressive and non-aggressive insulinoma after surgical treatment, CM levels did not change significantly and in individual patients they could both increase and decrease, including those patients with the expression of the corresponding marker in tumor tissue. It was shown that CART was expressed only in the tumor (in 4/10 of cases), while MTNR1b and rGLP1 were expressed in the tumor (in 6/10 and 10/10, respectively) and the islets of Langerhans (in 5/9 and 9/9, respectively). The association of marker expression with the aggressiveness of the course of insulinoma has not been revealed. CONCLUSION:The markers CART, MTNR1b and rGLP1 are of primary interest for further study in a larger sample of patients with insulinoma. Other markers (TPD52, XgB, NESP55, melatonin, AA-NAT) have not been shown to be associated with an insulin-producing tumor, therefore they are not promising for future researches. At the same time, it is necessary to continue research aimed at finding new both circulating and IHC markers in order to early diagnose the manifestation of the disease and its recurrence, and more accurately determine the malignant and proliferative potential of the tumor.

Hyperandrogenism is the most prevalent cause of menstrual cycle abnormalities and infertility in women. Here, we present a case of a 32-year-old woman with a 7-year history of menstrual irregularity and infertility. Laboratory test results revealed elevated 17-hydroxyprogesterone, progesterone 21-deoxycortisol. Abdominal computed tomography found a 3,9х2,9х2,6 cm left adrenal tumor. Non-classic congenital adrenal hyperplasia was diagnosed initially, however, treatment with supraphysiological doses of dexamethasone proved ineffective and progesterone and 17-hydroxyprogesterone levels remained high. Genetic testing revealed no 21-hydroxylase deficiency. Laparoscopic adrenalectomy was performed with subsequent pathological report being compatible with an adrenal cortical adenoma. 17-hydroxyprogesterone, progesterone 21-deoxycortisol levels returned to the normal range postoperatively and the patient’s menstrual cycle normalized without additional medication. Steroid metabolites producing adenomas are also extremely rare with only a few cases found in patients without previous CAH diagnosis. When a patient with clinically and biochemically diagnosed NCCAH demonstrate no typical features and shows poor response to steroid therapy, the patient should receive multisteroid LC–MS/MS assay for glucocorticoids and androgens, adrenal and ovarian imaging and undergo CYP21A2 gene mutation analysis.

The main treatment option of prolactin-secreting pituitary adenomas is dopamine agonist therapy, which demonstrates prolactin level normalizing and reducing the size of an adenoma in the majority of cases. However, significant amount of patients - about 20% - poorly responds even to high doses of dopamine agonists that is explained by the resistance to therapy. The occurrence of pharmacodynamic characteristics is one of the causes responsible for the development of resistance to typical therapy. Clinical manifestations of persistent hyperprolactinemia are due to following pathological factors: hormonal hypersecretion and the mass-effect of pituitary adenoma. Prevention of irreversible changes is possible only with timely detection of resistance and determination of the optimal personalized treatment algorithm.We report a clinical case of dopamine-agonist resistant microprolactinoma. Patient's health stabilisation, normal level of prolactin and reduction in size of adenoma were achieved due to administration of combined treatment with tamoxifen and dopamine agonists. Hyperprolactinaemia occurring because of prolactin-secreting pituitary adenoma and associated adverse effects are significant problem, decreasing quality of life and demographics in general. This underlines the importance of figuring out causes and identifying predictors of the therapy resistance.The results of the study, illustrated by a clinical example, are presented in the present paper.

Tumors of the thyroid gland are extremely common. The incidence of malignant thyroid neoplasms has increased rapidly in recent decades, although it is unclear whether this is a true increase or the result of widespread use of screening ultrasound. The standard diagnostic procedure for determining the risk of malignancy and indications for surgical treatment of thyroid neoplasms is fine-needle aspiration biopsy followed by cytologic examination of the cellular aspirate. Despite the fact that in the majority of cases it is possible to make a differential diagnosis between thyroid cancer and benign thyroid masses, there is a diagnostic problem with intermediate categories of cytologic findings according to Bethesda, which makes it necessary to search for alternative solutions. This determines the need to expand preoperative diagnostic possibilities. One of the key directions of work on its realization is the study of proteomic data in various thyroid pathologies. The study of the proteome of thyroid tumors opens the possibility of identifying specific protein markers or mechanisms that play a key role in the oncogenesis and metastasis of thyroid tumors, as well as potential targets for new methods of diagnosis and treatment of these diseases. All this determines the relevance and practical importance of studying thyroid pathology at the molecular level, taking into account the potential of proteins as markers.

BACKGROUND: It is important to evaluate the effect of androgen replacement therapy on glycemic control.AIMS: Assessment of the effect of transdermal testosterone therapy on glycemic control in men with hypogonadism and T2DM.MATERIALS AND METHODS: The prospective, comparative study included 300 men aged 55[49;61] years: men receiving both glucose-lowering therapy (GLT) and transdermal testosterone; and patients receiving only GLT. Observation period 1 year. A medical history, a questionnaire for symptoms of androgen deficiency, and measurement of total testosterone and glycated hemoglobin were carried out. Groups were compared using nonparametric methods.RESULTS: Patients receiving androgen replacement therapy in combination with GLT at both 6 and 12 months from the moment of inclusion in the study, they had a statistically significantly higher level of total testosterone and less severity of symptoms of androgen deficiency. When assessing the magnitude of changes in the studied parameters, it was found that patients receiving testosterone replacement therapy (TRT) were characterized by a statistically significantly more pronounced decrease in the level of glycated hemoglobin (average difference 0,3%). In 29 (20,4% (95% ДИ 13,8–27,0)) men who received only GLT, hypogonadism was eliminated. In 3 patients from the TRT group, a pathological increase in the level of total blood PSA was observed, and therefore TRT was discontinued. An increase in hemoglobin above the reference value (>172 g/l) was detected in 8% and 1,3% of men on TRT and without correction of hypogonadism, respectively, p=0,011.CONCLUSIONS: The combination of transdermal TRT and GLT has a positive effect on glycemic control, which is manifested by a decrease in glycated hemoglobin to a greater extent than when using GLT alone. Normalization of testosterone levels leads to a decrease in the symptoms of androgen deficiency, which is accompanied by an improvement in quality of life. Elimination of hypogonadism only with GLT is possible in a small number of cases.

BACKGROUND:Deficiency of 17β-hydroxysteroid dehydrogenase type 3 (HSD17B3) is a rare variant of 46,XY disorders of sex development (DSD). AIM:To give clinical, hormonal and molecular genetic characteristics of cases of 46,XY DSD associated with variants in the HSD17B3 gene. MATERIALS AND METHODS:The study included 310 patients with 46,XY DSD for the period from 2015 to 2019. The patients underwent a comprehensive examination, including a study of the steroid profile by high-performance liquid chromatography with tandem mass spectrometric detection, as well as a molecular genetic analysis using NGS. RESULTS:According to the results of molecular genetic studies, biallelic nucleotide substitutions in the HSD17B3 gene were detected in 13 cases, which accounted for 4.2% of the total number of patients with 46,XY DSD. All 13 patients with biallelic variants in the HSD17B3 gene were registered as females. The ratio of androstenedione/testosterone concentrations in the blood in this group ranged from 1.4 to 8.9. 2 variants in the HSD17B3 gene were found in several patients: c.277+4A>T (on 6 chromosomes) and c.729_735del:p.V243fs (on 9 chromosomes). 4 novel variants have been identified. Monoallelic nucleotide substitutions in the HSD17B3 gene were detected in 7 cases, which accounted for 2.3% of the total number of patients with 46,XY DSD. External genitalia in this group corresponded to Prader stages 3-4. In 1 patient, a pathogenic variant c.277+4A>T was detected in the HSD17B3 gene, in other cases variants with uncertain significance were detected. CONCLUSION:In the structure of 46,XY DSD, patients with biallelic variants in the HSD17B3 gene were identified in 4.2% of cases, with monoallelic variants - in 2.3% of cases. 4 novel variants were found in the HSD17B3 gene.

ЦЕЛЬ: выявить особенности стероидогенеза и артериальной гипертензии при «физиологической» гиперандрогении у мужчин. МАТЕРИАЛЫ И МЕТОДЫ: вплошное одномоментное исследование. Сравнивались группы мужчин с гиперандрогенией, обусловленной повышением общего тестостерона (n=34) и гиперандрогенией, обусловленной повышением дигидротестостерона (ДГТ) (n=66). При определении типа гиперандрогении и распределении пациентов по группам уровни ДГТ и общего тестостерона определялись методом усиленной хемилюминесценции. В группе пациентов с гиперандрогенией, обусловленной повышением общего тестостерона, проводилось сравнение подгрупп мужчин с артериальной гипертензией и без таковой. У всех пациентов оценивались индекс массы тела, величина окружности талии, величины систолического и диастолического артериального давления, оценивался пульс, определялись показатели уровней ЛГ, ГСПГ, эстрадиола, мультистероидного анализа крови (методом тандемной масс-спектрометрии), глюкозы, липидного спектра крови, мочевой кислоты, креатинина, ренина, калия, натрия, хлорида крови. Пациентам с артериальной гипертензией дополнительно проводились суточное мониторирование АД, оценка альбуминурии, электрокардиография, осмотр глазного дна. Базовый пороговый уровень значимости p<0,05. При множественных сравнениях проводился расчет уровня значимости p с применением поправки Бонферрони. РЕЗУЛЬТАТЫ: были выявлены статистически значимые различия в уровнях 17-гидроксипрегненолона, 17-гидроксипрогестерона и андростендиона, которые были выше у мужчин с повышенным уровнем общего тестостерона. Статистически значимых различий в других лабораторных параметрах выявлено не было. Случаев повышения артериального давления в группе мужчин с повышением ДГТ выявлено не было. В группе мужчин с повышением уровня общего тестостерона было выявлено 23,5% мужчин с артериальной гипертензией без поражения органов мишеней, с гиперандрогенией было ассоциировано 17,6% случаев. Для артериальной гипертензии, ассоциированной с гиперандрогенией, был характерен подъем артериального давления в ранние утренние часы. Уровни эстрадиола, оставаясь в пределах нормы, были статистически значимо ниже у пациентов с артериальной гипертензией в сравнении с мужчинами с повышенным тестостероном, но без гипертензии. ВЫВОДЫ: при «физиологической» гиперандрогении, обусловленной повышением уровня ДГТ случаев артериальной гипертензии не наблюдается, в то время как частота ее встречаемости при «физиологической» гиперандрогении, обусловленной повышением общего тестостерона, составила 23,5%. Особенностями стероидогенеза являлась повышенная выработка 17-гидроксипрегненолона, 17-гидроксипрогестерона и андростендиона у мужчин с тестостероновой гиперандрогенией, и сниженная выработка эстрадиола у пациентов с артериальной гипертензией в сравнении с пациентами без таковой при тестостероновой гиперандрогении.

AIM. To reveal the peculiarities of steroidogenesis and arterial hypertension in «physiological» hyperandrogenism in men. MATERIALS AND METHODS . One-stage simultaneous study. The groups of men with hyperandrogenism caused by increased total testosterone (n=34) and those with hyperandrogenism caused by increased dihydrotestosterone (DHT) (n=66) were compared. In determining the type of hyperandrogenism and allocating patients to groups, DHT and total testosterone levels were determined by enhanced chemiluminescence. Subgroups of men with and without arterial hypertension were compared in the group of patients with hyperandrogenism due to an increase in total testosterone. Body mass index, waist circumference, systolic and diastolic blood pressure, pulse, and LH, SBHG, estradiol, blood multisteroid levels by isotope dilution liquid chromatography/tandem mass spectrometry, glucose, blood lipid spectrum, uric acid, creatinine, renin, potassium, sodium, and blood chloride were assessed in all patients. Patients with arterial hypertension additionally underwent daily BP monitoring, albuminuria assessment, electrocardiography, ocular fundus examination. The baseline threshold level of significance was p<0.05. For multiple comparisons, the p significance level was calculated using the Bonferroni correction. RESULTS . Statistically significant differences were found in the levels of 17-hydroxypregnenolone, 17-hydroxyprogesterone, and androstenedione, which were higher in men with elevated levels of total testosterone. No statistically significant differences in other laboratory parameters were found. No cases of increased blood pressure were detected in the group of men with elevated DHT. In the group of men with elevated total testosterone, 23,5% of men with arterial hypertension without targetorgan lesions were identified, while hyperandrogenism was associated with 17,6% of cases. Arterial hypertension associated with hyperandrogenism was characterized by a rise in blood pressure in the early morning hours. Estradiol levels, while remaining within normal limits, were statistically significantly lower in patients with arterial hypertension compared with men with elevated testosterone but without hypertension. CONCLUSION. No cases of arterial hypertension were observed in «physiological» hyperandrogenism due to elevated DHT levels, whereas its incidence in «physiological» hyperandrogenism due to elevated total testosterone was 23,5%. The features of steroidogenesis were increased production of 17-hydroxypregnenolone, 17-hydroxyprogesterone, and androstenedione in men with testosterone hyperandrogenism and decreased estradiol production in patients with arterial hypertension compared with patients without testosterone hyperandrogenism.

A new quantification method is proposed for the determination of serum estrone (E1), estradiol (E2), and estriol (E3) based on high-performance liquid chromatography–tandem mass spectrometry combined with differential ion mobility separation (SelexION Technology). This approach allows determining analytes with limits of detection of 4.5, 18 and 45 fg on column for E1, E2, and E3, respectively. The lowest limits of quantification are 0.1, 0.5, and 0.5 pg mL–1 in serum for E1, E2, and E3, respectively. The suitability of proposed method for diagnostic purposes is demonstrated in an analysis of serum from 114 patients of different genders and ages.

Пролактиномы - наиболее распространенные гормонально-активные аденомы гипофиза, составляю- щие около 40% всех новообразований гипоталамо-гипофизарной области. Основным методом их лечения является медикаментозная терапия агонистами дофамина, которая в большинстве случаев позволяет до- биться нормализации уровня пролактина в сыворотке крови и уменьшения размеров аденомы. Однако, около 20% пациентов с пролактиномами неудовлетворительно реагируют даже на высокие дозы агонистов дофамина, что обусловлено резистентностью к терапии. Следует отметить, что на сегодняшний день отсут- ствуют клинические предикторы резистентности пролактин-секретирующих аденом к медикаментозной терапии, а причины резистентности - предмет научной дискуссии. ЦЕЛЬ: исследование абсорбции и метаболизма каберголина у пациентов с резистентными пролакти- номами. МАТЕРИАЛЫ И МЕТОДЫ: в исследование включено 14 пациентов: 7 - с резистентными пролактино- мами (отсутствие нормализации уровня пролактина в крови либо уменьшения размеров опухоли на 50% на фоне приема максимально переносимой дозы каберголина, но не менее 3,5 мг/неделю) и 7 пациентов с адекватной чувствительностью к препарату. Всем пациентам проведен специфический фармакокине- тический тест: 1) каберголин предварительно отменялся за 4 дня до исследования; 2) взятие крови осу- ществлялось утром в день исследования, затем через 30-, 60-, 90-, 120 минут и 4-, 12-, 24 часа после приема фиксированной дозы каберголина – 0,5 мг. Измерение концентрации каберголина в сыворотке крови проводилось с использованием метода высокоэффективной жидкостной хромато-масс-спектрометрии (ВЭЖХМС/МС). РЕЗУЛЬТАТЫ: выявлены значительные различия в концентрации каберголина в сыворотке крови у резистентных и чувствительных к терапии агонистами дофамина пациентов. Показано, что кривая кон- центрации каберголина у пациентов с резистентностью к лечению не демонстрирует ожидаемых фар- макокинетических пиков. Фармакокинетическая кривая 1 резистентного пациента обнаруживает пик на 30-й минуте с последующим быстрым снижением концентрации препарата до исходного уровня. Кривая концентрации каберголина у чувствительного пациента характеризовалась исходно значительной кон- центрацией каберголина, которая постепенно становилась выше, достигнув выраженного пика в конце периода тестирования. Статистически значимые различия в концентрации каберголина сохранялись между двумя группами пациентов в течение времени, достигая максимума к 120-й и 240-й минутам (р<0,05). Значения этих временных точек можно использовать в качестве порогового уровня для дифференциаль- ной диагностики резистентности пролактином к терапии. ВЫВОДЫ: особенности, характерные для пациентов с резистентными пролактиномами, выявляют общие патогенетические принципы резистентности и способствуют разработке алгоритма персонализиро- ванного ведения пациентов данной группы. In vivo исследования фармококинетических и метаболомных характеристик каберголина, позволяющие выявить случаи резистентности к терапии, имеют фундамен- тальную и клиническую значимость.