Immunohistochemical Analysis of the Expression of the Glycodelin Cytokine in Endometrial Tissue and the Endometrial Polyp, Before and After Hysteroscopy, in Infertile Female Patients

An endometrial polyp is most commonly a benign, localized proliferation of the glands and the endometrial stroma, covered with epithelium and protruding above the level of the mucosa. These polyps are most often diagnosed during investigation into the caus es of irregular menstrual bleeding or infertility. It is pro- duced in the highest concentration during the secretory phase of the endometrial cycle. The level of glycodelin reaches its peak 12 days after ovulation. The aim of this paper was to determine t he changes in the immunohistochemical expression of glycodelin at the level of the endometrium and in the tissue of the polyp, before and after hysteroscopic polypectomy, in infertile female patients with an endometrial polyp, and in the endometrial tissue of female patients without an endometrial polyp. The study included 82 in- fertile female patients. The infertile patients were divided into two groups. The first was the experimental group which included 56 infertile female patients who had an endometrial polyp. The sec- ond group was the control group, composed of 26 infertile female patients who did not have an endometrial polyp. The results ob- tained primarily indicate the existence of changes in the immuno- histochemical expression of the cytokine glycodelin in the female patients from both the experimental and the control group, not only prior to but also after hysteroscopy. A larger number of pa- tients who have an endometrial polyp show a lack of glycodelin expression, more pronouncedly so in the bioptate of the endome- trium than in the endometrial polyp.

Glycodelin , polyp, endometrium, infertility . SA Ž ETAK Polip endometrijuma je naj č e šć e benigna lokalizovana prolif- eracija ž lezda i strome endometrijuma pokrivena epitelom iznad ravni sluzoko ž e . Naj č e šć e se dijagnostikuju za vreme ispitivinja uzroka neurednog krvarenja iz materice ili neplodnosti . U najvećoj koncentraciji se produkuje tokom sekretorne faze endo- metrijalnog ciklusa. Nivo glikodelina dostiže svoj vrhunac 12 dana nakon ovul acije. Cilj ovog rada je bio da utvrdi promene u imunohistohemijskoj ekspresiji Glikodelina na nivou endometri- juma i u tkivu polipa, pre i posle histeroskopske polipektomije kod infertilnih pacijentkinja sa endometrijalnim polipom i u tkivu en- dometrijuma k od pacijntkinja bez endometrijalnog polipa. Istraživanjem je bilo obuhvaćno 82 infertilne pacijentkinje. Infer- tilne pacijentkinje su bile podeljenje u dve grupe. Prva grupa je označena kao eksperimentalna grupa pacijentkinja i činilo je 56 infertilnih paci jentkinja sa polipom endometrijuma. Druga grupa je označena kao kontrolna grupa pacijentkinja i činilo je 26 infer- tilnih pacijentkinja bez polipa endometrijuma. Dobijeni rezultati prvenstveno ukazuju na postojanje promena u imunohisto- hemijskoj ekspresiji c itokina Glikodelina kod pacijentkinja ek- sperimentalne i kod pacijentkinja kontrolne grupe, kako pre his- teroskopije tako i nakonhisteroskopije. Veći broj pacijentkinja sa polipom endometrijuma ne pokazuje ekspresiju Glycodelina, i to izraženije u bioptatu endometrijuma nego u polipu endometri- juma. Ključne reči : Glycodelin , polip, endometrijum, infertilitet . ABBREVIATIONS kDa - kilodalton ( atomic mass unit ) 1k Da = 1 , 000 dalton s Da - One d alton is defined as 1/12 of the m a s s of a single carbon - 12 atom . NK - n atural killer cells SI S - sonohysterography HE - hematoxylin - eosin PBS - p hosphate b uffered s aline UK NEQAS - UK National External Quality Assess- ment Scheme for Immunocytochemistry WHO - World Health Organization IH C – immunohistochemistry

According to the World Health Organization (WHO), in- fertility is a disease of the reproductive system defined by the failure to achieve a clinical pregnancy after a year or more of regular unprotected sexual intercourse ( 1) . In the world, around 10% of cou ples are infertile, and in Serbia the per- centage of infertile couples is 15% (1 ). Endometrial polyps are thickenings of the uterine mucosa consist ing of endome- trial glands, stroma and blood vessels (2 ). A polyp is formed as a focal proliferation of the endometrial basal layer , while thickened blood vessels can be found in the base of the polyp ( 3, 4 ). The rate of recurrence of endometrial polyps in the overall population is around 24%. Pathological uterine bleed- ing is the most common clinical symptom of an endometrial polyp. In infertile women, the diagnosis of an endometrial polyp is usually a coincidental finding (4) . The diagnosis of endometrial polyp is established on the basis of histopathology and sonographic findings. Hyster- osc opy is the golden standard in the diagnosis and therapy of endometrial polyps ( 5 , 6 , 7 ) . Cytokines are polypeptides and glycopeptides with a molecular mass of 6 - 10 kDa. Cyto- kines demonstrate their effects through specific receptors within the cell or in the cell membrane (8) . With the activa- tion of specific receptors , certain genes are activated, and as a result of this activation, phenotypic and functional changes occur in the target cell. Cytokines may be positive or negative regulators of immune response. The basic function of cyto- kines is in intercellular communication (9) . The activity of cytokines depends on their concentration. Depending on the influence that they h ave on the inflammatory reaction , cyto- kines are divided into proinflammatory and anti - inflamma- tory cytokines ( 10 ). Glycodelin, placental protein - 14 or oste- opontin ( 8 ) is a glycoprotein deposited in the glandular and epithelial cells of the endometrium (1 1, 12 ). It affects the en- dometrium through different cells (13) . In the human endometrium, during ovulation, the level of glycodelin is low 6 days before and 5 days a fter ovulation ( 14, 15) . This can facilitate or allow fertilization to occur (16) . During implantation the level of glycodelin signifi- cantly increases, prevents the action of NK cells, prepares the endometrium for implantation, and, to a certain extent, pr otects the embryo from the harmful effect of NK cell activ- ity (17 ).

The research was conducted in the form of a randomized cross - sectional study on a sample of 82 infertile female pa- tients , in the generative period. The first, experimental group comprised 56 infertile female patients with a diagnosis of en- dometrial polyp. The second, control group , consisted of 26 infertile patients who did not have an endometrial polyp. The study was carrie d out at the Obstetrics and Gynecology Clinic Narodni Front in Belgrade and at the Center for Mo- lecular Medicine and Stem Cell Research of the Faculty of Medical Sciences, University of Kragujevac . The criteria for inclusion in the study were: infertility lasting at least one year, age between 20 and 42 years, regular menstrual cycle, no use of any type of hormonal contraceptives or other hor- mon e products in the six months prior to the study, the fi nd- ing of an endometrial polyp confirmed by transvaginal 2D ultrasound examination or by SIS (for the experimental group , only ) , no other endometrial pathology confirmed by transvaginal ultrasound examination (4) . The criteria for ex- clusion from the study, for both groups of patients , were: the presence of submucosal myoma, endometriosis, endometrial carcinoma, uterine anomalies, patients who had undergone surgery of the uterus and fallopian tubes, patients with previ- ous unsuccessful ovulation stimula tion, and patients aged over 42 years. The study was approved by the Ethics Com- mittee of the Obstetrics and Gynecology Clinic Narodni Front, No. 04 - 24 / 3 - 1i and by the Ethics Committee of the Faculty of Medical Sciences, University of Kragujevac, No: 01 - 3 593 (4). Surgical procedures were performed on infertile patients in the first phase of the menstrual cycle, when the endometrial mucosa is the thinnest and when there is the least probability of interfering with early undiagnosed pregnancy ( 4, 7 ) . Research Methodology For the purpose of im m un o histochemical analysis, t he tis- sue of the endometrium and the polyp were fixed for 24 hours in 4% neutral buffered formalin, at room temperature . Then, the tissue sections were dehydrated, cleared in xylol and infiltrated with paraffin in the apparatus for automatic tissue sample fixation – the Leica TP1020 T issue Processor and were then embedded into paraffin blocks . P araffin molds ob- tained in this way were sectioned in the automatic Leica RM2155 Rotary Microto me into 4µm sections; they were then immersed in water at 56 °C and were finally placed on Superfrost glass microscope slides (18) . Hematoxylin and Eosin S taining (HE) The staining of paraffin tissue sections was performed b y the application of Heidenhain’s h ematoxylin - eosin

and in keeping with Gurr’s recommendations ( 19, 20 ) . S l ides with tissue sections obtained in this way were buffered in for- maldehyde buffer for 10 seconds, rinsed with running water and finally immersed for two minutes in Mayer’s hematoxy- lin (Merck). The tissue sections were then rinsed for one mi- nute in running wa ter and stained with alcoholic eosin (Merck) for one minute. After dehydration, tissue sections were dipped in a series of increasing ethanol concentrations, after which the clearing procedure was carried out, when the sections were immersed for 50 seconds in a mixture of xylol and ethanol (ratio 1: 1), and then twice in pure xylol, 50 sec- onds each time. A drop of Canada balsam (Centrohem, Ser- bia) was placed on the tissue sections . The tissue sections were covered with cover slips. After drying for 24 hours, the preparations were analyzed under a light microscope ( 21, 2 2 ) . T issue sections were heated at 56 ºC for a period of 60 minutes and were mounted onto adherent SuperFrost® slides. The slides with tissue sections were immersed in xy- lol, and then in a series of decreasing concentrations of etha- nol. For the purpose of antigen unmasking, tissue sections were h eated in a microwave oven in citrate buffer (pH = 6.0) for a period of 21 minutes. After cooling down, the prepara- tions were rinsed in distilled water and were then incubated three times, 10 minutes each time, in p hosphate b uffered s a- line (PBS ). For the pu rpose of fixation and permeabilization of the tissue sections, the slides with tissue sections were im- mersed in icy cold acetone, at 4 ºC, for 10 minutes. They were then rinsed in PBS. For the purpose of blocking the activity of endogenous peroxidase, seve ral drops of Hydrogen Perox- ide Block ing Reagent were applied on them. After ten minutes of incubation , the preparations were rinsed twice with PBS and then several drops of Protein Block were ap- plied on them . A fter a ten - minute incubation period, the Pro- tein Block was rinsed out, once , in PBS. Immunohistochemical staining Tissue sections were incubated overnight with primary antibodies in a humidity chamber at 4 ºC. The appropriate mouse monoclonal antibodies (Ab17247, m onoclonal 001 - 13, provided by Abcam) were used in the appropriate attenu- ation (1:200) dissolved in PBS (22). Tissue sections were rinsed in PBS, and appropriate commercial detection kits were used for antigen visualization (4, 23, 24). The repara- tions were covered with glycerol and a cover slip. Immuno- histochemical staining was performed, with control for quality and specificity of staining, in keeping with the UK NEQAS (UK National External Quality Assessment Scheme for Immunocytochemistry) standards, and w ith the use of positive and negative controls (20 ). Different expression of epithelial endometrial cells was obtained with staining, in proportion to the binding of the dye to the cells (21) . Statistical data processing Sample size was determined on the basis of the formula for calculating large samples , implemented in the PASS 11.0 software. All the data obtained were processed using descrip- tive statistics methods (25) . All results were processed with the aid of the SPSS 22.0 software p ackage ( 2 6 ) . The results obtained were compared to those of other authors available in literature (27) .

were made on the basis of the

obtained (4, 22) . For values that had a normal distri- bution, we used the Student’s t test and Student’s paired t test, and for other distributions nonparametric Mann - Whitney and Wilcoxon test is used. All statistical analyzes were performed with a 95% confidence interval. If the probability level of the null hypothesis is <5%, and if the significance of the test is p <0.05 the results of the statistical analysis were accepted as statistically significant.

This segment presents the analysis of the expression of the cytokine glycodelin obtained through the application of the immunohistochemistry method. Immunohistochemical expression of glycodelin Table 1 and Graph 1 show the immunohistochemical (IHC) expre ssion of the cytokine glycodelin in the polyp and the endometrium of the patients belonging to the experi- mental group and the endometrium of the patients from the control group. Table 1 . Immunohistochemical expression of glycodelin, in polyp tissue, endometrial biopsy of patients with polyp and without polyp Experimental group N % Pol yp 9 18 Biop tate of the endometrium 1 2 Control group Biop tate of the endometrium 1 3.8 N – number of patients % – number of patients in percentages Graph 1 . Immunohistochemical expression of glycodelin, in polyp tissue, endometrial biopsy of patients with polyp and without polyp Statistical processing of the data shown in Table 1 and Graph 1 found that a very highly statistically significant ly greater number of patients from the experimental group did not have IHC expression of glycodelin in the polyp (χ2 = 20.480; p<0.001) , as compared to the patients within the group who did have IHC expression of glycodelin in the polyp . In the endometrial bioptate of the same patient group a very highly statistically significant ly greater number of pa- tients did not demonstrate IHC expression of glycodelin (χ2 = 46.080; p<0.001) as compared to those within this group who did demonstrate IHC expression in the endometrial bi- optate . In the control group of infertile female patients there was also a very highly statistically significant ly larger n um- ber of patients who did not demonstrate IHC expression of glycodelin in the endometrial bioptate as compared to the number of patients in whom IHC expression of glycodelin was found in the endometrial bioptate (χ2 = 22.154; p<0.001). When IHC glycodelin expression was compared between the polyp and the endometrial bioptate of the experimental group of patients, a statistically significant difference in IHC glycodelin expression was found (Z = - 2.530; p<0.05). Signal intensity of immunohistochemical glycod elin expression Table 2 and Graph 2 show the signal intensity of gly- codelin immunohistochemical expression in the polyp and the endometrium of the patients from the experimental group and the endometrium of the patients from the control group. As shown in Table 2 and Graph 2, in most subjects, an IHC signal of glycodelin expression was registered neither in the polyp or the endometrial bioptate of the experimental pa- tient group, nor in the endometrial bioptate of the control group of patients . In the polyp of the experimental group, in patients who demonstrated glycodelin expression, a low - in- tensity signal was registered in 5 (10%) of patients, a me- dium - intensity signal was registered in three (6%) patients, and a high - intensity signal was registered in one (2%) patient . In the endometrial bioptate of the experimental group, a low - intensity signal of ICH glycodelin expression was registered in only one (2%) patient , which was also the case in the co n- trol group of patients – a low - intensity signal was registered in one (3,8%) patient . Table 2 . Signal intensity of IHC glycodelin expression, in polyp tissue, endometrial biopsy of patients with polyp and without polyp Experimental group Low - in- tensity signal (%) Medium - intensity signal (%) High - in- tensity sig- nal (%) Polyp 10 6 2 Biop tate of the endometrium 2 0 0 Control group Biop tate of the endometrium 3.8 0 0 Signal intensity : low - intensity signal, medium - intensity signal, high - intensity signal Graph 2. Signal intensity of IHC glycodelin expression, in polyp tissue, endometrial biopsy of patients with polyp and without polyp Statistical processing of the data shown in Table 2 and Graph 2 found a very highly statistically significant differ- ence in the signal intensity of IHC glycodelin expres sion, both in the polyp (χ2 = 87.280; p<0.001) and in the endome- trial bioptate (χ2 = 46.080; p<0.001) of the experimental group of patients. The same was true for the control group. The frequency of no signal registered was very highly statis- tically signif icantly greater than the registering of a low - in- tensity signal (χ2 = 22.154; p<0.001). In the experimental group of patients, t he distribution of IHC glycodelin expres- sion signal intensity was statistically significantly different between the endometrial b ioptate and the polyp (Z = - 2.511; p<0.05). The signal intensity of IHC glycodelin expression in the polyp of the experimental group of patients and the en- dometrial bioptate of the control group of patients d id not show a statistically significant differen ce (Z = - 1.753; p>0.05) , as was the case within the control group of patients among the endometrial bioptate specimens . (Z = 0.474; p>0.05). Microscopic view of immunohistochemical glycodelin expression Image 1 shows the microscopic view of the signal inten- sity of IHC glycodelin expression in the endometrial bioptate of the patients from the control group, at x 10 magnification. Images 2 and 3 represent microscopic views of the signal in- tensity of IHC glyc odelin expression in the endometrial polyp of the experimental patient group, at x 10 and x 20 mag- nification , respectively. Image 4 shows a microscopic view of the signal intensity of IHC glycodelin expression in the endometrial bioptate of patients from the control group, at x 10 magnification . Image 5 shows a microscopic view of IHC glycodelin expression in the tissue of the placenta and the epithelium of the chorionic villi, at x 20 magnification (ex- ternal positive control of immunohistochemical staining). Image 1 . Microscopic view of IHC glycodelin expression in the endometrial bioptate of the infertile patients without a verified endometrial polyp ( x10 magnification) Image 2 . Microscopic view of IHC glycodelin expression in the tissue of the endometrial polyp of infertile patients with- out a verified endometrial polyp ( x10 magnification) Image 3. Microscopic view of IHC glycodelin expression in the tissue of the endometrial polyp of infertile patients with a verified endomet rial polyp ( x20 magnification) Image 4 . Microscopic view of IHC glycodelin expression in the endometrial bioptate of infertile patients without a verified endometrial polyp( x10 magnification) Image 5. Microscopic view of IHC glycodelin expression in the tissue of the placenta and the epithelium of the chorionic villi (external positive control of immunohistochemical staining, x20 magnification) LITERATURE 1. Zegers - Hochschild F, Adamson GD, de Mouzon J, Ishihara O, Mansour R, Nygren K, Sullivan E, Van der Poel S. The international committee for monitoring assisted reproductive technology (ICMART) and the world health organization (WHO) revised glossary on ART terminology. Hum Reprod. 2009 Oct 4;24(11):2683 - 7. 2. Bassil S. 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