Diagnostic Value of Autoantibodies against Steroidogenic Enzymes and Hormones in Infertile Women with Premature Ovarian Insufficiency.

The objective of the study was to evaluate the profile and diagnostic significance of serum autoantibodies in infertile patients with premature ovarian insufficiency (POI). The pilot study included 26 patients of reproductive age with POI and diminished ovarian reserve who received complex treatment using new surgical technologies (Group 1) and 18 patients without POI (Group 2). The profile of serum autoantibodies, including anti-ovarian antibodies, antibodies against thyroid peroxidase (TPO), steroidogenic enzymes, and steroid and gonadotropic hormones, was studied using modified ELISAs and human recombinant steroidogenic enzymes (CYP11A1, CYP19A1, CYP21A2). Patients in Group 1 had higher levels of IgG autoantibodies against steroidogenic enzymes, estradiol, progesterone, and TPO than those in Group 2. Tests for IgG antibodies against CYP11A1, CYP19A1, and CYP21A2 exhibited high sensitivity (65.4-76.9%), specificity (83.3-89.9%), and AUC values (0.842-0.910) for POI, the highest in the first test. Three-antibodies panel screening showed higher diagnostic accuracy (84.1% versus 75-79.6%). The levels of these antibodies correlated with menstrual irregularities and a decrease in the antral follicle count. Thus, antibodies against CYP11A1, CYP19A1, and CYP21A2 have a high diagnostic value for POI. Three-antibody panel screening may improve the accuracy of POI diagnosis and be useful for identifying high-risk groups, early stages of the disease, and predicting POI progression.

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.

Novel Approaches to Possible Targeted Therapies and Prophylaxis of Uterine Fibroids.

Uterine leiomyomas are the most common benign tumors in women of childbearing age. They may lead to problems of conception or complications during the gestational period. The methods of treatment include surgical (myomectomy and hysterectomy, embolization of arteries) and therapeutic treatment (ulipristal acetate, leuprolide acetate, cetrorelix, goserelin, mifepristone). Both approaches are efficient but incompatible with pregnancy planning. Therefore, there is a call for medical practice to develop therapeutical means of preventing leiomyoma onset in patients planning on becoming pregnant. Based on the analysis of GWAS data on the search for mononucleotide polymorphisms associated with the risk of leiomyoma, in meta-transcriptomic and meta-methylomic studies, target proteins have been proposed. Prospective therapeutic treatments of leiomyoma may be based on chemical compounds, humanized recombinant antibodies, vaccines based on markers of the uterine leiomyoma cells that are absent in the adult organism, or DNA and RNA preparations. Three different nosological forms of the disease associated with driver mutations in the MED12, HMGA2, and FH genes should be considered when developing or prescribing drugs. For example, synthetic inhibitors and vaccines based on matrix metalloproteinases MMP11 and MMP16 are expected to be effective only for the prevention of the occurrence of MED12-dependent nodules.

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.

Familial Predisposition to Leiomyomata: Searching for Protective Genetic Factors.

In order to determine genetic loci associated with decreasing risk of uterine leiomyomata (UL), a genome-wide association study (GWAS) was performed. We analyzed a group of patients with a family history of UL and a control group consisting of patients without uterine fibroids and a family predisposition to this pathology. Six significant single nucleotide polymorphisms were selected for PCR-genotyping of a large data set of patients with UL. All investigated loci (rs3020434, rs11742635, rs124577644, rs12637801, rs2861221, and rs17677069) demonstrated the lower frequency of minor alleles within a group of women with UL, especially in a subgroup consisting of patients with UL and a familial history of leiomyomata. We also found that the minor allele frequencies of these SNPs in our control group were higher than those across the Caucasian population in all. Based on the obtained data, an evaluation of the common risk of UL was performed. Further work will pave the way to create a specific SNP-panel and allow us to estimate a genotype-based leiomyoma incidence risk. Subsequent studies of genetic variability in a group of patients with a familial predisposition to UL will allow us to make the prediction of the development and course of the disease more individualized, as well as to give our patients personalized recommendations about individual reproductive strategies.

Prolonged cyclical and continuous regimens of dydrogesterone are effective for reducing chronic pelvic pain in women with endometriosis: results of the ORCHIDEA study.

OBJECTIVE: To compare the effectiveness of two different treatment regimens of dydrogesterone in the management of endometriosis-related chronic pelvic pain. DESIGN: Observational, prospective cohort study over six months. SETTING: Twenty gynecology clinics in the Russian Federation. PATIENT(S): Three hundred fifty women from 18 to 45 years of age with endometriosis and chronic pelvic pain with or without dysmenorrhea. INTERVENTION(S): Dydrogesterone 10 mg 2 or 3 times daily, either between the 5th and 25th days of the menstrual cycle (prolonged cyclical treatment regimen) or continuously (continuous treatment regimen). For all patients, the data cutoff was at six months of treatment. MAIN OUTCOME MEASURE(S): Intensity of chronic pelvic pain on the 11-point numerical rating scale (after 6 months). RESULT(S): A marked reduction in chronic pelvic pain was observed with both the prolonged cyclical and continuous treatment regimens (mean ± standard deviation change from baseline -3.3 ± 2.2 and -3.0 ± 2.2, respectively), with no significant difference between the two groups. With both regimens, patients experienced significant improvements in the intensity of chronic pelvic pain, number of days in which analgesics were required, severity of dysmenorrhea, sexual well-being, and health-related quality-of-life parameters. A favorable safety profile of dydrogesterone was confirmed, and no serious adverse drug reactions were reported during the study. CONCLUSION(S): Prolonged cyclical and continuous treatment regimens of dydrogesterone therapy both demonstrated a pronounced and similar reduction in the severity of chronic pelvic pain and dysmenorrhea and led to marked improvements in all study parameters related to quality of life and sexual well-being. REGISTRATION NUMBER: NCT03690765.

The Inhibiting Effect of Dinitrosyl Iron Complexes with Thiol-containing Ligands on the Growth of Endometrioid Tumours in Rats with Experimental Endometriosis.

The possibility that binuclear dinitrosyl iron complexes with glutathione and cysteine (DNIC-GSН and B-DNIC-Cys) have a strong cytotoxic effect on the growth of endometrioid tumours (EMT) in rats with surgically induced experimental endometriosis established in our previous studies has been supported with experimental data. The increase in the DNIC-GSН or B-DNIC-Cys dose from 10 (in our previous studies) to 20 µmol/kg (after i/p administration to experimental rats) fully suppressed the growth of uterine tissues implanted onto the inner surface of the abdominal wall. At 2 µmol/kg DNIC-GSН, the median value of EMT volume increased from 0 to 15 mm3, while the mean size of EMT-from 55 to 77 mm3 (data from EMT measurements in 10 experimental rats). After treatment of animals with B-DNIC with N-acetyl-L-cysteine (10 µmol/kg) known for its ability to penetrate easily through the cell membrane, the inhibiting effect on EMT growth diminished as could be evidenced from the transformation of ~30% of the implants into large-size EMT. Possible reasons for this phenomenon are discussed.

Direct Mass Spectrometry Differentiation of Ectopic and Eutopic Endometrium in Patients with Endometriosis.

STUDY OBJECTIVE: To introduce a method for the rapid assessment of endometriotic tissues using direct mass spectrometry (MS)-based lipidomics. DESIGN: A prospective observational cohort study (Canadian Task Force classification II2). SETTING: Department of Operative Gynecology of the Research Centre for Obstetrics, Gynecology and Perinatology. PATIENTS: Fifty patients with ovarian cysts and peritoneal endometriosis who underwent laparoscopic surgery between 2014 and 2016. INTERVENTION: Differences in mass spectrometric profiles of ectopic endometria (endometriosis) and eutopic endometria were analyzed for each patient in combination with morphohistologic evaluation. The lipidomic approach was applied using a direct high-resolution MS method. MEASUREMENTS AND MAIN RESULTS: Of 148 metabolites, 15 showed significant differences between endometriotic tissue and a healthy endometrium of the same patient, considered as a control in this study. The main lipids prevalent in endometriotic tissues were phosphoethanolamine (PE O-20:0), sphingomyelin (SM 34:1), diglycerides (DG 44:9), phosphatidylcholines (PC 32:1, PC O-36:3, PC 38:7, PC 38:6, PC 40:8, PC 40:7, PC 40:6, PC 40:9, and PC O-42:1), and triglycerides (TG 41:2, TG 49:4, and TG 52:3). Using partial least squares discriminant analysis models, MS showed that the lipidomic profile of endometriotic tissue (peritoneal endometriosis and ovarian endometriomas) was clearly separated from the eutopic endometrium, indicating tissue-type differentiation. CONCLUSION: Our results suggest that direct MS may play an important role for endometriotic tissue identification. Such an approach has potential usefulness for real-time tissue determination and differentiation during surgical treatment. Lipids of 3 important classes, sphingolipids, phospholipids, and the fatty acids (di- and triglycerides), were identified. Validation is required to determine whether these lipids can be used to discriminate between patients with endometriosis and those with other gynecologic diseases.

Endometriosis foci differentiation by rapid lipid profiling using tissue spray ionization and high resolution mass spectrometry.

Obtaining fast screening information on molecular composition of a tissue sample is of great importance for a disease biomarkers search and for online surgery control. In this study, high resolution mass spectrometry analysis of eutopic and ectopic endometrium tissues (90 samples) is done using direct tissue spray mass spectrometry in both positive and negative ion modes. The most abundant peaks in the both ion modes are those corresponding to lipids. Species of three lipid classes are observed, phosphatidylcholines (PC), sphingomyelins (SM) and phosphoethanolamines (PE). Direct tissue analysis gives mainly information on PC and SM lipids (29 species) in positive ion mode and PC, SM and PE lipids (50 species) in negative ion mode which gives complementary data for endometriosis foci differentiation. The biggest differences were found for phospholipids with polyunsaturated acyls and alkils. Although, tissue spray shows itself as appropriate tool for tissue investigation, caution should be paid to the interpretation of mass spectra because of their higher complexity with more possible adducts formation and multiple interferences must be taken into account. The present work extends the application of direct tissue analysis for the rapid differentiation between endometriotic tissues of different foci.

The effect of dinitrosyl iron complexes with glutathione and S-nitrosoglutathione on the development of experimental endometriosis in rats: a comparative studies.

It has been established that intraperitoneal bolus administration of S-nitrosoglutathione (GS-NO) (12.5µmoles/kg; 10 injections in 10 days), beginning with day 4 after transplantation of two 2-mm autologous fragments of endometrial tissue onto the inner surface of the abdominal wall of rats with surgically induced (experimenta) endometriosis failed to prevent further growth of endometrioid (EMT) and additive tumors, while treatment of animals with dinitrosyl iron complexes (DNIC) with glutathione (12.5µmoles/kg, 10 injections in 10 days) suppressed tumor growth virtually completely. The histological analysis of EMT samples of GS-NO-treated rats revealed pathological changes characteristic of control (non-treated with GS-NO or DNIC) rats with experimental endometriosis. EPR studies established the presence of the active form of ribonucleotide reductase, a specific marker for rapidly proliferating tumors, in EMT samples of both control and GS-NO-treated animals. Noteworthy, in small-size EMT and adjacent tissues of DNIC-treated rats the active form of ribonucleotide reductase and pathological changes were not found.

Dinitrosyl iron complexes with glutathione suppress experimental endometriosis in rats.

Dinitrosyl iron complexes (DNIC) with glutathione exert a cytotoxic effect on endometrioid tumours in rats with surgically induced experimental endometriosis. Intraperitoneal treatment of rats (Group 1) with DNIC (12.5µmoles/kg, daily, for 12 days), beginning with day 4 after the surgical operation (implantation of two 2mm-thick uterine fragments onto the abdominal wall) followed by 14-day keeping of animals on a standard feeding schedule (without medication) resulted in complete inhibition of the growth of endometrioid implants (EMI) in the majority of experimental animals. The ratio of mean EMI volumes in control and experimental rats of Group 1 was 14:1. In Group 2 rats, the use of a similar treatment protocol 4 weeks after surgery changed this ratio to 1.4:1. Noteworthy, the decrease of this ratio was irrelevant to deceleration of EMI growth at later periods after surgery. The histopathological analysis of EMI samples from experimental rats of Group 2 demonstrated complete disappearance of endometrial cysts suggesting a cytotoxic effect of DNIC on the tumours. The data obtained demonstrate that DNIC with glutathione and, probably, with other thiol-containing ligands hold considerable promise in the design of drugs for treating endometriosis in female patients.

Effects of adding small amounts of oxygen to a carbon dioxide-pneumoperitoneum of increasing pressure in rabbit ventilation models.

OBJECTIVE: To evaluate the metabolic consequences of the addition of oxygen to the CO(2)-pneumoperitoneum. DESIGN: Prospective randomized study in rabbits. After 30 minutes of ventilation pneumoperitoneum was maintained for 90 minutes with pure CO(2) or CO(2) with 2% or 6% of oxygen. The intraperitoneal pressure was increased from 10 to 15 and 20 mm Hg every 30 minutes. Ventilation rate was either fixed or a progressive hyperventilation. End points were changes in arterial blood gases (Pco(2), Po(2)), pH, acid-base balance (actual base excess [ABE], standard bicarbonate [SBC], standard base excess [SBE], hydrogen carbonate [HCO(3)(-)], concentration of total carbon dioxide [Tco(2)]); oxygen and oximetry (oxyhemoglobin [O(2)Hb], oxygen saturation [So(2)], reduced hemoglobin [RHb], total oxygen concentration [To(2)], and oxygen tension at half saturation assessing hemoglobin oxygen affinity [p50]); and lactate concentrations assayed every 15 minutes. SETTING: University research center. ANIMALS: Twenty-four adult female New Zealand white rabbits. INTERVENTION(S): Anesthesia, mechanical ventilation, and pneumoperitoneum. RESULT(S): The effects of CO(2)-pneumoperitoneum on all end points increased with the elevated intraperitoneal pressure and were more pronounced when ventilation was fixed. Changes were less when 2% or 6% of oxygen had been added to the CO(2)-pneumoperitoneum. With use of logistic regression, the addition of oxygen, intraperitoneal pressure, and ventilation were found to be independent variables affecting Pco(2), pH, ABE, SBE, HCO(3)(-), O(2)Hb, So(2), p50, and end-tidal CO(2). CONCLUSION(S): The metabolic consequences of the combined effect of increased intraperitoneal pressure and CO(2)-pneumoperitoneum were less when 2% to 6% of oxygen was added or when animals were hyperventilated. We suggest that metabolic and mesothelial hypoxemia caused by CO(2) absorption can be reduced by adding small amounts of oxygen and by hyperventilation.

Pathogenesis of CO(2) pneumoperitoneum-induced metabolic hypoxemia in a rabbit model.

STUDY OBJECTIVE: To investigate the effects of carbon dioxide (CO(2)) pneumoperitoneum-induced changes in blood gases, acid-base balance, and oxygen homeostasis in rabbits. DESIGN: Prospective, randomized, controlled study (Canadian Task Force classification I). SETTING: University training and teaching center. SUBJECTS: Twenty-six adult female New Zealand white rabbits. INTERVENTION: Anesthesia and pneumoperitoneum. MEASUREMENTS AND MAIN RESULTS: In anesthetized rabbits arterial blood gases, acid-base balance, oxygenation values, and lactate concentrations were assayed during 2 hours. Spontaneous breathing, superficial and optimal ventilation without pneumoperitoneum, and with pneumoperitoneum at low (6 mm Hg) and higher (10 mm Hg) insufflation pressures were compared. The CO(2) pneumoperitoneum profoundly affected blood gases, acid-base balance, and oxygen homeostasis. Carboxemia with increasing end-tidal CO(2) and partial pressure of CO(2) (p <0.001), acidosis with decreasing pH (p <0.001), and base deficiency with decreasing actual base excess (p <0.001), standard base excess and standard bicarbonate and acid excess with increasing hydrogen bicarbonate (p <0.05 and <0.01) were found. Desaturation (p <0.01) with decreasing oxyhemoglobin p <0.05) and hemoglobin oxygen affinity (p <0.01) were also found. Carboxemia with acidosis was more pronounced with higher (p <0.01) than with lower (p >0.05) intraperitoneal pressures, and also with spontaneous breathing (p <0.05) and superficial ventilation (p <0.001) than with optimal ventilation, resulting in metabolic hypoxemia. CONCLUSION: In superficially ventilated and spontaneously breathing rabbits, CO(2) pneumoperitoneum profoundly affected blood gases, acid-base balance, and oxygen homeostasis, resulting in metabolic hypoxemia. With optimal ventilation and low intraperitoneal pressure carboxemia, respiratory acidosis, and changes in oxygen metabolism were minimal.

Reduction of CO(2)-pneumoperitoneum-induced metabolic hypoxaemia by the addition of small amounts of O(2) to the CO(2) in a rabbit ventilated model. A preliminary study.

BACKGROUND: CO(2)-pneumoperitoneum used in endoscopic surgery induces system effects by CO(2) absorption. This study investigated the effect of the addition of O(2) to CO(2)-pneumoperitoneum, upon CO(2) absorption. METHODS: The effect of a pneumoperitoneum using 100% CO(2) or 94% CO(2) + 6% O(2) upon arterial blood gases, acid base and O(2) homeostasis was evaluated. In series A suboptimal ventilation and a pneumoperitoneum pressure (PP) of 10 mmHg was used. In series B adequate ventilation and PP of 6 mmHg was used. RESULTS: CO(2)-pneumoperitoneum profoundly affected blood gases and acid base homeostasis i.e. increasing pCO(2), HCO(3)(P < 0.001) and lactate concentrations (P < 0.05) and decreasing pH, actual base excess and standard bicarbonate (P < 0.001), resulting in metabolic hypoxaemia with desaturation, lower pO(2) (P < 0.001) and O(2)Hb (P < 0.05). These effects were more pronounced with higher PP and suboptimal ventilation. CONCLUSION: CO(2)-pneumoperitoneum profoundly affected blood gases and acid base homeostasis resulting in metabolic hypoxaemia. The addition of 6% of O(2) to the CO(2)-pneumoperitoneum prevented these effects to a large extent. If these preliminary data are confirmed in the human, the addition of a few percent of O(2) to CO(2) could become important for endoscopic surgery of long duration, especially in obese patients with limited cardiorespiratory adaptation and steep Trendelenburg.