Psarris A, et al., J Reprod Med Gynecol Obstet 2019, 4: 027 DOI: 10.24966/RMGO-2574/100027 HSOA Journal of Reproductive Medicine, Gynaecology & Obstetrics Original Research variant (627bp) was expressed in all the women of the study (both The Correlation between CGB study group and control group). Gene Splice Variants in Women Keywords: CGB splice variants; HCG genes; Recurrent miscar- with Recurrent Spontaneous riage; Recurrent spontaneous abortions Abortions Introduction Human Chorionic Gonadotrophin (hCG) is a heterodimer glyco- Alexandros Psarris*, Chrisa Lourida, Sofoklis Stavrou, Despi- protein which is mainly produced by the syncytiotrophoblast cells of na Mavrogianni, Dimitris Loutradis and Peter Drakakis the placenta during pregnancy and it is involved in a variety of bio- 1st Department of Obstetrics and Gynecology, “Alexandra” General Hospital, logical procedures [1]. The synthesis of the β-subunit of Human Cho- National and Kapodistrian University of Athens, Athens, Greece rionic Gonadotropin (βhCG) begins shortly after fertilization (βhCG been detected in the 2-cell stage embryo [2]) and it reaches its peak in the maternal blood stream at 9-10 weeks of pregnancy. HCG has many functions during normal pregnancy as it is involved in delaying Abstract the apoptosis of the corpus luteum [3], modulating the implantation Background: Human Chorionic Gonadotrophin (hCG) is a heterod- of the blastocyst [4,5], regulating the placentation and angiogenesis imer glycoprotein which is mainly produced by the syncytiotropho- [6-8] and developing maternal immunotolerance [9]. blast cells of the placenta during pregnancy and it is involved in a variety of biological procedures. The β-subunit of hCG is coded by HCG is composed of two subunits α and β. Subunit α is common CGB, CGB5, CGB8 genes. Most of βCGB transcripts are produced while subunit β is hormone specific. Subunit α is encoded by a sin- by CGB5 and CGB8 genes, while CGB1 and CGB2 are considered gle gene located on chromosome 6q12-q21, while a cluster of genes to be pseudogenes, coding a hypothetical protein, involved in im- on chromosome 19q13.3 are responsible for encoding the β subunit plantation stage. Discrepancies in the expression levels of these [10]. There are six Chorionic Gonadotropin Beta (CGB) genes all genes are associated with a higher risk of abortion or ectopic preg- originating from the gene responsible for coding the β subunit of the nancy. The presence of polymorphisms in these genes, could act as Luteinizing Hormone (LH) via duplication during primate evolution a prognostic marker in women at risk for recurrent miscarriage. [11]. The beta subunit of hCG is a 163 amino acid protein coded by Objectives: The purpose of this study is to detect any correlation be- CGB, CGB5, CGB7 and CGB8 genes which share 97-99% of their tween CGB1 and CGB2 gene expression levels and the probability sequence identity [10]. of recurrent miscarriage. CGB1 and CGB2 genes share an 85% identity with the other genes Method: 163 Caucasian women with recurrent miscarriages and 87 ethnically matched controls with at least one successful pregnan- in the cluster and they encode a hypothetical protein of 132 amino cy were included in this study. DNA from cells of peripheral blood acids that does not have any homology with the functional β subunit was isolated using and subjected to PCR amplification to determine and does not correspond to any known protein from the GenBank the presence of specific for CGB1 and CGB2 splice variants using database [10]. This change has been caused by an inserted DNA frag- appropriate gene specific primers. The obtained product from each ment (736 bp for CGB1 and 724 bp for CGB2) that replaced the 52- patient was visualized using agarose gel and acrylamide gel elec- bp sequence at the proximal end of the promoter, and also the entire trophoresis. 5’-UTR of the ancestral hCG β-subunit coding gene fragment, which Conclusion: The splice variant of CGB1 (573bp) was not expressed is still present in classical CGB genes [12,13]. Despite our lack of neither in the RSA group nor in the control group. The CGB2 splice knowledge regarding the role of this protein, earlier studies have de- tected mRNA from CGB1 and CGB2 in the placenta as well as the *Corresponding author: Alexandros Psarris, 1st Department of Obstetrics and pituitary proving the functionality of these genes [12,14]. Gynecology, “Alexandra” General Hospital, National and Kapodistrian University of Athens, Vasilissis Sofia 80 and Lourou Street, 11528 Athens, Greece, Tel: In this study we aim to compare the presence of CGB1 and CGB2 +0030 6979232977; E-mail: [email protected] gene splice variants in women with normal pregnancies and women Citation: Psarris A, Lourida C, Stavrou S, Mavrogianni D, Loutradis D, et al. with recurrent spontaneous abortions. (2019) The Correlation between CGB Gene Splice Variants in Women with Re- current Spontaneous Abortions. J Reprod Med Gynecol Obstet 4: 027. Materials and Methods Received: August 05, 2019; Accepted: September 03, 2019; Published: Sep- The study group included 163 Caucasian women with at least two tember 10, 2019 miscarriages of unexplained aetiology, before the 20th week of gesta- Copyright: © 2019 Psarris A, et al. This is an open-access article distributed tion who visited the recurrent miscarriage outpatient clinic of Alex- under the terms of the Creative Commons Attribution License, which permits andra Hospital. Women with a history of thromboembolic, infectious, unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. autoimmune, endocrine or chromosomal disorders were excluded Citation: Psarris A, Lourida C, Stavrou S, Mavrogianni D, Loutradis D, et al. (2019) The Correlation between CGB Gene Splice Variants in Women with Recurrent Spontaneous Abortions. J Reprod Med Gynecol Obstet 4: 027. • Page 2 of 5 • Statistical Analysis from the study. The control group included 87 Caucasian women of proven fertility, with no history of pregnancy loss. The study protocol The statistical analysis was performed via IBM SPSS Statistics was approved by the Alexandra Hospital scientific committee and the for Windows, Version 25.0. Statistical significance was set to p<0.05. experiments were conducted in the Molecular Biology laboratory of P-values were calculated using chi-squared and Mann-Whitney tests. the IVF unit of Alexandra Hospital. Results Peripheral blood (2-3ml) was collected from all the women of the study. The DNA isolation was performed using the PureLink Genom- In our study the splice variant of CGB1 (573bp) was not expressed ic DNA kit by Invitrogen Life Technologies. The isolated DNA is neither in the RSA group nor in the control group. However, the stored in a buffer solution at -20oC. The DNA samples were subject- CGB2 splice variant (627bp) was expressed in all the women of the ed to PCR amplification to determine the presence of a specific for study (both study group and control group). Hence, this study failed to CGB1 and CGB2 splice variant, using appropriate gene specific prim- demonstrate a significant difference in the presence of either of these ers which have been previously reported by Burczynska et al., [15]. splice variants between women with recurrent spontaneous abortions The primers used for the detection of CGB1 and CGB2 were CGBF and the control group. However, our study demonstrates the expres- 5’ CgTCCAACACCCTCACTCCCGBR ggCAgCCCTCCTTCTC- sion of CGB2 splice variant in the Greek women population as well CAC. The conventional PCR protocol used in our study included as the absence of expression of the CGB1 splice variant. There was adding 10Χ PCR Buffer minus Mg2+, 10 mM dNTP mixture, 1μl 50 no statistically significant relationship between the expression pattern mM MgCl2, 1μl Primer Sense mix, 1μl Primer Antisense mix, 1μl and the number of first or second trimester abortions, parity regardless Template DNA, 0,3μl Taq DNA polymerase, 17,2μl of distilled water. of smoking habits, BMI and age (Tables 1 and 2). The PCR conditions are 95oC for 10 minutes and 95oC for 1 minute, 65oC for 1 minute, 72oC for 1 minute for 29 cycles, with a final exten- Discussion/Conclusion o o sion step at 72 C for 10 minutes. Then the tubes are incubated at 72 C The possible association of recurrent spontaneous abortions with for 10 minutes. The splice variants were detected using agarose gel different CGB variants and polymorphisms has not been fully inves- and acrylamide gel electrophoresis (Figure 1). tigated. Rull et al., has demonstrated the presence of protective single nucleotide polymorphisms in the CGB5 and CGB8 genes in the Finish and Estonian populations [16]. Furthermore, Rull et al., discovered the protective effect of 4 CGB5 promoter variants [16]. The presence of a protective SNP in the CGB5 gene was also demonstrated by Yong Sun et al., in Chinese women with RSA [17]. CGB1 and CGB2 genes appear to have a role in implantation and early pregnancy. Rull et al., demonstrated that in the first trimester of pregnancy CGB1 and CGB2 genes provided only 1/1000 to 1/5000 of the CGB mRNA transcripts and during the second and third trimester of pregnancy their contribu- tion was even lower (1/10.000) [18]. The expression profile of CGB genes in the trophoblastic tissue of women with ectopic pregnancies differs from normal pregnancies as it is characterized by higher ex- pression levels of CGB1/CGB2 and lower transcriptional activity of the other CGB genes [18]. CGB1/CGB2 contributed 1/500 to 1/3000 of mRNA CGB transcripts [18]. Rull et al., demonstrated the higher transcriptional activity of CGB1/CGB2 in cases of ectopic pregnancy [10]. Moreover, Rull et al., found a complete lack of expression of CGB1 and CGB2 genes in women with RSA suggestive of an eti- Figure 1: Detection of CGB splice variants.