INTRODUCTİON Recurrent pregnancy loss (RPL) �s defined as at least 2 consecut�ve spontaneous abort�ons before the 20th week of pregnancy w�th the same partner [1, 2]. The Amer�can Soc�ety of Reproduct�ve Med�c�ne (ASRM) has defined RPL as 2 or more consecut�ve pregnancy losses [3], whereas �n the most recent gu�del�nes of the Reproduct�ve Health and Infert�l�ty Assoc�at�on, RPL �s defined as 3 or more consecut�ve pregnancy losses [4]. In a study by W�lcox et al, a strong relat�onsh�p was shown between early pregnancy loss �n part�cular and endometr�al recept�v�ty [5]. These receptors play a role �n the control mechan�sm dur�ng embryo �mplantat�on and th�s recept�v�ty �s thought to be �map�red �n pat�ents w�th RPL [6]. Implantat�on of the blastocyst to the endometr�um may only be poss�ble for a very short per�od and th�s �s known as the �mplantat�on w�ndow. As th�s �s a very compl�cated event, both the embryo and the endometr�al receptors must be well-synchron�sed [7]. Of these receptors exam�ned �n the current study, Muc�n-1 (MUC-1) has been prev�ously �nvest�gated �n pat�ents w�th recurrent �mplantat�on d�sorder appl�ed w�th �nv�tro fert�l�sat�on and both the t�ssue and blood levels were found to be lower compared to the control group [8]. In the same study, Glycodel�n-A (GdA) levels were also found to be s�gn�ficantly lower both �n t�ssues and blood �n compar�son w�th the control group. In a study of endometr�al b�ops�es of RPL pat�ents, Leukem�a Inh�b�tory Factor (LIF) was shown to be lower �n the endometr�um compared to a control group, but the d�fference was not reported to be stat�st�cally s�gn�ficant [9]. In another study that exam�ned LIF and Interleuk�n-15 (IL-15) �n the endometr�al b�ops�es of pat�ents w�th recurrent �mplantat�on d�sorder, lower levels were observed �n the pat�ents than �n the control group [10]. Granulocyte Colony St�mulat�ng Factor (G-CSF) �s a hematopo�et�c cytok�ne wh�ch st�mulates granulocyte prol�ferat�on and d�fferent�at�on, produces the maternofetal �nterface dur�ng embryo �mplantat�on and �s thought to have a role �n dec�dual and placental funct�ons [11, 12]. Some stud�es have shown that the appl�cat�on of G-CSF to pat�ents w�th recurrent �mplantat�on d�sorder and RPL pat�ents could be useful �n respect of pregnancy outcomes [13-15]. From the start�ng po�nt of the above-ment�oned stud�es, the a�m of th�s study was to compare the serum levels of MUC-1, GdA, LIF, IL-15 and G-CSF �n RPL pat�ents and �n pat�ents who had not exper�enced any pregnancy loss and had at least one l�ve b�rth, and to be able to establ�sh an alternat�ve treatment approach based on the factors w�th the greatest effect. MATERİALS AND METHODS Pat�ent Select�on The study was conducted �n the Obstetr�cs and Gynaecology Cl�n�c of Evl�ya Çeleb� Tra�n�ng and Research Hosp�tal of Dumlup�nar Un�vers�ty, Kutahya, Turkey. A total of 87 pat�ents were �ncluded �n the study as 42 RPL pat�ents and 45 healthy pat�ents. Approval for the study was granted by the Eth�cs Comm�ttee of Ayd�n Adnan Menderes Un�vers�ty Med�cal Faculty Non-Intervent�onal Eth�cs Comm�ttee (Dec�s�on no: 11/11/2016-11). Pat�ents present�ng at the Obstetr�cs and Gynaecology Cl�n�c of Dumlup�nar Un�vers�ty Med�cal Faculty w�th a h�story of recurrent pregnancy loss were �nformed about the study, and those who w�shed to part�c�pate and met the �nclus�on cr�ter�a were �ncluded as the study group. The �nclus�on and exclus�on cr�ter�a are presented below �n Sect�on 2-2. The control group was formed of pat�ents selected from those who presented at the polycl�n�c, met the study �nclus�on cr�ter�a and w�shed to part�c�pate. Demograph�c data of age, abortus, par�ty and grav�da were recorded. Med�cal h�stor�es were taken �nclud�ng med�cat�on use and chron�c d�seases. Inclus�on and Exclus�on Cr�ter�a Inclus�on cr�ter�a for the RPL group: 1. At least 2 consecut�ve abortus, THE EFFECT OF MUCİN-1, GLYCODELİN-A, LEUKEMİA INHİBİTORY FACTOR, INTERLEUKİN-15 AND GRANULOCYTE COLONY STİMULATİON FACTOR IN THE UNEXPLAINED RECURRENT PREGNANCY LOSS - A CASE CONTROL STUDY Original Research Paper Murat Polat Department of Obstetrics and Gynecology Faculty of Medicine, Dumlupınar University, Kütahya, Turkey. Gynaecology Purpose: To exam�ne the effect on recurrent pregnancy loss of Mucın-1, Glycodel�n-a, Leukem�a Inh�b�tory Factor, Interleuk�n-15 and Granulocyte Colony St�mulat�on Factor blood serum levels and for those w�th the greatest effect to be able to contr�bute to alternat�ve treatment approaches. Mater�als and Methods: The study was conducted between November 2016 and December 2016 �n the Obstetr�cs and Gynaecology Cl�n�c of Evl�ya Celeb� Tra�n�ng and Research Hosp�tal, Dumlup�nar Un�vers�ty Med�cal Faculty. A total of 87 pat�ents were �ncluded, compr�s�ng 42 pat�ents who had exper�enced at least 2 consecut�ve pregnancy losses before the 12th week, and a control group of 45 pat�ents w�th at least 1 l�ve b�rth and no pregnancy loss. Exam�nat�on was made of the blood plasma levels of Mucın-1, Glycodel�n-a, Leukem�a Inh�b�tory Factor, Interleuk�n-15 and Granulocyte Colony St�mulat�on Factor. Results: In the pat�ent and control groups the Muc�n-1 plasma levels were determ�ned as 0.88±0.45 ng/ml and 1.17±0.57 ng/ml (p=0.008) respect�vely, Glycodel�n-a as 23.73 ng/ml and 25,92 ng/ml (p=0.006). Leukem�a Inh�b�tory Factor as 64.59 pg/mL, and 72.14 pg/mL (p=0,011), Interleuk�n-15 as 38.57 ng/ml and 31.44 ng/ml (p=0.013), and Granulocyte Colony St�mulat�on Factor as 13.07 pg/mL and 14.11 pg/mL (p=0.056). Conclus�on: Accord�ng to the results of th�s study, Mucın-1, Glycodel�n-a, Leukem�a Inh�b�tory Factor, and Interleuk�n-15 play an �mprtant role �n the pathology of RPL. Further stud�es conducted on these factors could be successful �n the treatment of RPL. ABSTRACT KEYWORDS : Recurrent pregnancy loss, Muc�n-1, Glycodel�n-a, Leukem�a Inh�b�tory Factor, Interleuk�n-15, Granulocyte Colony St�mulat�on Factor Nadi Keskin Department of Obstetrics and Gynecology Faculty of Medicine, Dumlupınar University, Kütahya, Turkey. Ghanim Khatib* Department of Obstetrics and Gynecology Faculty of Medicine, Çukurova University, Adana, Turkey. *Corresponding Author 56 X GJRA - GLOBAL JOURNAL FOR RESEARCH ANALYSIS VOLUME-7, ISSUE-12, DECEMBER-2018 • PRINT ISSN No 2277 - 8160 2. Aged at least 18 years, 3. Max�mum age of 35 years to d�scount reduced ovar�an reserve, 4. No prev�ous l�ve b�rth. Inclus�on cr�ter�a for the control group 1. No h�story of pregnancy loss, 2. Age 18 years – 35 years, 3. At least one l�ve b�rth. Exclus�on cr�ter�a for both the study group and the control group: 1. Uter�ne anomaly (septate, b�cornate, un�corn uterus etc.), 2. Myoma uter�s 3. Endometr�al polyps 4. Endocr�nolog�cal d�sorders (d�abetes, polycyst�c ovary syndrome, thyro�d funct�on d�sorder) 5. Known chromosome anomaly that could cause RPL 6. C�garette smok�ng and alcohol consumpt�on 7. Act�ve �nfect�on (as �t could affect the molecule levels �n the study) 8. Ol�gomenorrhea -amenorrhea w�th a h�story of abnormal uter�ne bleed�ng wh�ch could affect endometr�al recept�v�ty, or use of �ntra-uter�ne dev�ce or oral contracept�ves 9. At least one d�agnos�s of thromboph�l�a Blood Sample Collect�on As the molecules to be stud�ed are at the h�ghest amount �n the �mplantat�on w�ndow, blood samples were taken from all the part�c�pants �n th�s per�od. The blood samples were taken one week before the expected menstruat�on as LH �s at a peak 7-10 days later. After collect�on, the samples were centr�fuged at 10,000 rpm/m�n for 10 m�ns and serums were obta�ned and placed �n 2ml, ster�le, clear Eppendorf cryo tubes. The samples were stored at -20˚C out of d�rect l�ght unt�l assay. Pat�ents w�th hemorrhag�c, �cter�c or l�pem�c serums were excluded from the study. B�ochem�cal Analys�s The samples were exam�ned us�ng the Enz yme -l �nked Immunosorbent Assay (ELISA) method �n a pr�vate b�ochem�stry laboratory by a med�cal b�ochem�stry spec�al�st. MUC-1 was exam�ned w�th the ELISA k�t of Elabsc�ence B�otechnology Co. Ltd., wh�ch uses the Sandw�ch-ELISA method. In th�s k�t, the m�cro ELISA plate �s first covered w�th ant�body spec�fic to human MUC-1. Standards or samples are added to the appropr�ate m�cro ELISA plate wells and comb�ned w�th spec�fic ant�body. Then a determ�nant ant�body w�th b�ot�n, spec�fic to human MUC-1 and Av�d�n-Horserad�sh Perox�dase (HRP) conjugate �s added to each m�cro-plate and �ncubated. Free elements are washed and substrate solut�on �s added to each well. Wells conta�n�ng only human MUC-1, determ�nant ant�body w�th b�ot�n and Av�d�n-HRP conjugate are seen w�th a blue colour. The enzyme- substrate react�on �s term�nated by add�ng sulphur�c ac�d solut�on and the colour becomes yellow. Opt�c dens�ty (OD) �s measured spectrophotometr�cally at a wavelength of 450 nm± 2nm. The OD value �s proport�onal to the human MUC-1 concentrat�on. The colour formed �s measured spectrophotometr�cally. Then the concentrat�ons of the samples exam�ned are calculated from the drawn cal�brat�on curve. GdA was exam�ned w�th the ELISA k�t of Elabsc�ence B�otechnology Co.Ltd, wh�ch uses the Sandw�ch-ELISA method. In th�s k�t, the m�cro ELISA plate �s first covered w�th ant�body spec�fic to GdA. Standards or samples are added to the appropr�ate m�cro ELISA plate wells and comb�ned w�th spec�fic ant�body. Then a determ�nant ant�body w�th b�ot�n, spec�fic to GdA and Av�d�n-Horserad�sh Perox�dase (HRP) conjugate �s added to each m�cro-plate and �ncubated. Free elements are washed and substrate solut�on �s added to each well. Wells conta�n�ng only GdA, determ�nant ant�body w�th b�ot�n and Av�d�n-HRP conjugate are seen w�th a blue colour. The enzyme- substrate react�on �s term�nated by add�ng sulphur�c ac�d solut�on and the colour becomes yellow. Opt�c dens�ty (OD) �s measured spectrophotometr�cally at a wavelength of 450 nm± 2nm. The OD value �s proport�onal to GdA concentrat�on. The colour formed �s measured spectrophotometr�cally. Then the concentrat�ons of the samples exam�ned are calculated from the drawn cal�brat�on curve. LIF was exam�ned w�th the ELISA k�t of Elabsc�ence B�otechnology Co.Ltd, wh�ch uses the Sandw�ch-ELISA method. In th�s k�t, the m�cro ELISA plate �s first covered w�th ant�body spec�fic to LIF. Standards or samples are added to the appropr�ate m�cro ELISA plate wells and comb�ned w�th spec�fic ant�body. Then a determ�nant ant�body w�th b�ot�n, spec�fic to GdA and Av�d�n-Horserad�sh Perox�dase (HRP) conjugate �s added to each m�cro-plate and �ncubated. Free elements are washed and substrate solut�on �s added to each well. Wells conta�n�ng only LIF, determ�nant ant�body w�th b�ot�n and Av�d�n-HRP conjugate are seen w�th a blue colour. The enzyme- substrate react�on �s term�nated by add�ng sulphur�c ac�d solut�on and the colour becomes yellow. Opt�c dens�ty (OD) �s measured spectrophotometr�cally at a wavelength of 450 nm± 2nm. The OD value �s proport�onal to LIF concentrat�on. The colour formed �s measured spectrophotometr�cally. Then the concentrat�ons of the samples exam�ned are calculated from the drawn cal�brat�on curve. IL-15 was exam�ned w�th the ELISA k�t of Elabsc�ence B�otechnology Co.Ltd, wh�ch uses the Sandw�ch-ELISA method. In th�s k�t, the m�cro ELISA plate �s first covered w�th ant�body spec�fic to IL-15. Standards or samples are added to the appropr�ate m�cro ELISA plate wells and comb�ned w�th spec�fic ant�body. Then a determ�nant ant�body w�th b�ot�n, spec�fic to IL-15 and Av�d�n-Horserad�sh Perox�dase (HRP) conjugate �s added to each m�cro-plate and �ncubated. Free elements are washed and substrate solut�on �s added to each well. Wells conta�n�ng only IL-15, determ�nant ant�body w�th b�ot�n and Av�d�n-HRP conjugate are seen w�th a blue colour. The enzyme- substrate react�on �s term�nated by add�ng sulphur�c ac�d solut�on and the colour becomes yellow. Opt�c dens�ty (OD) �s measured spectrophotometr�cally at a wavelength of 450 nm± 2nm. The OD value �s proport�onal to IL-15 concentrat�on. The colour formed �s measured spectrophotometr�cally. Then the concentrat�ons of the samples exam�ned are calculated from the drawn cal�brat�on curve. G-CSF was exam�ned w�th the ELISA k�t of Elabsc�ence B�otechnology Co.Ltd, wh�ch uses the Sandw�ch-ELISA method. In th�s k�t, the m�cro ELISA plate �s first covered w�th ant�body spec�fic to human G-CSF. Standards or samples are added to the appropr�ate m�cro ELISA plate wells and comb�ned w�th spec�fic ant�body. Then a determ�nant ant�body w�th b�ot�n, spec�fic to human G-CSF and Av�d�n-Horserad�sh Perox�dase (HRP) conjugate �s added to each m�cro-plate and �ncubated. Free elements are washed and substrate solut�on �s added to each well. Wells conta�n�ng only G-CSF, determ�nant ant�body w�th b�ot�n and Av�d�n-HRP conjugate are seen w�th a blue colour. The enzyme-substrate react�on �s term�nated by add�ng sulphur�c ac�d solut�on and the colour b e c o m e s y e l l o w . O p t � c d e n s � t y ( O D ) � s m e a s u r e d spectrophotometr�cally at a wavelength of 450 nm± 2nm. The OD value �s proport�onal to G-CSF concentrat�on. The colour formed �s measured spectrophotometr�cally. Then the concentrat�ons of the samples exam�ned are calculated from the drawn cal�brat�on curve. Stat�st�cal Analys�s All analyses of the study data were performed us�ng SPSS vn 21 software. Cont�nuous quant�tat�ve var�ables were stated as number, mean and standard dev�at�on and qual�tat�ve var�ables as number, med�an, 25th and 75th percent�les. For cont�nuous var�ables from �ndependent measurements that d�d not show normal d�str�but�on, the Mann Wh�tney Rank Sum Test was appl�ed. A value of p<0.05 was accepted as stat�st�cally s�gn�ficant. RESULTS As a result of the analys�s of the demograph�c data of the study pat�ents, the mean age was determ�ned as 30.41±3.76 years �n the whole group and 30.44±3.91 �n the RPL pat�ent group and 30.40±3.68 years �n the control group (p=0,972). X 57GJRA - GLOBAL JOURNAL FOR RESEARCH ANALYSIS VOLUME-7, ISSUE-12, DECEMBER-2018 • PRINT ISSN No 2277 - 8160 Mean grav�da was determ�ned as 3.83±1.61 �n the RPL group and 1.80±0.73 �n the control group. Mean par�ty was 0 �n the RPL group and 1.80±0.73 �n the control group. The number of abortus was 3.86±1.20 �n the RPL group and 0 �n the control group. These results were summar�zed �n Table 1. Table 1: compar�son of the pregnancy results between the groups. The MUC-1, GdA, LIF, IL-15 and G-CSF plasma levels of the pat�ents were determ�ned and compared between the groups. The MUC-1 level of the RPL group was found to be stat�st�cally s�gn�ficantly lower than that of the control group (0.88±0.45 ng/ml vs. 1.17±0.57 ng/ml, p=0.008). The GdA level of the RPL group was found to be stat�st�cally s�gn�ficantly lower than that of the control group (23.73±3.72ng/ml vs. 25.92±4.20 ng/ml, p=0.006). The LIF level of the RPL group was found to be stat�st�cally s�gn�ficantly lower than that of the control group (64.59±21.13 pg/ml vs. 72.14±20.89 pg/ml, p=0.011). The IL-15 level of the RPL group was found to be stat�st�cally s�gn�ficantly h�gher than that of the control group (38.57±55.70 ng/ml vs. 31.44±43.65 ng/ml, p=0.013). No stat�st�cally s�gn�ficant d�fference was determ�ned between the RPL group and the control group �n respect of the G-CSF levels (13.07±3.84 pg/mL vs 14.11±3.62 pg/mL, p=0.056). All these find�ngs were summar�zed �n Table 2. Table 2: Compar�son between the groups accord�ng to MUC-1, GdA, LIF, IL-15 and G-CSF values. DİSCUSSİON Recurrent pregnancy loss (RPL) �s 2 or more consecut�ve losses before the 20th week of pregnancy. However, there are d�fferent defin�t�ons of th�s d�sease [16]. For example, �n the 2016 gu�del�nes of the Reproduct�ve Health and Infert�l�ty Assoc�at�on, RPL �s defined as 3 or more consecut�ve pregnancy losses before the 20th week of pregnancy [4]. The Amer�can Soc�ety of Reproduct�ve Med�c�ne (ASRM) has defined RPL as 2 or more pregnancy losses, but these do not need to be consecut�ve [3]. Although no clear consensus has been reached on the defin�t�on of RPL, several causes have been held respons�ble �n the et�ology. However, �n approx�mately half of pat�ents, the et�ology cannot be fully clar�fied. Desp�te great efforts made by phys�c�ans �n the d�agnos�s and treatment of pat�ents where the et�ology cannot be fully establ�shed, no correspond�ng reasons can be found �n most cases. Thus, there �s no chance of treat�ng approx�mately half of pat�ents when the et�ology cannot be determ�ned. The only current method for RPL of unknown causes �s close follow-up and mon�tor�ng. When �t �s cons�dered that 0.5%-2% of women are affected by th�s d�sease [16], there can be seen to be a great burden �n respect of the healthcare costs of d�agnos�s and treatment. Moreover, these pat�ents may develop psycholog�cal problems and the marr�age may even be at r�sk [17]. Pregnanc�es wh�ch cont�nue �n women w�th a h�story of RPL are generally seen to be del�vered by caesarean sect�on. When the �nd�cat�ons for caesarean del�very are exam�ned, the most common reason has been found to be that �t �s a valuable pregnancy [18, 19]. As there are d�fferent defin�t�ons of RPL made by prest�g�ous soc�et�es �n the field of obstetr�cs and gynaecology and et�olog�cal causes have not been clar�fied �n approx�mately half of cases, th�s suggests that there are several sc�ent�fic po�nts that are unknown and requ�re clar�ficat�on. In th�s context, the a�m of th�s study was to �nvest�gate poss�ble endometr�al �mplantat�on d�sorders among the as yet unexpla�ned et�olog�cal causes of RPL and to evaluate the plasma levels of a ser�es of cytok�nes wh�ch are effect�ve �n �mplantat�on. Successful embryo �mplantat�on requ�res both a healthy embryo and a healthy endometr�um. There has to be correct and healthy commun�cat�on between the embryo and the endometr�um for the �mplantat�on to be successful. In IVF pat�ents w�th recurrent fa�lure of �mplantat�on, one of these two elements �s usually seen to be �mpa�red. Several stud�es have shown �mpa�red endometr�al recept�v�ty �n IVF pat�ents w�th recurrent �mplantat�on fa�lure [8, 10, 20]. Consequently, there may be a s�gn�ficant d�fference between pat�ent and control groups �n plasma concentrat�ons that re�ect the endometr�al express�on levels of a ser�es of markers that are requ�red for �mplantat�on. W�th th�s hypothes�s, the a�m of the current study was to compare RPL pa�tents and a control group �n respect of blood plasma levels of MUC-1, GdA, LIF, IL-15 and G-CSF, wh�ch play a role �n endometr�al recept�v�ty and �mplantat�on and have been prev�ously stud�ed �n IVF pat�ents w�th recurrent �mplantat�on fa�lure. The results of the study demonstrated that the MUC-1, GdA and LIF mean plasma values were stat�st�cally s�gn�ficantly lower �n the pat�ent group than �n the control group. The blood plasma IL-15 level was determ�ned to be stat�st�cally s�gn�ficantly h�gher �n the pat�ent group than �n the control group. Although the G-CSF blood plasma values were lower �n the pat�ent group than �n the control group, th�s d�fference was not stat�st�cally s�gn�ficant (p=0.56). As prev�ously ment�oned, MUC-1 �s both an ant�-adhes�on molecule and a molecule that has �mmunomodulator effects on T- lymphocytes [21, 22]. The �mplanted embryo �s protected aga�nst fore�gn agents w�th the �mmunomodulator effect. Although �t has been suggested �n prev�ous stud�es that a relat�vely low level of MUC-1 �n the �mplantat�on w�ndow, �s �mportant for �mplantat�on, pat�ents w�th a h�story of RPL have been shown to have a s�gn�ficantly lower MUC-1 level than the normal populat�on [8, 23- 24]. Cons�stent w�th the find�ngs of prev�ous stud�es, the MUC-1 level of the RPL pat�ents �n the current study was found to be lower than that of the control group. Th�s paradox�cal s�tuat�on can be attr�buted to the fact that although a reduct�on �n MUC-1 levels dur�ng the �mplantat�on w�ndow �s �mportant �n respect of �mplantat�on of the blastocyst to the endometr�um, the excess�ve reduct�on �n MUC-1 levels �n RPL pat�ents cannot funct�on to protect the embryo and therefore leaves the embryo defenceless aga�nst external factors [(22, 25]. It has been proposed that by st�mulat�ng apoptos�s �n pro- �n�ammatory monocytes, GdA could have a role �n ma�nta�n�ng the ant�-�n�ammatory env�ronment that �s necessary for pregnancy [26]. In a study by Tulppala et al, the GdA serum levels �n the �mplantat�on w�ndow were compared between pat�ents w�th a h�story of RPL and a healthy control group, and the GdA level was found to be lower �n the RPL group [27]. Dalton et al also compared pat�ents w�th a h�story of RPL and a control group, through the exam�nat�on of the GdA level �n �u�d adm�nstered to the uter�ne cav�ty as 10 ml ster�le sal�ne and then re-asp�rated dur�ng the �mplantat�on w�ndow. The GdA level was determ�ned to be lower �n the RPL group than �n the control group [28]. In another study conducted on pat�ents w�th �mplantat�on fa�lure, GdA levels both �n the serum and �n endometr�al t�ssue samples were found to be lower when compared to a healthy control group [8]. In the current study, the blood plasma GdA levels were found to be lower �n the RPL pat�ents than �n the control group, wh�ch was cons�stent w�th find�ngs �n l�terature. Var�able Pat�ent group (n=42) mean±SD Control group (n=45) mean±SD P Grav�da 3,83±1,61 1,80±0,73 <0,001 Par�ta 0±0 1,80±0,73 <0,001 Abortus 2,86±1,20 0±0 <0,001 Parameter Total (n=87) mean±SD Pat�ent group (n=42) mean±SD Control group (n=45) mean±SD P MUC-1 (ng/l) 0,88±0,45 1,17±0,57 0,008 GdA (µg/l) 24,86±4,09 23,73±3,72 25,92±4,20 0,006 LIF(pg/l) 68,49±21,22 64,59±21,13 72,14±20,89 0,011 IL-15 (ng/l) 34,88±49,67 38,57±55,70 31,44±43,65 0,013 G-CSF (pg/l) 13,60±3,73 13,07±3,84 14,11±3,62 0,056 58 X GJRA - GLOBAL JOURNAL FOR RESEARCH ANALYSIS VOLUME-7, ISSUE-12, DECEMBER-2018 • PRINT ISSN No 2277 - 8160 Several prev�ous stud�es have exam�ned the relat�onsh�p between LIF and embryo �mplantat�on. It �s known that �mmed�ately before �mplantat�on, LIF synthes�s �n the glandular ep�thel�um of the endometr�um reaches a max�mum level [29-30]. In the per�- �mplantat�on stage, the effects of LIF reach�ng peak levels has been shown w�th trophoblast funct�on and placental vascular format�on [30, 31]. In a study by La�rd SM et al, the LIF concentrat�on �n the uter�ne �rr�gat�on �u�d of pat�ents w�th �mplantat�on fa�lure was found to be lower than that of the normal populat�on [32]. In contrast, Xu B et al found no d�fference between pat�ents w�th a h�story of RPL and a control group �n respect of endometr�al express�on of LIF [9]. However, �n another study, LIF levels were found to be s�gn�ficantly lower �n both endometr�al t�ssue and blood plasma �n pat�ents w�th a h�story of RPL compared to a control group [33]. In the current study, the LIF levels of the RPL pat�ents were determ�ned to be stat�st�cally s�gn�ficantly lower than those of the control group. These results were cons�stent w�th the find�ngs of several stud�es �n l�terature that LIF levels, wh�ch have a very �mportant role �n the �mplantat�on process, can be expected to be lower �n pat�ents w�th a h�story of RPL. In a study by Cheg�n� et al, endometr�al IL-15 synthes�s was shown to be �ncreased �n pat�ents w�th a h�story of RPL [143]. Mar�ee et al also showed �ncreased levels of endometr�al IL-15 synthes�s �n pat�ents w�th recurrent �mplantat�on fa�lure compared to a control group [10]. In the current study, the blood plasma levels of the RPL pat�ent group were determ�ned to be h�gher than those of the control group, wh�ch was cons�stent w�th the find�ngs of prev�ous stud�es that have measured endometr�al levels. IL-15 �s known to be effect�ve on the prol�ferat�on of uNK cells and �ncreases the cytotox�c�ty of these cells [34]. It has been suggested that IL-15 plays an �mportant role �n the transformat�on of NK cells to uNK cells [35, 36] and the number of uNK �s known to be �ncreased �n pat�ents w�th a h�story of RPL [37, 38, 39]. From th�s po�nt, �t can be thought that the greater transformat�on of NK cells to uNK cells could be a reason for �mpa�red �mplantat�on. Th�s could even be a reason for the loss of �mplanted healthy embryos. In a recent, random�sed, placebo-controlled study, pat�ents w�th a h�story of RPL for whom IVF was planned were separated �nto 3 groups. One group was adm�n�stered w�th �ntrauter�ne G-CSF, one group w�th low-molecular we�ght hepar�n, acetylsalıcyl�c ac�d and predn�solone and one group w�th a placebo. The results showed that the rates of l�ve b�rths were h�gher and pregnancy losses were lower �n the G-CSF group than �n the other groups [13]. In the current study, �t was a�med to evaluate how the G-CSF serum values d�ffered �n the RPL pat�ents from the control group. The results of the study showed that although the serum G-CSF values were lower �n the RPL group than �n the control group, the d�fference was not stat�st�cally s�gn�ficant. Strong aspects of the current study can be sa�d to be that �n l�terature the stud�es made on the concept of endometr�al recept�v�ty and the molecules that have a funct�on �n that, have generally been conducted on pat�ents who have undergone IVF w�th �mplantat�on fa�lure. There �s a l�m�ted number of stud�es that have exam�ned these molecules �n pat�ents w�th a h�story of RPL. In l�terature, although MUC-1 has been prev�ously exam�ned �n RPL, �t has been exam�ned �n uter�ne �rr�gat�on �u�d and endometr�al b�ops�es. To the best of our knowledge, there has been no prev�ous study that has exam�ned blood plasma MUC-1 concentrat�ons �n RPL compared w�th a healthy populat�on. Therefore, the current study �s the first �n th�s respect. However, there �s a need for further stud�es to be made of the MUC-1 blood plasma levels �n RPL pat�ents to support the results of th�s study. The only study �n l�terature to have stud�ed GdA serum levels �s that by Tulppala et al [27]. The s�m�lar results obta�ned �n the current study suggest that th�s molecule has an �mportant role �n RPL. Nevertheless, further stud�es are requ�red on th�s subject to support the results. Only 1 study could be found that has exam�ned LIF blood plasma levels �n RPL, the results of wh�ch were s�m�lar to those of the current study. Further stud�es are requ�red on th�s subject. Another strong aspect of the current study �s that �t �s the first to have exam�ned IL-15 blood plasma levels �n RPL pat�ents. In the 2 prev�ous stud�es that evaluated the relat�onsh�p between IL-15 and RPL, express�on levels were exam�ned �n endometr�al t�ssues. Although the results of the current study are cons�stent w�th the find�ngs of those 2 stud�es, there can st�ll be cons�dered a need for further research to shed l�ght on th�s subject. As th�s study exam�ned the molecules �n the plasma levels obta�ned from venous blood samples rather than us�ng an �nvas�ve method such as endometr�al b�opsy, th�s can be cons�dered a useful method for the exam�nat�on of endometr�al recept�v�ty �n RPL. Although there are dozens of molecules that funct�on �n embryo �mplantat�on, the plasma levels of only 5 were exam�ned �n th�s study because of t�me and financ�al restra�nts. If a study could be conducted wh�ch could evaluate all these molecules, �t could be determ�ned wh�ch molecules were related to RPL and could be used as pred�ct�ve markers. CONCLUSİON Accord�ng to the results obta�ned �n th�s study, stat�st�cally s�gn�ficantly low levels of MUC-1, GdA and LIF were determ�ned �n RPL pat�ents, wh�ch was cons�stent w�th prev�ous find�ngs �n l�terature, and these could be cons�dered for use at both the d�agnos�s and treatment stage �n future cl�n�cal pract�ce. S�m�larly, that the IL-15 values were found to be h�gher �n the RPL pat�ents than �n the control group �s of cl�n�cal value. Although the G-CSF levels of the RPL pat�ents were lower than those of the control group, the d�fference was not of a stat�st�cally s�gn�ficant level, and as �t has been shown �n l�terature that the use of th�s molecule �mproved results �n cases of recurrent IVF fa�lure, there can be cons�dered to be a need for further more extens�ve stud�es of the effects of th�s molecule on pat�ents w�th a h�story of RPL. The et�ology of RPL has not been fully clar�fied and therefore a pat�ent group of approx�mately 50% who cannot be d�agnosed w�th known causes do not have full access to treatment opt�ons. In th�s context, �t can be cons�dered that further descr�pt�ve and random�sed, controlled stud�es w�ll s�gn�ficantly contr�bute to �mplementat�ons �n cl�n�cal pract�ce. Endometr�al recept�v�ty �s an �mportant part of embryo �mplantat�on and can be thought to play an �mportant role �n the clar�ficat�on of the et�ology of th�s d�sease. Generally, samples are taken w�th endometr�al b�opsy �n the �mplantat�on w�ndow to evaluate endometr�al recept�v�ty. The samples taken are usually exam�ned under electron m�croscope or w�th �mmunoh�stochem�cal methods. These exam�nat�ons are both expens�ve and �mpract�cal and �nclude the �nvas�ve method of b�opsy. The use of venous blood samples to evaluate endometr�al recept�v�ty, as �n the current study, can be cons�dered a more pract�cal, cheaper and less �nvas�ve method. Further s�m�lar stud�es to th�s �nvest�gat�ng the et�ology of RPL, conducted on more extens�ve pat�ent ser�es, are necessary and �mportant �n respect of contr�but�ng to l�terature and the development of new d�agnost�c and treatment methods for th�s d�sease. Compl�ance w�th Eth�cal Standards: Confl�ct of �nterest: All authors (Nad� Kesk�n, Murat Polat, and Ghan�m Khat�b) declare that there �s no confl�ct of �nterest. X 59GJRA - GLOBAL JOURNAL FOR RESEARCH ANALYSIS VOLUME-7, ISSUE-12, DECEMBER-2018 • PRINT ISSN No 2277 - 8160 Eth�cal approval: All procedures performed �n th�s study �nvolv�ng human part�c�pants were �n accordance w�th the eth�cal standards of the �nst�tut�onal research comm�ttee and w�th the 1964 Hels�nk� declarat�on and �ts later amendments or comparable eth�cal standards. Informed consent: Informed consent was obta�ned from all �nd�v�dual part�c�pants �ncluded �n the study. REFERENCES 1- Stirrat GM. (1990) Recurrent miscarriage. Lancet 336(8716): 673-5. 2- Berry CW, Brambati B, Eskes TK, et al., (1995) The Euro-Team Early Pregnancy (ETEP) protocol for recurrent miscarriage. Hum Reprod 10(6): 1516-20. 3- American Society for Reproductive Medicine (2016) De�nitions of infertility and Recurrent Pregnancy Loss: a Committee Opinnion. https://www.asrm.org/ uploadedFiles/ ASRM_Content/News_and_Publications/Practice_ Guidelines/ Committee_Opinions/ De�nitions_of_infertility.pdf. Accessed 1 December 2016 4- Turkish Society of Reprocuctive Medicine (2016) Evidence Based Medicine Guideline of Recurrent Pregnancy Loss. [Tekrarlayan Gebelik Kayıplarında Kanıta Dayalı Yaklaşım Rehberi]. http://www.tsrm.org.tr/pro/dosyalarimiz Accessed 1 December 2016 5- Wilcox AJ, Baird DD, Weinberg CR. (1999) Time of implantation of the conceptusand loss of pregnancy. N Engl J Med 340: 1796–9. 6- Teklenburg G, Salker M, Heijnen C, Macklon NS, Brosens JJ. (2010) The molecular basis of recurrent pregnancy loss: impaired natural embryo selection. Mol Hum Reprod 16: 886–95. 7. Van Mourik MS, Macklon NS, Heijnen CJ. (2009) Embryonic implantation: cytokines, adhesion molecules, and immune cells in establishing an implantation environment. J Leukoc Biol 85: 4–19. 8. Bastu E, Mutlu MF, Yasa C, et al. (2015) Role of Mucin 1 and Glycodelin A in recurrent implantation failure. Fertil Steril 103(4): 1059-64.e2. 9. Xu B, Sun X, Li L, et al. (2012) Pinopodes, leukemia inhibitory factor, integrin-β3, and mucin-1 expression in the peri-implantation endometrium of women with unexplained recurrent pregnancy loss. Fertil Steril 98(2): 389-95. 10. Mariee N, Li TC, Laird SM. (2012) Expression of leukaemia inhibitory factor and interleukin 15 in endometrium of women with recurrent implantation failure after IVF; correlation with the number of endometrial natural killer cells. Hum Reprod 27(7): 1946-54. 11. Eftekhar M, Sayadi M, Arabjahvani F. (2014) Transvaginal perfusion of G-CSF for infertile women with thin endometrium in frozen ET program: A non-randomized clinical trial. Iran J Reprod Med 12: 661. 12. Barad DH, Yu Y, Kushnir VA, et al. (2014) A randomized clinical trial of endometrial perfusion with granulocyte colony-stimulating factor in in vitro fertilization cycles: impact on endometrial thickness and clinical pregnancy rates. Fertil Steril 101: 710–15. 13. Santjohanser C, Knieper C, Franz C, et al. (2013) Granulocyte-colony stimulating factor as treatment option in patients with recurrent miscarriage. Arch Immunol Ther Exp 61: 159–64. 14. Scarpellini F, Sbracia M. (2012) G-CSF treatment improves IVF outcome in women with recurrent implantation failure in IVF. J Reprod Immunol 94: 103. 15. Würfel W. (2015) Treatment with granulocyte colony-stimulating factor in patients with repetitive implantation failures and/or recurrent spontaneous abortions. J Reprod Immunol 108: 123–35. 16- Wilcox AJ, Weinberg CR, O’Connor JF. (1988) Incidence of early loss of pregnancy. N Engl J Med 319: 189–94. 17- Kolte AM, Olsen LR, Mikkelsen EM, Christiansen OB, Nielsen HS. (2015) Depression and emotional stress is highly prevalent among women with recurrent pregnancy loss. Hum Reprod 30(4): 777-82. 18- Jivraj S, Anstie B, Cheong YC, Fairlie FM, Laird SM, Li TC. (2001) Obstetric and neonatal outcome in women with a history of recurrent miscarriage: a cohort study. Hum Reprod 16: 102-6. 19- Sheiner E, Levy A, Katz M, Mazor M. (2005) Pregnancy outcome following recurrent spontaneous abortions. Eur J Obstet Gynecol Reprod Biol 118: 61-5. 20- Scarpellini F, Sbracia M. (2009) Use of colony-stimulating factor for thetreatment of unexplained recurrent miscarriage: a randomized con-trolled trial. Hum Reprod 11: 2703–8. 21- Serle E, Aplin JD, Li TC, et al. (1994) Endometrial differentiation in the peri- implantation phase of women with recurrent pregnancy loss: a morphological and immunohistochemical study. Fertil Steril 62: 989–96. 22- DeSouza MM, Surveyor GA, Price RE, et al. (1999) MUC1/episialin: a critical barrier in the female reproductive tract. J Reprod Immunol 45: 127–58. 23- Li TC, Tuckerman EM, Laird SM. (2002) Endometrium factors in recurrent pregnancy loss. Hum Reprod Update 8: 43–52. 24- Aplin JD, Hey NA, Li TC. (1996) MUC1 as a cell surface and secretory component of endometrial epithelium: reduced levels in recurrent pregnancy loss. Am J Reprod Immunol 35: 261–6. 25- Meseguer M, Pellicer A, Simon C. (1998) MUC1 and endometrial receptivity. Mol Hum Reprod 4:1089–98. 26- Alok A, Mukhopadhyay D, Karande AA. (2009) Glycodelin A, an immunomodulatory protein in the endometrium, inhibits proliferation and induces apoptosis in monocytic cells. Int J Biochem Cell Biol 41: 1138–47. 27- Tulppala M, Julkunen M, Tiitinen A, Stenman UH, Seppala M. (1995) Habitual abortion is accompanied by low serum levels of placental protein 14 in the luteal phase of the fertile cycle. Fertil Steril 63: 792–5. 28- Dalton CF, Laird SM, Estdale SE, Saravelos HG, Li TC. (1998) Endometrial protein PP14 and CA-125 in recurrent miscarriage patients; correlation with pregnancy outcome. Hum Reprod 13: 3197–202. 29- Bhatt H, Brunet LJ, Stewart CL. (1991) Uterine expression of leukemia inhibitory factor coincides with the onset of blastocyst implantation. Proc Natl Acad Sci USA 88: 11408–12 30- Cullinan EB, Abbondanzo SJ, Anderson PS, Pollard JW, Lessey BA, Stewart CL. (1996) Leukemia inhibitory factor (LIF) and LIF receptor expression in human endometrium suggests a potential autocrine/paracrine function in regulating embryo implantation. Proc Natl Acad Sci USA 93: 3115–20. 31- Aghajanova L, Stavreus-Evers A, Nikas Y, Hovatta O, Landgren BM. (2003) Coexpression of pinopodes and leukemia inhibitory factor, as well as its receptor, in human endometrium. Fertil Steril 79 (1): 808–14. 32- Laird SM, Tuckerman EM, Dalton CF, Dunphy BC, Li TC, Zhang X. (1997) The production of leukaemia inhibitory factor (LIF) by human endometrium: presence in uterine �ushings and production by cells in culture. Hum Reprod 12: 569–74. 33- Comba C, Bastu E, Dural O, et al. (2015) Role of in�ammatory mediators in patients with recurrent pregnancy loss. Fertil Steril 104 (6): 1467-74.e1. 34- Carson WE, Giri JG, Lindemann MJ, et al. (1994) IL-15 is a novel cytokine that activates human natural killer cells via components of the IL-2 receptor. J Exp Med 180: 1395–403. 35- Croy BA, Esadeg S, Chantakru S, et al. (2003) Update on pathways regulating the activation of uterine natural killer cells, their interactions with decidual spiral arteries and homing precursors to the uterus. J Reprod Immunol 59: 175–91. 36- Manaster I, Mizrahi S, Golman-Wohl D, et al. (2008) Endometrial NK cells are special immature cells that await pregnancy. J Immunol 181: 1869–76. 37- Clifford K, Flanagan AM, Regan L. (1999) Endometrial CD56+ natural killer cells in women with recurrent miscarriage. Hum Reprod 14: 2727–30. 38- Quenby S, Bates M, Doig T, et al. (1999) Pre-implantation endometrial leukocytes in women with recurrent miscarriage. Hum Reprod 14: 2386–91. 39- Tuckerman E, Laird SM, Prakash A, Li TC. (2007) Prognostic value of the measurement of uterine natural killer (uNK) cells in the endometrium of women with recurrent miscarriage. Hum Reprod 22: 2208–13. 60 X GJRA - GLOBAL JOURNAL FOR RESEARCH ANALYSIS VOLUME-7, ISSUE-12, DECEMBER-2018 • PRINT ISSN No 2277 - 8160