Anna Borisova (Presenter)
Scientific Center for Obstetrics and Gynecology
Bio: In 2011- graduated from Siberian State Medical University (Tomsk, Russia). From 2011 to 2012- internship in Obstetrics and Gynecology in Siberian State Medical University (Tomsk, Russia). From 2012 to 2014 - residency in Obstetrics and Gynecology at the department of Operative Gynecology of the FSBE «Science Center of Obstetrics, Gynecology and Perinatology, named after academician V.I. Kulakov» (Moscow, Russia). From 2015 till now - PhD student at the department of Operative Gynecology of the FSBE «Science Center of Obstetrics, Gynecology and Perinatology, named after academician V.I. Kulakov» (Moscow, Russia).
Authorship: À. Borisova; V. Frankevich; V. Chagovets; N. Starodubtseva; A. Kozachenko; À. Kononikhin; A. Bugrova; V. Naumov; E.Kogan; Leila V. Adamyan.
Scientific Center for Obstetrics, Gynecology and Perinatology of the Ministry of Health of the Russian Federation.Moscow, Russia.
| Short Abstract We recruited 80 patients with ovarian cysts and with peritoneal endometriosis who then underwent laparoscopic surgery. The comparison group consisted of 40 patients with uterine fibroids and without endometriosis, which was proved by laparoscopy. Differences in the mass spectrometric profiles of the endometriotic tissues and comparison group were analyzed in combination with the morphological features of endometrioid heterotopy in order to find the specific metabolic biomarkers. Identified biomarkers were verified in the blood and peritoneal fluid of patients with endometriosis. A new possible non-invasive method for endometriosis diagnostic was proposed based on mass-spectrometric analysis of blood plasma. |
Long Abstract
Introduction
Endometriosis is a benign recurrent gynecological disorder characterized by presence of endometrial tissue outside the uterine cavity. It is one of the most common diseases in the gynecological field, affecting 10-15% of women in reproductive age. Endometriosis induces a chronic inflammatory process. The incidence of endometriosis in women with dysmenorrhea is up to 40–60%, whereas in women with subfertility is about 20–30%. The pathogenesis of endometriosis is still unknown and current therapies are only symptomatic. The main method of treatment is complete removal of endometriosis foci by surgical methods. An important task is the quality control of removal of the endometriosis foci and prediction of disease relapses. The percentage of recurrence of external genital endometriosis varies greatly in different studies (from 6 to 67%). On average, there is a 7-11 year delay in diagnosis following the onset of symptoms. According to the consensus in the World Endometriosis Society, the development of a reliable noninvasive test is one of the top research priorities in endometriosis.
Mass spectrometry (MS) is one of the most widely used techniques for the analysis of biological samples. In the past decade, a novel improvement in MS was the invention of ambient ionization (direct MS) which stands out owing to its unique capability of direct analysis of complex samples with no or minimal pretreatment. Direct analysis in real time mass spectrometry (DART-MS) has also become an established technique for rapid mass spectral analysis of a large variety of samples.
The aim of this research was the development of fast and robust MS method for analysis of endometriotic tissues and biological fluids. This method will be a key instrument for the early and non-invasive diagnosis of endometriosis.
Methods
All the samples were collected during surgical procedures in the department of Operative Gynecology of the FSBE «Science Center of obstetrics, gynecology and perinatology, named after academician V.I. Kulakov» (Moscow, Russia). On the case–control study we recruited 80 patients with ovarian cysts and with peritoneal endometriosis who then underwent laparoscopic surgery between 2015 and 2016. The diagnosis of endometriosis was confirmed histologically. The age of the patients ranged between 22 and 41 years (mean age 31.5). In our study we included 57 patients in late proliferative/early secretory of the menstrual cycle (days 8 to 21), 19 patients in the proliferative phase of the menstrual cycle (cycle days 1 to 7), and 4 patients in the secretory phase (cycle days 22 to 30).
The main complaints of the patients were infertility (61 patients), chronic pelvic pain (non-cyclical) (59 patients), dysmenorrhea (68 patients), miscarriage (12).
Staging the disease was accomplished according to the American Society for Reproductive Medicine classification. According to this classification, 40 patients had I-II stage of endometriosis, the other 40 patients - III-IV stage. The primary inclusion criteria were: reproductive age, absence of any severe somatic pathologies, inflammatory diseases. Also, the patients had not been receiving any hormone therapy in the last 6 months before the surgery. The comparison group consisted of 40 patients with uterine fibroids and without endometriosis, which was proved by laparoscopy.
The endometriotic lesions after excision (without thermal effect), the capsules of endometrioid cyst after cystectomy, the endometrium after curettage were immediately placed in liquid nitrogen, followed by freeze-up at – 800 Ñ. Differences in the mass spectrometric profiles of the endometriotic tissues and comparison group were analyzed in combination with the morphological features of endometrioid heterotopy in order to find the specific biomarkers. At the second stage previously identified lipids and free fatty acids were verified in the blood and peritoneal fluid of patients with endometriosis.
Direct-spray-from-tissue method was used for on-line molecular species extraction and simultaneously ionization. The solvent was constantly delivered to the sample to provide stable ion current. After ionization, charged analytes were sampled through the ion optics system into the mass analyzer for further MS analysis. All MS and MS2 spectra were acquired by electrospray ionization quadrupole time of flight mass spectrometer (Maxis Impact, Bruker, Germany).
Results
148 metabolites were analyzed. 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 O-42:1), triglycerides (TG 41:2, TG 49:4, TG 52:3) showed significant differences between the biological samples of the patients with endometriosis and those of the control group. Blood plasma and peritoneal fluid MS analyses showed the presence of some species from the above list. A new non-invasive MS method for endometriosis diagnostics could be developed based on these data.
Conclusion
For the first time the Direct MS approach was applied to analyze endometrium and endometriotic lesions of different localization. The combination of this method with lipidomic study of blood plasma and peritoneal fluid was performed in order to understand the dysregulation in the metabolism and fluxes of lipids. Our clinical lipidomics approach defines the net outcome of several imbalanced lipids and fatty acids which may be crucial for endometriosis pathophysiology. Two important classes of lipids, the phosphatidylcholine and sphingomyelin, have been suggested as possible biomarkers for endometriosis as these lipids are strongly related to apoptosis suppression. The results of these studies may bring us closer to the understanding of the pathobiology of the disease and make it possible to elaborate new ways of early diagnostics, treatment targeted at the pathophysiological mechanisms and intelligent surgery.
References & Acknowledgements:
This work was supported by grant No. 16-14-00029 of RSF, and N.S. acknowledge MERF grant No. ÌÊ-8484.2016.7.
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