Analysis of Glycodelin Levels Before and After Hysteroscopic Polypectomy in Infertile Patients

Glycodelin (or placental protein 14) is a glycoprotein located in the glandular and thin epithelium of the endometrium. It is considered an important factor in the implantation process, and its traces can be found in elevated concentrations in the uterine fl ushing obtained at the time of implantation, while in the prolif- erative phase of the cycle, its levels are low. A certain concentra- tion has been found to inhibit the binding of spermatozoids to the zona pellucida of the oocites therefore, it eff ects conception. It has a role in angiogenesis and is in high concentrations in the tissues of both benign and malignant gynaecological tumours. Th e aim of this study is to analyse and display the gly- codelin level changes before and after hysteroscopic polyp- ectomy in infertile patients in the uterine fl ushing fl uid and serum. Th is survey covers 80 infertile patients, who were divided into two groups. Th e fi rst group, the experimental group, consisted of 50 infertile patients with endometrial polyps, and a control group of 30 infertile patients without endometrial polyps was also included. Th e results primarily indicate the existence of changes in glycodelin levels preoperatively in the fl ushing and venous blood in infertile patients with endometrial polyps compared with the levels after surgery. In the control group of patients, no signifi cant change in the glycodelin levels was detected in the fl ushing and venous blood. When comparing these two groups, statistically signifi cant diff erences in the glycodelin levels in the fl ushing and venous blood were noted. We con- clude that the presence of endometrial polyps in the cavum uteri aff ects the increase in the glycodelin concentration in the fl ushing fl uid and in the plasma. Increased glycodelin concentrations complicate fertilization and implantation.

cytokine, glycodelin, endometrial polyp, fl ushing and serum. ANALYSIS OF GLYCODELIN LEVELS BEFORE AND AFTER HYSTEROSCOPIC POLYPECTOMY IN INFERTILE PATIENTS Marija Sorak1*and Ana Devic2* 1Department of Gynecology, Faculty of Medical Sciences, University of Kragujevac, Kragujevac, Serbia 2Clinical Hospital Center Zemun, Hospital for Gynecology and Obstretics, Serbia *Authors Marija Sorak and Ana Devic contributed equally to this study ANALIZA NIVOA GLIKODELINA PRE I POSLE HISTEROSKOPSKE POLIPEKTOMIJE KOD INFERTILNIH PACIJENTKINJA Marija Šorak1*i Ana Dević2* 1Katedra za ginekologiju, Fakultet medicinskih nauka, Univerzitet u Kragujevcu, Kragujevac, Srbija 2Kliničko bolnički centar Zemun, Bolnica za ginekologiju i akušerstvo, Srbija *Autori Marija Šorak i Ana Dević su podjednako učestvovali u izradi ove studije Received / Primljen: 13. 12. 2016. Accepted / Prihvaćen: 17. 01. 2017. SAŽ ETAK Glikodelin (ili placentni protein 14) je glikoprotein koji se nalazi u glandularnom i površinskom epitelu endome- trijuma. Smatra se da je jedan od bitnih faktora u proce- su implantacije i nalazi se u povišenim koncentracijama u ispirku uterusa dobijenom u vreme implantacije, dok su u proliferativnoj fazi ciklusa njegove vrednosti niske. U određenoj koncentraciji nađeno je da inhibira vezivanje spermatozoida za zonu pelucidu ovuuma, tako da utiče i na koncepciju. Ima ulogu u angiogenezi, te se nalazi u povišenim koncentracijama u tkivu i benignih i malignih ginekoloških tumora. Cilj ovog rada je analiza i prikaz promena nivoa gliko- delina, pre i posle histeroskopske polipektomije kod infertil- nih pacijentkinja, u ispirku uterusa i serumu. Istraživanje obuhvata 80 infertilnih pacijentkinja, koje su podeljene u dve grupe. Prvu grupu, eksperimentalnu, čini 50 infertilnih paci- jentkinja sa polipom endometrijuma, a kontrolnu grupu 30 infertilnih pacijentkinja bez polipa endometrijuma. Dobijeni rezultati prvenstveno ukazuju na postojanje promena nivoa g likodelina preop erativno, i u ispirku i u venskoj krvi, kod infertilnih pacijentkinja sa endometrijal- nim polipom u odnosu na nivoe nakon operativnog zahvata. U kontrolnoj grupi pacijentkinja nema značajnih promena nivoa glikodelina u ispirku i venskoj krvi. Poređenjem ove dve grupe prisutne su statistički značajne razlike u nivoima glikodelina i u ispirku i u venskoj krvi. Možemo zaključiti da prisustvo endometrijalnog polipa u kavumu uterusa utiče na porast koncentracije g likodelina, i u ispi rku i u plazmi. Povećana koncentracija glikodelina otežava i oplodnju i im- plantaciju. Ključne reči: citokin, glikodelin , endometrijalni polip, ispirak i serum. 248

Infertility is a problem that affects approximately 80 million couples worldwide. It is believed that approximate- ly 15% of the general population is affected by this prob- lem. Endometrial polyps occur when the endometrium is hypertrophic, due to oestrogen stimulation (1). A diagno- sis is based on sonographic, hysteroscopic or hysterosono- graphic findings, when a polyp is spotted in the cavum uteri (2). Cytokines are a large family of protein molecules that function as a mediator and regulator of cellular communi- cations, in both physiological and pathological conditions (3). Different cells produce a variety of cytokines, which act on the chemotaxis, activation, proliferation and dif- ferentiation of other cells. Cells that secrete the highest amounts of cytokines are leukocytes. Glycodelin (or pla- cental protein 14) is a cytokine and is a 28kDa glycoprotein that contains 180 amino acids (4). Glycodelin is encoded by a single gene that is located in the chromosomal region 9q34; it is derived from the endometrium. It consists of a single (GaINAcβ1-4GlcNAc) oligosaccharide series and is located in the glandular and surface epithelium of the endometrium. Glycodelin is the primary product during the secretion phase of endometrial cells. Maximum lev- els are produced in the secretory endometrium and early pregnancy decidual. It is produced in the lumen of the en- dometrial glands and can be detected in circulation. The highest concentration of glycodelin is produced during the secretory phase of the endometrial cycle, while at the early proliferative phase, its concentration is low. There are strong indicators that, in conjunction with other cytokines, glycodelin represents an important factor that facilitates implantation (5). It is found in elevated concentrations in the uterine flushing obtained at the time of implantation. During conception, glycodelin levels remain elevated. Gly- codelin has an immunosuppressive effect and probably has a protective role in the preservation of embryos from natural killer (NK) cell destruction. Additionally, it has been found that in certain concentrations, glycodelin inhibits the bind- ing of sperm to the zona pellucida of the oocites therefore, it effects conception. Recently, it has been shown that it plays a role in angiogenesis and is elevated in the tissues of both benign and malignant gynaecological tumours (6).

The survey was conducted as a study of 80 infertile pa- tients. The two groups of female patients were compared. The first group of patients, the experimental group, con- sisted of 50 infertile patients diagnosed with endometrial polyps. The second group was a control group of 30 in- fertile patients without endometrial polyps. The research was carried out at the Gynaecology and Obstetrics Clinic “National Front” in Belgrade, from May 2012 to November 2013, and in the Center for Molecular Medicine and Test- ing of Stem Cells, Faculty of Medical Sciences, in Kragu- jevac. This study was conducted on patients with childbear- ing potential (aged 20-43 years) who were diagnosed with endometrial polyps as a cause of infertility. The polyp diag- noses were determined by transvaginal ultrasound exami- nations during the first phase of the cycle, by hysterosono- graphic examinations, or during an actual hysteroscopy in patients with suspected endometrial polyps, based on anamnesis. In addition to the group who participated in the study, there was a control group of patients of the same age who were treated for infertility did not have endome- trial polyps. The patients were evaluated for one month after the intervention in terms of peripheral venous blood sampling, cytokine level determinations, and control ultra- sound examinations. Diagnostic and operative hysteroscopies were per- formed under general anaesthesia in the operating room with appropriate equipment and instruments. The preop- erative preparation included a complete diagnosis and the necessary routine analyses including cervical and vaginal smears, chlamydia, mycoplasma and ureaplasma evalua- tions, Pap test, ultrasound, blood typing, blood count and biochemical analysis with an anaesthetic examination and surgical treatment approval. Surgical procedures were per- formed in infertile patients to mid-proliferative phase im- mediately after menstrual bleeding. Hysteroscopic examination and intervention-polyp- ectomy are done during the first phase of the menstrual cycle (9). During hysteroscopy, the uterine cavity is viewed at a 30-degree angle, which allows for visualization of both mouths of the fallopian tube, fundus, anterior and poste- rior wall of the uterus as well as the lateral sides of the uter- ine cavity (10). At the height of the internal uterine mouth with the hysteroscope, a panoramic image of the uterine cavity is displayed, and the presence of any pathological findings, such as endometrial polyps (which are often ob- served), submucosal fibroids, septa, and adhesions, can be easily visualized. Polyps are removed with hysteroscopic graspers and hysteroscopic scissors followed by electroco- agulation of their base (11). To determine the glycodelin (PP14) concentration in the uterus flushing during hysteroscopic polypectomy, the operation sequence was as follows: 10 ml injection of sa- line into the cavum uteri and immediate aspiration with- out contamination. Then, the flushing was centrifuged at 2500 rpm for 10 minutes. The supernatant was collected and stored at -20° C. The glycodelin concentration was de- termined with the ELISA method at the Center for Mo- lecular Medicine and Testing of Stem Cells, at the Faculty of Medical Sciences in Kragujevac (15). To determine the glycodelin (PP14) serum concen- tration, approximately 5 ml of peripheral venous blood was taken from patients in both groups, before hystero- scopic surgery and one month after surgery. The sample processing methodology is as follows: the blood was col- lected in a vacutainer with heparin and centrifuged for 249 10 minutes at 2500 rpm. The supernatant was collected and stored at -20 ° C. Detection and determination of the glycodelin concentration was then determined with the ELISA method (16). The study inclusion criteria were women with endo- metrial polyps who were verified as 20-43 years of age and treated for primary or secondary infertility as well as infertile women without endometrial polyps of the same age (16). Criteria for exclusion from the study for both groups of respondents were the existence of submucosal fibroids, endometriosis, endometrial cancer, uterine anomalies, and patients who had surgery on their uterus and tubes, as well as patients with a previous failed ovulation following stimulation. Statistical data analysis was based on use of the t-test. As the sample distribution in this study (with a minimum group of 30 patients) was mildly leptokurtic and skewed, for this study, t-tests were successfully implemented, and even for similar mean values of the sample, the t-test pro- vided more accurate results than the Wilconxon Mann- Whitney test (17).

We analysed the glycodelin levels in the uterus flush- ing and venous blood from both test and control group pa- tients (8). The levels of this cytokine were examined before and one month after hysteroscopic polypectomy. Statistical analysis of the patient groups was based on testing the null hypothesis with t-tests of group differences according to the mean value and variance (7). The groups were significantly different if p <0.05. Figure 1 shows the glycodelin values in the uterine flush- ing from patients with endometrial polyps compared with the control group during surgery. It can easily be seen that the glycodelin levels in the uterine flushing were signifi- cantly decreased in the control group subjects compared with the experimental group. Using the t-test and analysis of the obtained results, it can be concluded that there was a statistically significant difference (t=1.992; p=0.0144). Analysis of the venous blood glycodelin levels in the en- dometrial polyp patients is shown in Figure 2. Notably, the venous blood glycodelin levels that were collected a month after hysteroscopic polypectomy were significantly lower. By applying the t-test, it can be concluded that the groups differ statistically (t=2.01; p=0.00017). The venous blood glycodelin values in the endometrial polyp and control group patients before hysteroscopic pol- ypectomy are shown in Figure 3. Applying a t-test leads to the conclusion that the groups differ statistically and that the glycodelin levels were significantly higher in the endo- metrial polyp patients (t=1.996; p=0.0036). Figure 4 provides the venous blood glycodelin values in the control group before and one month after hysteros- copy. By applying the t-test, it can be concluded that the groups did not differ statistically, i.e., the glycodelin levels did not significantly differ in the control group before and after hysteroscopy (t=2.048; p=0.851). The venous blood glycodelin values in patients one month after hysteroscopic polypectomy and in the control group one month after hysteroscopy are shown in Figure 5. These levels were found to not be significantly different using the t-test (t=2.004; p=0.838).

The glycodelin concentrations in the flushing and se- rum were significantly higher in patients with endometrial polyps in comparison with the control group. A month af- Figure 1. Glycodelin average values in the uterine fl ushing from patients with endometrial polyps compared with control group patients during hysteroscopy. Th ese are the t-test parameters, the critical average values for accepting the null hypothesis. 250 Figure 2. Venous blood glycodelin average values in patients with endometrial polyps before hysteroscopic polypectomy and one month after surgery. glycodelin (pg/mol) Before After Hysteroscopic polypectomy Figure 3. Venous blood glycodelin average values in the endometrial polyp and control group patients before hystero- scopic polypectomy. glycodelin (pg/mol) Еxperimental group Control group Before hysteroscopic polypectomy ter hysteroscopic polypectomy, the serum glycodelin levels were reduced in the experimental group and reached values very similar to the those of the control group. These results were confirmed in most studies that examined glycodelin levels in the uterine flushing and plasma of infertile patients. Endometrial polyps are common in infertile patients who are preparing for in vitro fertilization, and their pres- ence does not affect pregnancy rates, but there is a higher risk of pregnancy loss in these patients (18, 19). Polyps are endometrial tissue growths covered with epithelium. They have glands, stroma and blood vessels that contrib- ute to increased glycodelin secretion in the cavum uteri (12, 13). The common assumption is that glycodelin may “leak” from blood vessels near polyps and dissolve in sa- line, which is used during hysteroscopy uterine flushing (14, 20). Glycodelin levels in uterus flushing fluid are then determined using ELISA measurements, and in most sur- veys, it was confirmed that the values were significantly higher in uterine flushing with endometrial polyp or sub- mucosal fibroids compared with the control group (21). 251 Figure 5. Venous blood glycodelin average values in the endometrial polyp and control group patients one month after surgery. Еxperimental group Control group After hysteroscopic polypectomy Figure 4. Th e venous blood glycodelin average values in the control group patients before hysteroscopy and one month after surgery. glycodelin (pg/mol) Before After Hysteroscopic polypectomy 252 Glycodelin contributes to polyp growth by encouraging neovascularization and thus allowing a better flow of the nutrients necessary for polyp growth. It can be detected in the circulation; therefore, in various studies, its concen- tration in plasma was often determined. In most of these studies, elevated glycodelin levels in the plasma of patients with endometrial polyps was observed compared with control group patients (22). It has been proven that glycodelin inhibits sperm and oocyte binding as well as NK cell activity (23). Glycodelin values are very low in the period 6 days before and 5 days after ovulation (periovulatory period). Thus, low glycode- lin levels allow for fertilization. Then, 6 days after ovula- tion, glycodelin secretion increases significantly and be- comes a key to the formation of a receptive endometrium and suppresses NK cell activity. The results of this study indicate that the glycodelin levels measured during the middle and late proliferative phase of the menstrual cycle were elevated when they should have been at their lowest values, or even absent; however, they significantly affected fertilization and altered endometrial receptivity in patients with endometrial polyps (20). Changes in glycodelin concentrations are registered in the endometrium, or uterine flushing and are correlated with the values obtained in the serum of the experimental group patients. We conclude that changes in the glycodelin values can be tracked by just specifying the venous blood in patients in whom a hysteroscopic polypectomy was per- formed and can thus monitor the success of the operational procedures and limit possible polyp recurrences by avoid- ing administering complicated uterus flushings, which require preoperative preparation and general anaesthesia during a hysteroscopy.

Two groups of infertile patients were analysed, one with endometrial polyps and a second control group that did not have polyps. The glycodelin values in the uterus flushing fluid and in the venous blood before surgery were significantly higher in the patients with endome- trial polyps. The uterus flushing fluid and venous blood glycodelin values in the polyp group were significantly decreased one month after surgery. The glycodelin levels in the serum of the control group patients who were de- tected preoperatively remained similar, even one month after surgery. Additionally, there was no significant dif- ference in the glycodelin levels one month after surgery in a group of patients where the polyps were removed com- pared with the control group patients. The results of this study clearly indicate that the gly- codelin levels in infertile patients with endometrial polyps were elevated; therefore, we can safely conclude that gly- codelin is a cytokine that has a crucial impact on endome- trial miles and can significantly disturb homeostasis at the molecular level, thus affecting fertility. Аcknowledgement This work was supported by the Faculty of Medical Sci- ences, University of Kragujevac, Serbia (Grants JP 11/14 (13-2015) “Cytokine levels before and after hysteroscopic polypectomy in infertile patients).

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