Altered Glycolysis, Mitochondrial Biogenesis, Autophagy and Apoptosis in Peritoneal Endometriosis in Adolescents.

Energy metabolism plays a pivotal role in the pathogenesis of endometriosis. For the initial stages of the disease in adolescents, this aspect remains unexplored. The objective of this paper was to analyze the association of cellular and endosomal profiles of markers of glycolysis, mitochondrial biogenesis, apoptosis, autophagy and estrogen signaling in peritoneal endometriosis (PE) in adolescents. We included 60 girls aged 13-17 years in a case-control study: 45 with laparoscopically confirmed PE (main group) and 15 with paramesonephric cysts (comparison group). Samples of plasma and peritoneal fluid exosomes, endometrioid foci and non-affected peritoneum were tested for estrogen receptor (Erα/ß), hexokinase (Hex2), pyruvate dehydrogenase kinase (PDK1), glucose transporter (Glut1), monocarboxylate transporters (MCT1 and MCT2), optic atrophy 1 (OPA1, mitochondrial fusion protein), dynamin-related protein 1 (DRP1, mitochondrial fission protein), Bax, Bcl2, Beclin1, Bnip3, P38 mitogen-activated protein kinase (MAPK), hypoxia-inducible factor 1 (Hif-1α), mitochondrial voltage-dependent anion channel (VDAC) and transforming growth factor (TGFß) proteins as markers of estrogen signaling, glycolysis rates, mitochondrial biogenesis and damage, apoptosis and autophagy (Western-Blot and PCR). The analysis identified higher levels of molecules associated with proliferation (ERß), glycolysis (MCT2, PDK1, Glut1, Hex2, TGFß and Hif-1α), mitochondrial biogenesis (OPA1, DRP1) and autophagy (P38, Beclin1 and Bnip3) and decreased levels of apoptosis markers (Bcl2/Bax) in endometrioid foci compared to non-affected peritoneum and that in the comparison group (p < 0.05). Patients with PE had altered profiles of ERß in plasma and peritoneal fluid exosomes and higher levels of Glut1, MCT2 and Bnip3 in plasma exosomes (p < 0.05). The results of the differential expression profiles indicate microenvironment modification, mitochondrial biogenesis, estrogen reception activation and glycolytic switch along with apoptosis suppression in peritoneal endometrioid foci already in adolescents.

Androgen Insensitivity Syndrome with Bilateral Gonadal Sertoli Cell Lesions, Sertoli-Leydig Cell Tumor, and Paratesticular Leiomyoma: A Case Report and First Systematic Literature Review.

Androgen insensitivity syndrome (AIS) is a rare Mendelian disorder caused by mutations of the androgen receptor (AR) gene on the long arm of the X chromosome. As a result of the mutation, the receptor becomes resistant to androgens, and hence, karyotypically male patients (46,XY) carry a female phenotype. Their cryptorchid gonads are prone to the development of several types of tumors (germ cell, sex cord stromal, and others). Here, we report a 15-year-old female-looking patient with primary amenorrhea who underwent laparoscopic gonadectomy. Histologically, the patient's gonads showed Sertoli cell hamartomas (SCHs) and adenomas (SCAs) with areas of Sertoli-Leydig cell tumors (SLCTs) and a left-sided paratesticular leiomyoma. Rudimentary Fallopian tubes were also present. The patient's karyotype was 46,XY without any evidence of aberrations. Molecular genetic analysis of the left gonad revealed two likely germline mutations-a pathogenic frameshift deletion in the AR gene (c.77delT) and a likely pathogenic missense variant in the RAC1 gene (p.A94V). Strikingly, no somatic mutations, fusions, or copy number variations were found. We also performed the first systematic literature review (PRISMA guidelines; screened databases: PubMed, Scopus, Web of Science; ended on 7 December 2023) of the reported cases of patients with AIS showing benign or malignant Sertoli cell lesions/tumors in their gonads (n = 225; age: 4-84, mean 32 years), including Sertoli cell hyperplasia (1%), Sertoli cell nodules (6%), SCHs (31%), SCAs (36%), Sertoli cell tumors (SCTs) (16%), and SLCTs (4%). The few cases (n = 14, 6%; six SCAs, four SCTs, two SLCTs, and two SCHs) with available follow-up (2-49, mean 17 months) showed no evidence of disease (13/14, 93%) or died of other causes (1/14, 7%) despite the histological diagnosis. Smooth muscle lesions/proliferations were identified in 19 (8%) cases (including clearly reported rudimentary uterine remnants, 3 cases; leiomyomas, 4 cases). Rudimentary Fallopian tube(s) were described in nine (4%) cases. Conclusion: AIS may be associated with sex cord/stromal tumors and, rarely, mesenchymal tumors such as leiomyomas. True malignant sex cord tumors can arise in these patients. Larger series with longer follow-ups are needed to estimate the exact prognostic relevance of tumor histology in AIS.

Pelvic Pain, Mental Health and Quality of Life in Adolescents with Endometriosis after Surgery and Dienogest Treatment.

BACKGROUND: Diagnostic and treatment delays have caused significant impacts on the physical and emotional well-being of adolescents with endometriosis, though such research is limited. This study aimed to assess the effects of one-year dienogest therapy on the clinical picture, pain patterns, psycho-emotional status, and quality-of-life indicators in adolescents with endometriosis after surgical treatment. METHODS: The study enrolled 32 girls aged 13-17 with peritoneal endometriosis to analyze one-year dynamics of the Visual Analog Scale (VAS), McGill Pain Questionnaire, Beck Depression Scale (BDI), Hospital Anxiety and Depression Scale (HADS), Spielberger State-Trait Anxiety Inventory (STAI) and SF-36 quality-of-life survey scores along with clinical and laboratory indicators before surgery and after one-year dienogest therapy. RESULTS: The therapy provided a significant alleviation of endometriosis-associated clinical symptoms, including dysmenorrhea, pelvic pain, gastrointestinal/dysuria symptoms, decreased everyday activity (<0.001), a decrease in anxiety/depression scores (BDI, HADS, STAI), and quality-of-life improvement (<0.001). These effects were accompanied by beneficial dynamics in hormone and inflammatory markers (prolactin, cortisol, testosterone, estradiol, CA-125, neutrophil/lymphocyte ratio; <0.005) within reference ranges. A low body mass index and high C-reactive protein levels were associated with higher VAS scores; a high estradiol level was a factor for anxiety/depression aggravation (<0.05). CONCLUSIONS: Dienogest, after surgical treatment, significantly improved quality of life and reduced pain symptoms while showing good tolerability and compliance, and reasoning with timely hormonal therapy in adolescents with endometriosis.

Endometriosis in Adolescents: Diagnostics, Clinical and Laparoscopic Features.

BACKGROUND: The early diagnosis of endometriosis in adolescents is not developed. OBJECTIVE: We aim to conduct clinical, imaging, laparoscopic and histological analyses of peritoneal endometriosis (PE) in adolescents in order to improve early diagnosis. METHODS: In total, 134 girls (from menarche to 17 years old) were included in a case-control study: 90 with laparoscopically (LS) confirmed PE, 44 healthy controls underwent full examination and LS was analyzed in the PE group. RESULTS: Patients with PE were characterized with heredity for endometriosis, persistent dysmenorrhea, decreased daily activity, gastrointestinal symptoms, higher LH, estradiol, prolactin and Ca-125 (<0.05 for each). Ultrasound detected PE in 3.3% and MRI in 78.9%. The most essential MRI signs are as follows: hypointense foci, the heterogeneity of the pelvic tissue (paraovarian, parametrial and rectouterine pouch) and sacro-uterine ligaments lesions (<0.05 for each). Adolescents with PE mostly exhibit initial rASRM stages. Red implants correlated with the rASRM score, and sheer implants correlated with pain (VAS score) (<0.05). In 32.2%, foci consisted of fibrous, adipose and muscle tissue; black lesions were more likely to be histologically verified (0.001). CONCLUSION: Adolescents exhibit mostly initial PE stages, which are associated with greater pain. Persistent dysmenorrhea and detected MRI parameters predict the laparoscopic confirmation of initial PE in adolescents in 84.3% (OR 15.4; <0.01), justifying the early surgical diagnostics and shortening the time delay and suffering of the young patients.

Assessment of the Level of Accumulation of the dIFN Protein Integrated by the Knock-In Method into the Region of the Histone H3.3 Gene of Arabidopsis thaliana.

Targeted DNA integration into known locations in the genome has potential advantages over the random insertional events typically achieved using conventional means of genetic modification. We investigated the possibility of obtaining a suspension cell culture of Arabidopsis thaliana carrying a site-specific integration of a target gene encoding modified human interferon (dIFN) using endonuclease Cas9. For the targeted insertion, we selected the region of the histone H3.3 gene (HTR5) with a high constitutive level of expression. Our results indicated that Cas9-induced DNA integration occurred with the highest frequency with the construction with donor DNA surrounded by homology arms and Cas9 endonuclease recognition sites. Among the monoclones of the four cell lines with knock-in studied, there is high heterogeneity in the level of expression and accumulation of the target protein. The accumulation of dIFN protein in cell lines with targeted insertions into the target region of the HTR5 gene does not statistically differ from the level of accumulation of dIFN protein in the group of lines with random integration of the transgene. However, one among the monoclonal lines with knock-in has a dIFN accumulation level above 2% of TSP, which is very high.

The Relevant Hormonal Levels and Diagnostic Features of Polycystic Ovary Syndrome in Adolescents.

RELEVANCE: The clinical picture of polycystic ovary syndrome (PCOS) is extremely polymorphic, especially in adolescence. At the same time, the diagnostic criteria of PCOS in adolescence are still under discussion, and the hormonal parameters, including anti-Mullerian hormone range and hyperandrogenism, are not determined. The aim of the present study was to characterize the pivotal clinical and hormonal features of PCOS in adolescents and to establish the age-specific thresholds of the most essential hormonal parameters. DESIGN: A case-control study. METHODS: The study included 130 girls with PCOS according to the complete Rotterdam criteria, aged 15 to 17 years. The control group consisted of 30 healthy girls with a regular menstrual cycle of the same age. A complete clinical and laboratory examination, hormonal assays, and ultrasound of the pelvic organs were performed. The serums anti-Mullerian hormone (AMH), follicle-stimulating hormone (FSH), luteinizing hormone (LH), LH/FSH, prolactin, estradiol, 17α-OH progesterone (17α-OHP), androstenedione, testosterone (T), dehydroepiandrosterone sulfate (DHEAS), sex hormone-binding globulin (SHBG), leptin, and free androgen index (FAI) were analyzed. The diagnostic accuracy of AMH, FAI, LH/FSH, T, and androstenedione levels in predicting PCOS in adolescents was established using a logistic regression model and calculating area under the receiver operator characteristic (ROC) curve (AUC). RESULTS: The serum levels of LH (9.0 (5.4-13.8) vs. 3.7 (2.5-4.7) IU/L; p < 0.0001), LH/FSH (1.6 (1.0-2.3) vs. 0.7 (0.5-1.1); p < 0.0001), 17α-OHP (4.1 (3.2-5.1) vs. 3.4 (2.7-3.8) nmol/L; p = 0.0071), cortisol (464.0 ± 147.6 vs. 284.0 ± 129.7 nmol/L; p < 0.0001), prolactin (266.0 (175.0-405.0) vs. 189.0 (142.0-269.0) mIU/L; p = 0.0141), T (1.9 (1.2-2.5) vs. 0.8 (0.7-1.1) nmol/L; p < 0.0001), androstenedione (15.8 (11.6-23.2) vs. 8.3 (6.5-10.8) ng/mL; p < 0.0001), AMH (9.5 (7.5-14.9) vs. 5.8 (3.8-6.9) ng/mL; p < 0.0001), FAI (5.5 (2.8-7.0) vs. 1.6 (1.1-2.3); p < 0.0001), SHBG (37.0 (24.7-55.5) vs. 52.9 (39.0-67.6) nmol/L; p = 0.0136), DHEAS (6.8 ± 3.2 vs. 5.1 ± 1.5 µmol/L; p = 0.0039), and leptin (38.7 ± 27.1 vs. 23.7 ± 14.0 ng/mL; p = 0.0178) were significantly altered in the PCOS patients compared to the controls. Multivariate analysis of all studied hormonal and instrumental parameters of PCOS in adolescents revealed as the most essential: AMH level > 7.20 ng/mL, FAI > 2.75, androstenedione > 11.45 ng/mL, total T > 1.15 nmol/L, LH/FSH ratio > 1.23, and the volume of each ovary > 10.70 cm3 (for each criterion sensitivity ≥ 75.0-93.0%, specificity ≥ 83.0-93.0%). The diagnostic accuracy of PCOS determination was 90.2-91.6% with the combined use of either four detected indexes, which was significantly higher than the use of each index separately. The accuracy of PCOS diagnostics reached 92% using AMH and leptin concentrations when the value of the logistic regression function [85.73 - (1.73 × AMH) - (0.12 × Leptin)] was less than 70.72. CONCLUSIONS: The results of the study estimate the threshold for AMH, FAI, androstenedione, testosterone, LH/FSH, and ovarian volume, which could be suggested for use in the PCOS diagnostics in adolescents with a high sensitivity and specificity. Moreover, the combination of either four determined indexes improved the diagnostic accuracy for the PCOS detection in adolescents.

Activation of Systemic Inflammation and Oxidative Stress in Adolescent Girls with Polycystic Ovary Syndrome in Combination with Metabolic Disorders and Excessive Body Weight.

RELEVANCE: Mitochondrial dysfunction and systemic inflammation are believed to play pivotal role in the pathogenesis of polycystic ovary syndrome (PCOS) and related complications of metabolic disorders in adult patients. Though such researches are limited or almost absent in adolescents. The aim of the study is to evaluate the impact of mitochondrial dysfunction and systemic inflammation on PCOS pathogenesis during adolescence with regard to body mass index and insulin resistance. DESIGN: a case-control study. METHODS: The study included 95 adolescent girls (15 to 17 years old inclusive) diagnosed with PCOS based on the Rotterdam criteria. The control group consisted of 30 healthy girls of the same age with a regular menstrual cycle. All participants were subjected to a full clinical and instrumental examination, as well as an assessment of the levels of leptin, C-reactive protein (CRP), and malondialdehyde (MDA) as oxidative stress marker. Serum levels of IL-6, IL-10, IL-18, TNF-α, and plasma concentrations of macrophage migration inhibitory factor (MIF), sFas, and sFasL were determined. Patients with PCOS were divided into groups according to the presence of metabolic disorders (MD) (impaired glucose tolerance and/or over insulin resistance) and normal weight or excessive weight (NW or OW). RESULTS: Patients with PCOS of NW in the absence of metabolic disorders (MD-/NW) had a lower concentration of MDA and a higher level of IL-10 compared to healthy girls (p < 0.05). The group (MD-/NW) was characterized with lower levels of CRP, leptin, MDA, and higher levels of sFasL, when compared to OW patients with PCOS in the absence of metabolic disorders (MD-/OW) (p < 0.05). Overweight adolescent girls with PCOS and metabolic disorders (MD+/OW) showed higher CRP, leptin, and a two-fold increase in IL-6 and IL-18 concentrations compared to the control group of healthy girls (p < 0.05 for all parameters). The group (MD+/OW) was also characterized with higher levels of CRP, leptin, MDA, IL-18, MIF (p < 0.05), when compared to overweight patients with PCOS in the absence of metabolic disorders (MD-/NW). In comparison with the MD-/OW group, the obese insulin resistant girls with PCOS (MD+/OW) had a highera level of IL-18 (p < 0.05). Moreover, the MD+/OW girls demonstrated a significant increase in CRP, MDA and IL-18 levels when compared to the MD+/NW group (p < 0.05). OW girls with PCOS without MD (MD-/OW) had lower concentrations of sFasL compared to healthy girls (p < 0.05), and higher levels of MDA compared to MD+/NW (p < 0.05). Adolescent girls of NW with PCOS and with MD (MD+/NW) had lower levels of MDA compared to the control group of healthy girls (p < 0.05). These data are confirmed by a correlation analysis and two-factor ANOVA test. CONCLUSIONS: Lean girls with PCOS demonstrate the protective mechanism of decrease in oxidative stress mediated by the activation of antioxidant defense, reduction of lipid peroxidation and systemic inflammation. Excessive weight and metabolic disorders in adolescents with PCOS are the most significant factors in reducing the capacity of antioxidant systems, activation of oxidative stress, mitochondrial dysfunction, and systemic inflammation.

Evaluation of resorbable polydioxanone and polyglycolic acid meshes in a rat model of ventral hernia repair.

The objective of this study was to evaluate physical, mechanical, and biological properties of the polydioxanone (PDO) monofilament meshes and polyglycolide (PGA) polyfilament meshes in comparison with Permacol® implants. In rat experimental model, a 1.5 × 2.0 cm defect in abdominal wall was reconstructed by using the Permacol surgical implant or knitted meshes produced from either PDO monofilament, or PGA multifilament. The implant sites were assessed for the tensile strength and the extents of material resorption, host inflammatory response and host tissue replacement on days 3, 10, 30, or 60 after the surgery. The PDO and PGA meshes were rapidly pervaded by the host connective tissue with elements of skeletal muscle histogenesis. The degree of adhesions was significantly higher in the Permacol group. All of the prostheses underwent resorption, which correlated with gradual decreases in the overall tensile strength of the site and the Col1a1 gene expression level. Elevated expression of Fgf2 gene maintained longer in the PDO group, and the Mmp9 gene expression level in this group was higher than in the other groups. Gene expression levels of inflammatory cytokines were higher in the Permacol group. The foreign body giant cell numbers were lower in the PDO and Permacol groups than in the PGA group. Minimal macrophage infiltration with predominance of M2 cells was observed in the PDO group. Overall, the PDO prosthesis turned out to be significantly better than the PGA or Permacol prostheses by a number of indicators of biocompatibility and efficacy. © 2018 The Authors Journal of Biomedical Materials Research Part B: Applied Biomaterials Published by Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 00B: 000-000, 2018. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 652-663, 2019.

The recombinant fusion protein CFP10-ESAT6-dIFN has protective effect against tuberculosis in guinea pigs.

Development of effective vaccine candidates against tuberculosis is currently the most important challenge in the prevention of this disease since the BCG vaccine fails to guarantee a lifelong protection, while any other approved vaccine with better efficiency is still absent. The protective effect of the recombinant fusion protein ESAT6-CFP10-dIFN produced in a prokaryotic expression system (Escherichia coli) has been assessed in a guinea pig model of acute tuberculosis. The tested antigen comprises the Mycobacterium tuberculosis (Mtb) proteins ESAT6 and CFP10 as well as modified human γ-interferon (dIFN) for boosting the immune response. Double intradermal immunization of animals with the tested fusion protein (2 × 0.5 µg) induces a protective effect against subsequent Mtb infection. The immunized animals do not develop the symptoms of acute tuberculosis and their body weight gain was five times more as compared with the non-immunized-infected animals. The animal group immunized with this dose of antigen displays the minimum morphological changes in the internal organs and insignificant inflammatory lesions in the liver tissue, which complies with a decrease in the bacterial load in the spleen and average Mtb counts in macrophages.

The recombinant fusion protein CFP10-ESAT6-dIFN has protective effect against tuberculosis in guinea pigs.

Development of effective vaccine candidates against tuberculosis (TB) is currently the most important challenge in the prevention of this disease since the BCG vaccine fails to guarantee a lifelong protection, while any other approved vaccine with better efficiency is still absent. The protective effect of the recombinant fusion protein CFP10-ESAT6-dIFN produced in a prokaryotic expression system (Escherichia coli) has been assessed in a guinea pig model of acute TB. The tested antigen comprises the Mycobacterium tuberculosis (Mtb) proteins ESAT6 and CFP10 as well as modified human γ-interferon (dIFN) for boosting the immune response. Double intradermal immunization of guinea pigs with the tested fusion protein (2 × 0.5 µg) induces a protective effect against subsequent Mtb infection. The immunized guinea pigs do not develop the symptoms of acute TB and their body weight gain was five times more as compared with the non-immunized infected guinea pigs. The animal group immunized with this dose of antigen displays the minimum morphological changes in the internal organs and insignificant inflammatory lesions in the liver tissue, which complies with a decrease in the bacterial load in the spleen and average Mtb counts in macrophages.

Transgenic carrot expressing fusion protein comprising M. tuberculosis antigens induces immune response in mice.

Tuberculosis remains one of the major infectious diseases, which continues to pose a major global health problem. Transgenic plants may serve as bioreactors to produce heterologous proteins including antibodies, antigens, and hormones. In the present study, a genetic construct has been designed that comprises the Mycobacterium tuberculosis genes cfp10, esat6 and dIFN gene, which encode deltaferon, a recombinant analog of the human γ-interferon designed for expression in plant tissues. This construct was transferred to the carrot (Daucus carota L.) genome by Agrobacterium-mediated transformation. This study demonstrates that the fusion protein CFP10-ESAT6-dIFN is synthesized in the transgenic carrot storage roots. The protein is able to induce both humoral and cell-mediated immune responses in laboratory animals (mice) when administered either orally or by injection. It should be emphasized that M. tuberculosis antigens contained in the fusion protein have no cytotoxic effect on peripheral blood mononuclear cells.

Oral Immunogenicity of plant-made Mycobacterium tuberculosis ESAT6 and CFP10.

Two lines of transgenic carrot plants producing Mycobacterium tuberculosis proteins (ESAT6 and CFP10) have been constructed. The target proteins are present in carrot storage roots at a level not less than 0.056% of the total storage protein (TSP) for ESAT6 and 0.002% of TSP for CFP10. As has been shown, oral immunization of mice induces both the cell-mediated and humoral immunities. These data suggest that the proteins in question are appropriate as a candidate edible vaccine against tuberculosis.