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>>�.�-�.�-�-�->���� 3��U��*�&t�-�.�/0�/�-5;)�5�-�-&5.��-�.�.�-�/��������������������������������������������������������������������5��:Fetuin A Concentration in the Amniotic Fluid of Fetuses with Down Syndrome 
Salih Burcin Kavak1, Ebru Celik Kavak2, Askin Sen3, Rasit Ilhan1, Murat Kaya4,Ekrem Sapmaz1
1:Salih Burcin Kavak, M.D. Assistant Professor, F1rat University, School of Medicine, F1rat Medical Center, Department of Obstetrics and Gynecology, Elaz1g, Turkey, burcinkavak@gmail.com.
2:Ebru Celik Kavak, M.D. Special Medical Park Hospital, Department of Obstetrics and Gynecology, Elaz1g, Turkey,  HYPERLINK "mailto:eckavak@gmail.com" eckavak@gmail.com.
3: Askin Sen, M.D. Assistant Professor, F1rat University, School of Medicine, F1rat Medical Center, Department of Medical Genetics, Elaz1g, Turkey,  HYPERLINK "mailto:askinsen107@hotmail.com" askinsen107@hotmail.com.
1:Rasit 0lhan, M.D. F1rat University, School of Medicine, F1rat Medical Center, Department of Obstetrics and Gynecology, Elaz1g, Turkey,  HYPERLINK "mailto:dr_rasitilhan@hotmail.com" dr_rasitilhan@hotmail.com.
4: Murat Kaya, M.D. Special In Vitro Lab, Department of Biochemistry, Elaz1g, Turkey, muratkaya@mail.com.tr.
1: Ekrem Sapmaz, M.D. Professor, F1rat University, School of Medicine, F1rat Medical Center, Department of Obstetrics and Gynecology, Elaz1g, Turkey, ekremsapmaz@hotmail.com
Corresponding Author: Salih Burcin KAVAK, MD, Assistant Professor, 
Firat University, School of Medicine, Firat Medical Center, 
Department of Obstetrics and Gynecology,
 23100, Elazig, Turkey.
Phone: +90 424 233 35 55-2124 , Fax: +90 424 237 91 38
E-mail: HYPERLINK "mailto:burcinkavak@gmail.com"burcinkavak@gmail.com,     HYPERLINK "mailto:burcinkavak@yahoo.com"burcinkavak@yahoo.com




ABSTRACT
Background: Fetuin-A is a plasma protein called Alfa 2-Heremans Schmid glycoprotein. During the fetal life, it is the major component of non-collagenous bone matrix. The aim of this study was to examine the probable variation of Fetuin A which is produced by the fetal liver and trophoblastic tissue and found in abundance in the amniotic fluid.
Methods: Twenty cases in which amniocentesis were performed were included in the study between December 2012 and December 2013. Amniotic fluid samples were collected from women who underwent amniocentesis in the second trimester of pregnancy. Conventional cytogenetic culture was performed and ten fetuses with DS were identified. They were classified as Group 1 and ten fetuses with normal karyotype were classified as Group 2. Levels of Fetuin-A in the amniotic fluid were measured in each group. 
Results: Age, gender, gestational age, obstetrics history and body mass index of the cases in Group 1 and Group 2 were similar. Mean amnion fluid Fetuin-A levels in Group 1 was 3.5�0.4 ng/mL and these values were detected to be 4.7�0.7 ng/mL in Group 2. There was statistically significant difference between the mean amniotic fluid Fetuin-A levels of two groups.  (P = 0.001 from the Mann-Whitney U test).
Conclusion: In the present study, amnion fluid Fetuin-A levels were found to be significantly lower in the presence of Down Syndrome. Decreased Fetuin-A levels may be effective on antenatal and postnatal developments of fetuses with DS. By the clarification of its importance treatment modalities may be developed. 
Keywords: Down Syndrome, amniocentesis, Fetuin-A.


INTRODUCTION
	Down Syndrome (DS) is a congenital autosomal abnormality characterized by growth and mental retardation and can be seen in 1 out of approximately 800-1000 births [1]. DS is 95% due to maternal (75% during meiosis 1, 25% during meiosis 2) chromosomal nondisjunction. The rest of the cases are the result of mosaism or translocation. When Down Syndrome is present, there are changes in the amount of the production of fetal metabolites.
	Fetuin-A is a plasma protein called Alfa 2-Heremans Schmid glycoprotein. Its molecular weight is about 60 kDa. It is a member of the cystatin superfamily of cysteine   protease inhibitors. While fetuin-A, a serum albumin like protein, is produced by many tissues during fetal development (placenta, kidney, choroid plexus and all other major organs), the liver is the major production site in adults [2,3]. It decreases during acute infection. It is considered an acute phase reactant because of this aspect [4]. In fetal life, it is the major component of non-collagenous bone matrix. It causes bone growth and remodeling by antagonizing the effects of TGF-b. It is the major inhibitor of calcium and phosphate precipitation [5]. 
	In the present study, we aimed to examine the probable variation of Fetuin A, which is produced in the fetal liver and trophoblastic tissue and found in abundance in the amniotic fluid, produced from the hemodynamically changed liver in the presence of chromosomal defect. 
MATERIALS AND METHOD
	The study was conducted in F1rat University School of Medicine Obstetrics and Gynecology Department Perinatology Unit between December 2012 and December 2013. 	Twenty cases in which amniocentesis were performed were included in the study.  Amniotic fluid samples were collected from women who underwent amniocentesis in the second trimester of pregnancy because of advanced maternal age, pathological sonographic features, or abnormal first/or second trimester biochemical screening for DS. For determination of gestational age, the date of last menstruation or 1st trimester crown-rump length (CRL) measurement was used. As a result of conventional cytogenetic culture, 10 DS detected cases were determined as Group 1 and 10 cases with normal karyotype were determined as Group 2. Group 2 (control group) was determined by creating a randomized block design for 10 normal karyotype cases. Cases in which intrauterine growth retardation, multiple pregnancy or fetal abnormality was detected with ultrasonography were excluded from the study. 
	The local research ethics committee approved the study protocol. Written informed consents were obtained in the clinic and randomization was made simply according to a sequence prior to operation upon admission to the hospital. 
	For Fetuin-A level measurement, after taking amniotic fluid into a plain dry tube and after incubation for 30 minutes at room temperature, it was centrifuged for 10 minutes at 5000 rpm. Samples of amniotic fluid were centrifuged, and supernatants were stored in polypropylene tubes at "80� Celsius until the date of quantitative determination of Fetuin-A. Total levels of Fetuin-A in the amniotic fluid were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (AssayPro Saint Charles, Missouri, USA). The sensitivity, intra-assay and inter-assay coefficient of variation for Fetuin-A were ~3ng/mL, 5.1%, and 7.1%, respectively. Analysts were blinded to the clinical information.
STATISTICAL ANALYSIS
	For statistical analysis, we applied the nonparametric Mann-Whitney test for comparison of Fetuin-A levels between the two groups, since the hormone measured was not normally distributed.
RESULTS
	Age, gender, gestational age, obstetrics history and body mass index of the cases in Group 1 and Group 2 were similar, and there wasn�t any statistically significant difference determined. Mean age of the mothers of the fetuses with DS was 32�3.8 years and only two patients were (20%) older than 35 years. The number of parities of the cases in Group 1 and Group 2 included in the study were 3.3�0,9 and 3.4�0.8, respectively, and these values reflect high birth rates   in the region where the study was conducted.
	Amnion fluid Fetuin-A levels were significantly lower in Group 1 and p<0,001. These values are shown in Table 1.
Table 1: Obstetric features of the patients and Fetuin A levels.
Group 1Group 2P Age (year)32�3.832�3.7NSGravidy (Number)3.3�0,93.4�0.8NSParity (Number)1.9�0.72�0.6NSAbortus (Number)0.4�0.50.4�0.5NSBMI23.7�0.523.6�0.4NSGestational Age (Week)17.9�0.618�0.6NSFetuin A (ng/mL)3.5�0.44.7�0.7*BMI: Body Mass Index,
NS: Not Significant
*:p<0,001

	Mean amnion fluid Fetuin-A levels in the Trisomy 21 group (Group 1) was 3.5�0.4 ng/mL and these values were detected to be 4.7�0.7 ng/mL in Group 2 (Control Group). Mean amnion fluid Fetuin-A levels were statistically significantly different between the two groups (P = 0.001 from the Mann-Whitney U test). Fetuin-A levels of both groups are demonstrated in Figure 1.
Figure 1: Graphic of Fetuin A concentrations (ng/mL) in fetuses with trisomy 21 and normal fetuses.

G1: Group 1
G2: Group 2



DISCUSSION
	In the present study conducted, amnion fluid Fetuin-A levels were found to be significantly lower in the presence of Down Syndrome than in the control group. Fetuin-A is produced largely from placental trophoblasts and the fetal liver during the intrauterine period and takes various metabolic roles. There are defective trophoblasts, due to chromosomal abnormalities, that exist with Down Syndrome, along with altered hemodynamics in the liver [6]. All of this can influence Fetuin-A levels.
	In various studies on Down Syndrome, changes in the levels of many molecules that are of fetal origin or fetal cellular contribution of its production have been detected and these have been the basis for genetic screening tests. 
	Maternal serum AFP levels are below normal values between the 15th and 20th gestational weeks in pregnancies with Down Syndrome. In pregnancies with Down Syndrome, maternal serum unconjugated estriol levels are significantly low and MoM values vary between 0.65 and 0.79. Amniotic fluid and placental tissue unconjugated estriol levels are significantly decreased. It is suggested that one reason for high hCG levels in maternal serum might be hCG production and secretion by the placenta. Relative incomplete maturation of the placenta supports these predictions. Thus, the placenta continues to release excessive amounts of hCG as it does in the first trimester [7,8]. Significantly high inhibin-A levels in the second trimester could be detected in maternal serum. Serum PAPP-A levels, first measured by Brambati et al in 1991 at the 8th and 13th weeks of pregnancies with Down syndrome, were observed to decrease significantly. Interestingly, serum PAPP-A values return to normal levels at the 17th and 19th weeks of the pregnancy [9,10]. Torsdottir et al reported that superoxide dismutase activities increased in patients with Down Syndrome than the control group and ceruloplasmin levels did not change [11]. In another study, seruloplasmin concentrations were reported to be similar both in healthy and children with Down syndrome [12]. 
	Fetuin-A is also an effective molecule on bone matrix development. It regulates bone growth and restructuring by antagonizing the effect of TGF-�, and decreased Fetuin-A levels in amniotic fluid of fetuses with DS can produce the intrauterine basis of postnatal defective bone development [13,14]. Fetuin-A also has other metabolic and anti-inflammatory roles besides this. In rats that have had Fetuin gene ablation, the congenital tendency towards calcification occurs and fatal calcifications occur in their kidneys, testicles, skin, heart and vessels [15]. Fetuin-A can inhibit insulin receptor tyrosine kinase activity and results of insulin resistance [16]. There is a converse relationship between Serum Fetuin-A and CRP and its levels decrease in the presence of inflammation [4,17]. Additionally, it has an anti-inflammatory effect by regulating the phagocytosis of apoptotic particles through macrophages [18].
	The limitation of the recent study is the limited number of cases. However, Fetuin has calcification, insulin metabolism and anti-inflammatory effects and decreased Fetuin-A levels may be effective on antenatal and postnatal developments of fetuses with DS. The issue needs to be clarified by larger studies.
Disclosure
This study was presented in the 12. Congress of National Obstetrics and Gynecology in
May 2012, in Turkey. 
CONFLICT OF INTEREST STATEMENT
The authors declare that they have no conflict of interest.


REFERENCES
1. Nussbaum RL, McInnes RR, Willard HF. Thompson & Thompson Genetics in Medicine, 6th edn. Philadelphia: W.B. Saunders Company. 2001: 157-162.
2. Dziegielewska KM, Matthews N, Saunders NR, Wilkinson G: Alpha2HS-glycoprotein is expressed at high concentration in human fetal plasma and cerebrospinal fluid. Fetal Diagn Ther. 1993;8:22�27. 
3. Terkelsen OB, Jahnen-Dechent W, Nielsen H, et al: Rat fetuin: distribution of protein and mRNA in embryonic and neonatal rat tissues. Anat Embryol. 1988;197: 125�133.
4. Lebreton JP, Joisel F, Raoult JP, Lannuzel B, Rogez JP, Humbert G: Serum concentration of human alpha 2 HS glycoprotein during the inflammatory process: evidence that alpha 2 HS glycoprotein is a negative acute-phase reactant. J Clin Invest. 1979; 64:1118-1129.
5. Heiss A, DuChesne A, Denecke B et al. Structural basis of calcification inhibition by alpha2-HS glycoprotein/fetuin-A. Formation of colloidal calciprotein particles. J Biol Chem. 2003;278(15):13333-13341.
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