SOLUBLE HUMAN LEUKOCYTE ANTIGEN-G EXPRESSION IN PREGNANCY SUCCESS AND EARLY PREGNANCY LOSS IN KORLE-BU TEACHING HOSPITAL BY IRENE SITSOFE BLEBU (10357539) DEPARTMENT OF PATHOLOGY UNIVERSITY OF GHANA MEDICAL SCHOOL COLLEGE OF HEALTH SCIENCES KORLE-BU THIS THESIS/DISSERTATION IS SUBMITTED TO THE UNIVERSITY OF GHANA, LEGON IN PARTIAL FULFILMENT OF THE REQUIREMENT FOR THE AWARD OF MPHIL IMMUNOLOGY DEGREE JULY, 2014 University of Ghana http://ugspace.ug.edu.gh i DECLARATION ALL THE WORK RECORDED IN THIS THESIS IS ORIGINAL, UNLESS OTHERWISE ACKNOWLEDGED IN THE TEXT OR BY THE REFERENCES CITED. THIS WORK HAS ALSO NOT IN ITS PRESENT FORM OR OTHERWISE BEEN SUBMITTED TO THIS OR ANY OTHER UNIVERSITY FOR THE AWARD OF A HIGHER DEGREE. ……………………………………………… IRENE SITSOFE BLEBU (CANDIDATE) ………………………………………………. PROF. ANDREW ANTHONY ADJEI (SUPERVISOR) ……………………………………………… DR. MICHAEL OFORI (SUPERVISOR) University of Ghana http://ugspace.ug.edu.gh ii DEDICATION THIS THESIS IS DEDICATED TO THE ALMIGHTY GOD, MY FAMILY AND TO THE MEMORY OF MY BELOVED SANELA SETUTSI ADZOTOR HOSU-PORBLEY. . University of Ghana http://ugspace.ug.edu.gh iii ACKNOWLEDGEMENTS My thanks, praises and worship to God most high, my tower of strength and sustenance, my God in whom is the depths of wisdom and knowledge. I am ever grateful to my supervisors for their support throughout the course of this work. The immense considerations, good thoughts and calm dispositions of Prof. Andrew Anthony Adjei, Director of Research, Office of Research, Innovation and Development, University of Ghana, encouraged me not to lose hope but fight out all the challenges that hindered the completion of this work. Dr. Michael Ofori of the Noguchi Memorial Institute of Medical Research, Department of Immunology was a key pillar to the completion of this work. Thank you for making the laboratories available at all times, the guidance, deep concerns and especially the financial support. My thanks go to Dr. Kareem of Obstetrics and Gynaecology, Korle-Bu Teaching Hospital for his invaluable assistance and selflessness. Prof. Yao Tettey was a pillar of confidence. I acknowledge a dedicated colleague, Mr. Mubarak Abdul-Rahman for his presence and assistance throughout the course of this work. Many thanks to the Department of Virology, Korle-Bu Teaching Hospital and the staff of Noguchi Memorial Institute of Medical Research, Department of Immunology for their assistance. University of Ghana http://ugspace.ug.edu.gh iv TABLE OF CONTENTS ACKNOWLEDGEMENTS .............................................................................................................. iii TABLE OF CONTENTS ................................................................................................................. iv LIST OF TABLES ........................................................................................................................... vi LIST OF FIGURES ......................................................................................................................... vi ABSTRACT ................................................................................................................................. vii CHAPTER ONE ............................................................................................................................. 1 1.0 INTRODUCTION ..................................................................................................................... 1 1.2 SPECIFIC OBJECTIVES ............................................................................................................ 3 PROBLEM STATEMENT .............................................................................................................. 4 JUSTIFICATION ............................................................................................................................ 5 CHAPTER TWO ............................................................................................................................ 7 2.0 LITERATURE REVIEW ............................................................................................................. 7 2.1 Major Histocompatibility Complex (MHC) Class II ................................................................ 8 2.3 Classical MHC Class I Molecules (HLA CLASS Ia) ................................................................. 11 2.4 Non-Classical MHC Class I Molecules (HLA Class Ib). .......................................................... 12 2.5 Human Leukocyte Antigen (HLA) –G .................................................................................. 13 2.6 Human Leukocyte Antigen (HLA) -G and Polymorphisms .................................................. 15 2.7 Membrane-bound and Soluble HLA-G ................................................................................ 17 2.8 Human Leukocyte Antigen (HLA) -G and Pregnancy .......................................................... 20 2.9 Human Leukocyte Antigen (HLA) -G and Diseases ............................................................. 26 2.10 Enzyme-Linked Immunosorbent Assay (ELISA) ................................................................. 30 CHAPTER THREE ........................................................................................................................ 33 3.0 MATERIALS AND METHODS ................................................................................................ 33 3.1 Study Site ............................................................................................................................ 33 3.2 Inclusion Criteria ................................................................................................................. 34 3.3 Exclusion Criteria ................................................................................................................ 34 3.4 Study Participants ............................................................................................................... 34 3.5 Collection of Blood Samples ............................................................................................... 35 3.6 Separating Plasma from Blood Samples ............................................................................. 35 3.7 Preparation of Plasma for ELISA ......................................................................................... 36 3.8 Measuring sHLA-G levels by ELISA ...................................................................................... 36 3.9 Statistical Analysis ............................................................................................................... 37 University of Ghana http://ugspace.ug.edu.gh v CHAPTER FOUR ......................................................................................................................... 38 4.0 RESULTS .............................................................................................................................. 38 4.1 Description of Study Participant ......................................................................................... 38 4.2 Levels of sHLA-G among study participants ........................................................................ 39 4.3 Effect of Gender on sHLA-G Levels ..................................................................................... 41 4.4 The impact of Gestation on sHLA-G levels .......................................................................... 42 4.5 Relationship between the levels of sHLA-G, maternal age, infant birth weight and contraception. .......................................................................................................................... 43 CHAPTER FIVE ........................................................................................................................... 45 5.0 DISCUSSION ........................................................................................................................ 45 CHAPTER SIX ............................................................................................................................. 49 6.0 CONCLUSION AND RECOMMENDATION ............................................................................ 49 6.1 CONCLUSION ....................................................................................................................... 49 6.2 RECOMMENDATION ........................................................................................................... 50 REFERENCES .............................................................................................................................. 51 APPENDICES .............................................................................................................................. 85 Appendix I: ................................................................................................................................ 85 Appendix II: ............................................................................................................................... 86 Appendix III: .............................................................................................................................. 87 Appendix IV: .............................................................................................................................. 88 Appendix V. ............................................................................................................................... 89 Appendix VI ............................................................................................................................... 90 Appendix VII .............................................................................................................................. 94 University of Ghana http://ugspace.ug.edu.gh vi LIST OF TABLES Table 1: Age Distribution of study participants. …………………………………….…......... 38 Table 2: Relationship between the levels of sHLA-G, maternal age; infant birth weight and contraception. ……………………………………………………………................................44 LIST OF FIGURES Figure 1: Structural differences in MHC class I and MHC class II molecules. ........................10 Figure 2: sHLA-G levels in spontaneous abortion patients (SA) and pregnant women ...........39 Figure 3: sHLA-G levels in spontaneous abortion patients (SA), pregnant women who had normal delivery (NPD), non-pregnant women (NPW). ............................................................40 Figure 4: Comparison of sHLA-G levels in non-pregnant women (NPW) and normal healthy male (NM). ................................................................................................................................41 Figure 5: Differences in the levels of sHLA-G of first and second trimesters of normal pregnant women who had normal delivery (PND) and women who had spontaneous abortion………………………………………………………………………………………...43 University of Ghana http://ugspace.ug.edu.gh vii ABSTRACT Background: Human Leukocyte Antigen (HLA) -G is a non-classical major histocompatibility complex (MHC) class I protein which has been described as being selectively expressed on the invasive trophoblast at the materno-foetal interface at the beginning of pregnancy. HLA-G has the potential role of protecting the trophoblast from cytotoxicity and enhancing maternal acceptance of the semi-allogeneic foetus by modulating the maternal immune system. HLA-G exerts several immunomodulatory effects, being beneficially implicated in embryo implantation and foetal survival. HLA-G inhibits the activation of the immune cells, and primes them into cytokine secretion profiles to control trophoblast invasion and maintain a local immunosuppressive environment for successful implantation and pregnancy survival. HLA-G has the ability to modulate the release of cytokines from human allogeneic peripheral blood mononuclear cells, and generate allogeneic cytotoxic T lymphocytes (CTLs) response in a concentration-dependent manner. Soluble isoforms of HLA-G has also been demonstrated to inhibit trophoblast invasion of the maternal decidua. Setting/Location: The study was conducted at the Department of Obstetrics and Gynaecology in the Korle-Bu Teaching Hospital. Korle-Bu Teaching Hospital is a leading teaching hospital and a major referral centre in Ghana. Aim: The aim of the study was to determine the role of soluble HLA-G in pregnancy success and early pregnancy loss in Korle-Bu Teaching Hospital. Methods: This study involved eighty participants made up of twenty eight (28) normal pregnant women who had normal delivery, thirty two (32) women who had spontaneous or recurrent abortion, ten non-pregnant women and ten healthy men. A semi-structured questionnaire was administered to each consented participant to document their sociodemographic characteristics, and the history of pregnancy was University of Ghana http://ugspace.ug.edu.gh viii obtained from the clinic folders. 5ml of venous blood samples were collected from each consented participant and the plasma used to determine sHLA-G levels by Enzyme-linked immunosorbent assay (ELISA). Results: The median sHLA-G levels were higher among women who had spontaneous abortions (66.5 U/ml) as compared to pregnant women who had normal delivery (49.53 U/ml), this was statistically significant. The first and second trimester sHLA-G levels of women who had spontaneous abortion are 66.53U/ml and 74.08U/ml respectively and was not statistically significant. The first and second trimester sHLA- G levels of pregnant women who had normal delivery are 39.73U/ml and 69.06U/ml respectively and were also not statistically significant. Healthy males had sHLA-G level (79.11 U/ml) as compared to healthy non-pregnant women (58.28 U/ml) but the difference was not statistically significant. Maternal sHLA-G levels was not statistically significant (P=0.26) in relation to maternal age and birth weight (P=0.38). Conclusion: The results indicate that high levels of sHLA-G may adversely affect pregnancy outcome whilst reduced sHLA-G expressions may enhance pregnancy survival. There was not a significance difference between gestation and sHLA-G levels of women who had spontaneous abortion and normal pregnancy in pregnant women who had normal delivery. The sHLA-G levels were not affected by gender; healthy males had higher sHLA-G level as compared to healthy non-pregnant women, who were all in normal conditions of health but the difference was not statistically significant. This suggests that high sHLA-G levels in healthy individual may play a role in immunosurveilance. The history of contraceptive use had no effect on sHLA-G levels of women who had spontaneous abortion. Finally, there was no relationship between maternal age and corresponding sHLA-G levels, which had no effect on infant birth weight. University of Ghana http://ugspace.ug.edu.gh 1 CHAPTER ONE 1.0 INTRODUCTION Human Leukocyte Antigen (HLA) -G is a class Ib HLA molecule which was first characterized by its expression at the materno–foetal interface (Meer et al., 2004). It shares structural properties of its classic counterparts HLA-A, B and C (Alvaro et. al., 2005). However, unlike its counterparts, it is characterized by limited tissue distribution in healthy conditions and by the expression of seven different isoforms that can be either membrane bound (G1–G4) or secreted (G5–G7) (Meer et al., 2004). HLA-G1 is a full length isoform encoding the complete molecule with α1, α2 and α3 domains, transmembrane region and the intra cellular region of the class I heavy chain. The other HLA-G isoforms are alternatively spliced shorter transcripts, lacking regions complementary to one or more entire exons. Thus, HLA-G2 lacks exon 3, corresponding to the α2 domain; HLA-G3 lacks exon 3 and 4 and thus, only has the α1 domain; and HLA-G4 lacks exon 4 hence, the α3 domain. HLA-G5 and -G6 are equivalent to HLA-G1 and -G2, respectively, but they retain intron 4 which contain a stop codon. The anchoring transmembrane region could not be transcribed resulting in the expression of a soluble protein. Hence, they are also known as soluble HLA-G1 and HLA-G2. A further splice variant of HLA-G (HLA-G7) has also been reported. This isoform contains intron 2, which also has a stop codon, so that the resulting G7 protein would be a soluble HLA-G comprised of only the α1 region (Sargent et al., 2005). HLA-G has suppressive effects on natural killer (NK) cells, CD4 and CD8 T cells, B lymphocytes and antigen presenting cells such as macrophages and dendritic cells (Ober et al., 2006). Although HLA-G was found to inhibit the activation of the immune cells, its primary role is thought to be the modulation of cytokine secretion by University of Ghana http://ugspace.ug.edu.gh 2 these cells to control trophoblast invasion and maintain a local immunosuppressive environment for successful implantation (Sargent et al., 2005). In normal successful pregnancies, the developing foetal placental unit acts as an allograft which presents the allogeneic paternal HLA antigens to the mother. This presentation is done by the extravillous cytotrophoblast by expressing HLA-G (Le Bouteiller et. al., 2000) and lowered or reduced HLA-C prior to implantation (Onno et al., 1994, Redman et al., 2000). These antigens have the potential role of protecting the trophoblast from cytotoxicity. However, HLA-G helps in the maternal acceptance of the semi allogeneic foetus by modulating the maternal immune system (Hviid, 2005). Expression of HLA class Ib genes in the trophoblast cells prevents cytolysis of trophoblast cells by resident NK cells. Resident natural killer (NK) (decidual) cells are a subpopulation of NK cells that reside in the decidua during pregnancy. Resident NK cells are poor killers of the usual NK cell targets and are programmed to recognise, attack and kill HLA null cells (Hunt, 2005). NK cells of unusual phenotype CD16ˉ56 bright are resident and abundant in the decidua in first and second trimester and reduce thereafter. HLA-G inhibits these NK cells through the NKAT3, CD96/NKG2A receptors preventing cytolysis and contributing to foetal survival (Munz et al., 1997; Perez-Villar et al., 1997; Soderstorm et al., 1997; Pende et al., 1997). NK cells also express immunoglobulin transcript (ILT2) and killer inhibitory receptor (KIR) 2DL4. ILT2 though expressed in low quantities on decidual NK cells (Trundley and Moffet, 2004; Navarro et al., 1999; Allan et al., 1999) binds HLA-G to prevent cell lysing. KIR2DL4 interaction with HLA-G may produce the cytokine interferon gamma (IFN- γ) at the materno-foetal interface (Kikuchi et al., 2003; Rajapalon et al., 2003). IFN-γ is a proinflammatory cytokine that can possibly drive immune cells into University of Ghana http://ugspace.ug.edu.gh 3 immunosuppressive profiles, hence, serving the role of an anti-inflammatory cytokine and aiding pregnancy success. The trophoblastic cells of the placenta were further observed to resist NK cell– mediated lysis independent of HLA class I molecules (Sivori et al., 2000; Kikuchi-Maki et al., 2003). HLA-G effects are highly concentration dependent and its soluble isoforms reduce the ability of T cells to function effectively in the pregnant uterus. According to Hunt et al., (2004), about ninety-one percent of mothers, nullipara women and men lack antibodies to HLA-G in sera but women who had been pregnant even with multiple successful pregnancies showed anti-HLA-G antibodies in sera, suggesting interactions with B cells. However, this did not show any damage to the foetus resembling other anti HLA-G antibodies. 1.1 GENERAL OBJECTIVE OF THE STUDY The aim of the study is to determine the role of sHLA-G expression in pregnancy success and early pregnancy loss in Korle–Bu Teaching Hospital (KBTH). 1.2 SPECIFIC OBJECTIVES • To measure soluble HLA-G in normal pregnant women, women undergoing spontaneous or recurrent abortion in KBTH. • To determine whether maternal sHLA-G plasma levels affect infant birth weight. University of Ghana http://ugspace.ug.edu.gh 4 PROBLEM STATEMENT Spontaneous abortion refers to a clinical condition that describes expulsion of products of conception before 20 weeks gestation (Ryan et al., 1999) without outside intervention. There are several deaths and disabilities’ resulting from spontaneous abortion (Ahman et al., 2000) but it has been estimated to have the risk of one maternal death per 100,000 events (Meer et al., 2004). Worldwide, a little over 79 million spontaneous abortions occur per year (Pop. Council, 2000) and about 500,000 estimated deaths from pregnancy-related causes in a year as a result of induced abortions (Odlind et al., 1997). Spontaneous abortion is a major public health concern in Ghana. A study conducted at KBTH in Accra found that 18% of gynaecology admissions in the year 2000 were related to complications of abortion (Schwandt et al., 2010). Furthermore, of the total of 105 maternal deaths recorded at KBTH, 14% were due to complications of abortion (Schwandt et al., 2010). Ghana Maternal Health Survey in 2007 reports that more than one in 10 maternal deaths result from complications of abortion (Schwandt et al., 2010). Recurrent spontaneous abortion (RSA) of unknown etiology is a frustrating and emotionally charged clinical problem and it is one of the least understood pathological processes in spite of it being one of the most common pregnancy complications (Meka and Reddy, 2006). The emotional issues surrounding pregnancy loss become magnified exponentially when miscarriage occurs on a repetitive basis. A study conducted by Meka and Reddy (2006) showed 4- 6% of all women attempting pregnancy experienced at least two miscarriages, of which 1-2% has three or more miscarriages and 12-14% of all pregnancies experienced clinical intrauterine pregnancy losses. The cause of repeated pregnancy loss is multifactorial and most women are not able to ascribe any reason for the University of Ghana http://ugspace.ug.edu.gh 5 incident of spontaneous abortion to themselves (Laird, 2003). The etiology in approximately 50% of cases of spontaneous abortion is unknown, but it has been postulated that a proportion of these repeated pregnancy losses may be due to immune causes (Vercammen, 2008). One of the problems in understanding the underlying etiology of this immune failure is that the mechanisms by which the foetus is protected from the maternal immune system during normal pregnancy are not fully understood (Vercammen, 2008). Human leukocyte antigen (HLA)-G is thought to play a role in implantation. This non-classical HLA class Ib molecule has been demonstrated in human preimplantation embryos at the mRNA and protein level as well as in their culture supernatant, where the presence of soluble HLA-G (sHLA-G) has been reported a prerequisite for implantation (Shankarkurmar, 2004). If soluble HLA-G has a role in pregnancy survival, then its expression may be altered in conditions with poor trophoblast invasion such as miscarriage and preeclampsia. These observations point the way to the use of soluble HLA-G measurements as a possible diagnostic tool for these disorders. JUSTIFICATION HLA-G is a class Ib HLA which has gained much attention due to its multiple functions on the immune system. HLA-G exerts several immunomodulatory effects, being beneficially implicated in embryo implantation and foetal survival but, conversely, being potentially detrimental in tumours and viral infections (Vercammen, 2008). This two-edged sword behaviour suggests that HLA-G expression is under tight University of Ghana http://ugspace.ug.edu.gh 6 regulation. However, to date, little is known about the regulation of this gene. All this features have made HLA-G an attractive target in different situations in which immune tolerance is involved, such as pregnancy and its complications, transplantation, cancer and viral infections, as well as in inflammatory and autoimmune diseases. sHLA-G has been shown to stimulate uterine natural killer (NK) cells to induce a unique profile of proinflammatory and proangiogenic mediators and cytokines favouring implantation and placentation (Vercammen, 2008). These observations point the way to the use of plasma soluble HLA-G measurements as a possible diagnostic tool or as a therapeutic tool and or target for predicting and achieving successful implantation and pregnancy outcome. University of Ghana http://ugspace.ug.edu.gh 7 CHAPTER TWO 2.0 LITERATURE REVIEW The term HLA refers to the Human Leucocyte Antigen Systems, which is located on and controlled by genes on the short arm of chromosome six (Messer et al., 1992). The HLA loci are part of the genetic region known as the Major Histocompatibility Complex (MHC) (Janeway et al., 2001). The MHC spans approximately 4Mb and encodes at least ~130 functional genes, of which more than 20% have functions in immunity, and it is the most gene-dense region of the human genome, as well as the region with the most disease associations (Sadki et al., 2011; Doherty and Zinkernagel, 1975). The MHC genes are traditionally divided into three classes: the MHC class I and class II genes, which encode the antigen-presenting MHC molecules; and the class III genes, which encode complement, hormones and other proteins (Sadki et al., 2011). The peptide antigen-presenting MHC molecules are known as classical MHC molecules. There are also structurally related molecules of class I and class II that do not function in the presentation of peptide antigens to T cells and these are known as non-classical MHC molecules. The expression of HLA molecules is different between HLA class I and class II (Sadki et al., 2011). HLA class I molecules are expressed on all nucleated cells, whereas class II molecules are expressed on antigen presenting cells (APCs): macrophages/monocytes, dendritic cells, Langerhans cells and B cells. Moreover, within the same class, different loci do not have the same level of tissue expression, HLA-C are naturally more weakly University of Ghana http://ugspace.ug.edu.gh 8 expressed than HLA-A or HLA-B in class I and in class II, HLA- DR is strongly expressed than HLA- DP and HLA-DQ (Berrih et al., 1985). 2.1 Major Histocompatibility Complex (MHC) Class II HLA-DP, -DQ and -DR loci are termed HLA Class II. Their function is to present antigens that originate from inside the cell (endogenous antigens) to cytotoxic T lymphocytes (CTLs). The tissue distribution of HLA Class II antigens is confined to antigen presenting cells, including B-lymphocytes, macrophages, dendritic cells, endothelial cells and activated T-lymphocytes (Sridhar). The expression of HLA Class II, on cells, which would not normally express them, is stimulated by cytokines like interferon γ (Janeway et al., 2001). HLA Class II molecules consist of two chains each encoded by genes in the HLA complex on Chromosome 6 (Janeway et al., 2001). MHC class II molecules comprise of non-identical and non-covalently associated polypeptide chains (α1, α2, β1 and β2) (Dahl and Hviid, 2011). These two chains have amino ends on the surface, a short transmembrane stretch and intra-cytoplasmic carboxyl ends, with the exception of the α1 domain, all domains are stabilized by disulphide bridges (Dahl and Hviid, 2011). The β chain is shorter than the α chain and contains the alloantigenic sites. A peptide binding groove is formed in between α1 and β1 domains with a beta pleated floor. The greatest polymorphic variability was found to occur in the amino acids sequence in the peptide binding region (Dahl and Hviid, 2011). This in turn determines the chemical structure of the groove and influences the specificity and affinity of peptide binding (Dahl and Hviid, 2011). University of Ghana http://ugspace.ug.edu.gh 9 2.2 Major Histocompatibility Complex (MHC) Class I Major Histocompatibility Complex (MHC) class I genes play an integral role in host defence against intracellular pathogens and tumours (Ribic, 2005). HLA Class I antigens are expressed on the surface of most nucleated cells (Ribic, 2005). Additionally, they are found in soluble form in plasma and are adsorbed onto the surface of platelets. Erythrocytes were also found to adsorb HLA Class I antigens to varying degrees (Janeway et al., 2001). HLA-B7, A28 and B57 are recognizable on erythrocytes and are termed “Bg” antigens (Janeway et al., 2001). HLA class I genes are composed of eight exons and seven introns. Exon 1 encodes the signal sequence, exons 2, 3, 4 respectively encode for the extracellular domain ( α 1, α 2, α3), exon 5 encodes the transmembrane portion and exons 6, 7, 8 encode the intra-cytoplasmic tail (Berrih et al.,1985). University of Ghana http://ugspace.ug.edu.gh 10 Figure 1: Structural differences in MHC class I and MHC class II molecules. Credit: Kuby Immunology (www.ask4biology.com) Class I MHC molecules contain two separate polypeptide chains, the heavier (44-47 KDa [kilodaltons]) alpha chain and the lighter (12 KDa) beta chain. The carboxyl end of α chain resides inside the cell while the amino end projects on the surface of cell with a short intervening hydrophobic segment traversing the membrane. The chain is coded by the MHC genes and has three globular domains α1, α2 and α3. β2- microglobulin is encoded by a gene on another chromosome. The α3 domain is non- covalently associated with the β2-microglobulin. Both α chain and β2-microglobulin are members of the immunoglobulin (Ig) superfamily. A peptide-binding groove is formed between α1 and α2 helices with beta-pleated sheet as its floor. A peptide of 8- 10 amino acids long can be presented in this groove. The alloantigenic sites that carry determinants specific to each individual are found in the α1 and α2 domains (Dahl and Hviid, 2011). The greatest variability in amino acids (or polymorphism) occurs in the α1 and α2 sequences that line the wall and floor of the groove that binds the peptides. University of Ghana http://ugspace.ug.edu.gh 11 The α1 and α2 domains also bind T cell receptor (TCR) of CD8 T lymphocytes. The parts of these domains that are in contact with TCR also show polymorphism. The immunoglobulin-like region of α3 domain is constant and non-covalently bound to β2- microglobulin. The CD8 molecules present on CD8 T lymphocytes binds to the conserved region of α3 (Dahl and Hviid, 2011). There are two groups of MHC class I molecules; classical (HLA class Ia) and non- classical MHC class I molecule (HLA class Ib) (Adams & Parham 2001). 2.3 Classical MHC Class I Molecules (HLA CLASS Ia) Classical MHC class I molecules are highly polymorphic, usually form trimmers on the cell surface and are mainly associated with antigen presentation. The cell surface glycopeptide antigens of the HLA-A, -B and -C series are called the classical HLA Class I antigens (Shankarkumar, 2004). HLA class Ia genes are among the most polymorphic genes described in the human genome. According to the IMGT/HLA data base, 1698 alleles of HLA-A, 2271 alleles of HLA-B and 1213 alleles of HLA-Cw have been identified. HLA-A, HLA-B and HLA-C also provide both stimulatory and inhibitory signals to natural killer (NK) cell immunoglobulin-like receptors (ILR). Although the three loci encode molecules with similar structure and function, the extent of their role in peptide presentation and ILR engagement varies across loci (Adams & Parham 2001). University of Ghana http://ugspace.ug.edu.gh 12 2.4 Non-Classical MHC Class I Molecules (HLA Class Ib). The non-classical class I genes, HLA- E, F, G, H, J, K and L are monomorphic and expressed on a more restricted set of cell types. They are not important as loci for peptide presenters (Shankarkumar, 2004). HLA-E, F and G were the first group of genes identified in the non-classical HLA family. HLA-E is located between HLA-C and HLA-A, whereas HLA-F is located near HLAG and HLA-A (The MHC sequencing Consortium, 1999). They display a high level of similarity with class Ia genes and their protein products associate with β2- microglobulin (β2m). In contrast with classical genes they are almost non-polymorphic and show a very restricted expression pattern (Martinez et al., 2001). Differences do exist between the 3’ cytoplasmic tail of class Ia and Ib genes (Heinrichs and Orr, 1990; Geraghty et al., 1987). HLA-G has a short cytoplasmic tail which is necessary for its much reduced spontaneous endocytosis (Davis et al., 1997). HLA-E is expressed on many different cells and tissues as the class 1a molecules (Lee et al., 1998). HLA-E binding groove has a unique amino acid substitution and a great affinity for signal peptides derived from HLA-G, an important role for the expression of HLA-E on trophoblast cells (Hviid, 2005). HLA-E was found to bind peptides derived from HLA- A, -B and -C leader sequences and in this manner forms ligands for the inhibitory CD94/NKG2A receptor of NK cells (Shankarkumar, 2004; Llano et al., 2003). The function for HLA-F has not been well defined; however it has been shown to be bound by ILT2 and ILT4, so it may play a role similar to that of other non-classical in regulation of cytolytic cells (Janeway et al., 2001). HLA-F is has been detected on the invasive cytotrophoblast cells of the placenta (Hviid, 2005). The invasive University of Ghana http://ugspace.ug.edu.gh 13 cytotrophoblast cells are the only cells of the placenta that express three class Ib molecules and may functionally substitute for one another (Ishitani et al., 2003). 2.5 Human Leukocyte Antigen (HLA) –G Human leukocyte antigen G (HLA-G) is a non-classical major histocompatibility complex (MHC) class Ib antigen characterized by a limited polymorphism. HLA-G primary transcript generates 7 alternative mRNAs that encode membrane-bound (HLAG1, G2, G3, G4) and soluble (HLA-G5, G6, G7) protein isoforms (Abediankenari et al., 2007). HLA-G was first detected on extravillous cytotrophoblast cells. In nonpathological situations, HLA-G expression is restricted to the materno- foetal interface of the extravillous cytotrophoblasts, placental chorionic endothelium, thymic epithelial cells, and erythropoietic lineage cells from the bone marrow, as well as other immuneprivileged tissues such as the cornea, nail matrix, and autologous tissues such as the pancreas (Cai et al., 2012). HLA-G expression was detected in various types of human malignancies, and has been correlated with certain clinicopathological parameters in gastric carcinoma, lymphoma, ovarian and endometrial carcinoma (Cai et al., 2012). This suggested that HLA-G may promote tumour progression by suppressing immune regulation within tumour microenvironment, and thus helping tumour cells escape from anti-tumour immune surveillance. HLA-G expression was also detected in transplantation, multiple sclerosis, inflammatory diseases, and viral infections (Carosella et al., 2012). HLA-G was found to bind to inhibitory receptors. Three HLA-G receptors have been described: ILT2/CD85j/LILRB1 (ILT2), ILT4/CD85d/LILRB2 (ILT4), and University of Ghana http://ugspace.ug.edu.gh 14 KIR2DL4/CD158d (KIR2DL4) (Carosella et al., 2012). ILT2 is expressed by B cells, some T cells, some NK cells, and all monocytes and dendritic cells, but ILT4 is myeloid specific and only expressed by monocytes and dendritic cells (Carosella et al., 2012). KIR2DL4 expression is mainly restricted to the CD56bright subsets of NK cells, which constitute a minority of peripheral NK cells, but a majority of uterine NK cells (Carosella et al., 2012). Through these differentially expressed receptors, HLA-G can interact with B cells, T cells, NK cells, and antigen-presenting cells (APCs). Functionally, HLA-G1 was found to inhibit the cytolytic function of uterine and peripheral blood NK cells, the antigen specific cytolytic function of cytotoxic T lymphocytes, the alloproliferative response of CD4 T cells, the proliferation of T cells and peripheral blood NK cells and the maturation and function of dendritic cells (Carosella et al., 2012). Soluble HLA-G5 or soluble HLA-G1, which is generated by proteasomal cleavage from the cell membrane, has similar functions. The other HLA- G isoforms have been less well studied, and little is known about their function except that membrane-bound HLA-G2, HLA-G3, and HLA-G4 can inhibit NK-cell and cytotoxic T lymphocyte cytolysis in vitro (Carosella et al., 2012). HLA-G1 is the full length isoform encoding the complete molecule which is the α1, α2 and α3 domains, the transmembrane region and the intracellular region of the class I heavy chain. The other HLA-G isoforms are alternatively spliced shorter transcripts lacking regions complementary to one or more entire exons. HLA-G2 lacks exon 3, corresponding to the α2 domain; HLA-G3 lacks exon 3 and 4 and thus, only has the α1 domain; and HLA-G4 lacks exon 4 and hence, the α3 domain. HLA-G5 and-G6 is equivalent to HLA-G1 and -G2, respectively, but are highly unusual in that, due to an incomplete splicing process, they retain intron 4 which contains a stop codon. This prevents the transcription of the anchoring transmembrane region, resulting in the University of Ghana http://ugspace.ug.edu.gh 15 expression of soluble proteins. Hence, they are also known as soluble HLA-G1 and HLA-G2 (Sargent et al., 2005). HLA-G is proposed the human functional homolog of murine Qa-2. Qa-2, the mouse Ped gene, is a MHC Class Ib protein with a defined function in regulation of preimplantation embryonic development (Comiskey et al., 2006). The Ped gene confers survival advantage to term and increases embryonic cleavage rates (Wu et al., 1999). Qa-2 positive mice exhibit enhanced birth and weaning weights (Warner et al., 2001). HLA-G and Qa-2 share structurally similar characteristics and interact with receptors of the innate and acquired immune systems (Comiskey et al., 2006). Similarities include expression of membrane-bound and soluble isoforms, increase in preimplantation growth rates and enhanced foetal survival, and have a shortened cytoplasmic tail (Comiskey et al., 2003). 2.6 Human Leukocyte Antigen (HLA) -G and Polymorphisms The limited polymorphisms in HLA-G are located outside its binding groove (Hviid, 2005). Major groups of HLA-G alleles arise from single–nucleotide polymorphisms that change the amino acid sequence of HLA-G proteins, even still, further specific allelic variants are seen that arise from silent nucleotide variations (Hviid, 2005). However, WHO-acknowledged HLA-G alleles are not defined as polymorphisms in the noncoding region the HLA-G gene. Two amino acid substitutions in exon 2, 3 and 4 of HLA-G respectively define alleles HLA-G 0102 and HLA-G 0103, HLA-G 0104, and HLA-G 0106 respectively (Hviid et al., 2001, 2002). Other existing alleles are: HLA– G 01010x group of alleles, as well as HLA-G0105N (a null alleles) with a third base deletion of codon 129 or the first base of codon 130 (Suarez et al., 1997; Hviid et University of Ghana http://ugspace.ug.edu.gh 16 al., 1997) in exon 3 leads to a conservative frame shift and is expected to be a non- functional protein and also responsible for decreased amounts of HLA-G1 isoforms (Pfeiffer et al., 2001). HLA-G0105N induces surface expression of HLA E which can interact with CD19/NKG2A in its implantation roles. These HLA-G alleles may be classified into three: high secretors, the in-betweens and the low secretors. HLA-G0104 is a high secretor allele, -G01013 and –G 0105N are low secretor alleles, with –G01011 and –G 01012 falling between the two extremes (Hviid, 2005). Hence, the presence of an allele in an individual may determine the blood levels of HLA-G in that individual. HLA-G alleles are distributed in different ethnic groups around the world. HLA–G 01010x group of alleles are predominant, with a frequency of about 80% in some Africans and 50-60% in Caucasians and Japanese. According to Ishitani et al., (1999), Ghanaians showed a high of 83% frequency of the allele HLA-G010101, 70% for African Americans; and a least of 10% for North Indians (Abbas et al., 2004). However, HLA-G010102 a member of the –G 01010x group of alleles which has a 14bp sequence in the 3’UTR of the gene is sparsely distributed in Africans (2.4%) but has a 30% frequency in other populations. Harrison et al., (1993) and Tamaki et al., (1993) stated that DNA sequence variations exist in the 5’URR (5’ustream regulatory region) in introns and also in the 3’UTR (3’untranslated region) of the HLA-G gene. In addition, Hviid, (2005) observed that sequences and polymorphisms in these same regions are important in HLA-G expression and regulation. Polymorphisms in position -725 are likely to be associated with the status of methylation and sHLA-G expression, and may have an association with spontaneous abortion (Ober et al., 2003). The HLA-G gene and its transcript possess a 14bp deletion/insertion polymorphism in exon 8 of the 3’UTR (Harrison et al., 1993). HLA-G 010101 with 14bp deletions have University of Ghana http://ugspace.ug.edu.gh 17 a significantly reduced expression compared to HLA-G010102 mRNA isoform having the 14bp sequence (Hviid, 2005). HLA-G010102 in transcription and alternate splicing showed additional mRNAs which lack the first 92bp of exon 8; these are variants of HLA-G1 and HLA-G5/G6. HLA-G 010103 alleles more closely resemble HLA-G 010101 alleles in their HLA-G mRNA expression. The HLA-G 010103 alleles showing this close resemblance to HLA-G 010101 have mRNA isoform with 14bp sequence and lack the first 92bp in exon 8, and are unique for HLA-G2 and possibly – G4 (Hviid, 2005). Rousseau et al., (2003) reports that HLA-G transcripts with the 92bp deletions are much more stable than the complete for the +14bp sequence in the 3’UTR (Hviid et al., 2002, 2004a) and women heterozygous for the 14bp polymorphism (Tripathi et al., 2004) risk having recurrent spontaneous abortion than their controls. 2.7 Membrane-bound and Soluble HLA-G Pregnancy maintenance is said to be the prime role of HLA-G molecules and evidence from many studies suggest different isoforms of HLA-G as well as membrane-bound HLA-G, the shedded sHLA-G1, the soluble HLA-G5 may play different roles in pregnancy (Dahl et al., 2013). HLA-G in peripheral blood is generated by alternative splicing of HLA-G transcript to produce soluble isoforms HLA-G5, G6, G7 (Hviid et al., 2000; Paul et al., 2000 ; Moreau et al., 1995; Fujii et al., 1994). HLA-G mRNAs retain intron 4 which has a stop codon are translated into sHLA-G isoforms (Hviid et al., 2000; Fujii et al., 1994). Membrane-bound isoforms may be shedded into peripheral blood to generate sHLA-G in a process requiring metalloproteinases (Park et al., 2004). University of Ghana http://ugspace.ug.edu.gh 18 Soluble HLA-G in peripheral blood is also found in amniotic fluid (Rebman et al., 1998; Hunt et al., 2000). Rebman et al., (1998) and LeFriec et al., (2004) also reported peripheral blood monocytes are predominant in the secretion of HLA-G5, which is the central HLA-G molecule in male reproductive system; and detected in the tissues of the testis, epididymis, prostate gland (Langat et al., 2006) including sHLA-G in seminal fluid (Dahl et al., 2014) and in peripheral blood of men and non-pregnant women (Rizzo et al., 2007; Fuzzi et al., 2002; Hunt et al., 2000). Suggestion are that the predominating soluble HLA-G isoform in non-pregnant female and in male are HLA-G5, and during pregnancy the rise in sHLA-G in the maternal blood might primarily be a result of shedded HLA-G1 from trophoblast cell membranes in the placenta (Dahl et al., 2014). Many studies have detected sHLA-G in the culture media of preimplantation embryos in in vitro fertilization IVF procedures after 46-72hrs before transfer, and in single embryo cultures (Noci et al., 2005). Levels of sHLA-G in blastocyst media from IVF have shown high sHLA-G levels correlates with fertility success which is clinical pregnancy (Rebman et al., 2010; Vercammen et al., 2008; Hviid et al., 2006). Yie et al., 2005 also reported 386 embryo culture supernatants of which 69.9% were positive for sHLA-G after 72 hours, and observed live births rate in women who had an HLA-Gpositive embryos transferred and was significantly higher than those who had HLA-G– negative embryo transfers. sHLA-G negative preimplantation embryos culture used in IVF treatments failed with no signs of implantation (Sher et al., 2004; Fuzzi et al., 2002). HLA-G mRNAs and protein expression was also detected in 2-16 –cell stage preimplantation embryos (Juriscova et al., 1996) and Hviid et al., (2006) confirmed HLA-G transcript have an association with increased cleavage rate when compared with those lacking HLA-G mRNAs. According to Hviid et al., (2006) expression of HLA-G is no guarantee for of University of Ghana http://ugspace.ug.edu.gh 19 implantation. Regarding the many publications of the above, contradictions arise where human embryo culture supernatants from 8-cell morula embryos or beyond had no detectable sHLA-G (van Lierop et al., 2002) and large studies also recorded IVF treatment failures with sHLA-G positive preimplantation embryos (Hviid et al., 2006). Yie et al., (2005) again reported pregnancy and live births with sHLA-G negative IVF cycles. One of the many receptors of HLA-G is KIR2DL4 and is mainly expressed on CD56bright NK cells which is absent in peripheral blood (Trowsdale and Moffet, 2008). Membrane-bound isoforms of HLA-G was found to induce inhibition of uterine NK cell mediated cytolysis through KIR2DL4 but not in peripheral NK cells which are devoid of this receptor (Ponte et al., 1999; Yu et al., 2006). Dahl et al., (2014) demonstrated peripheral NK cell inhibition by HLA-G1 in extravillous cytotrophoblast (EVT) cell lines and transfection in K562 cell lines showed HLA-G 5 is a more potent inhibitor of NK cell mediated cytolysis than HLA-G1. However, a study by Zhang et al., (2013) show that a combination of HLA-G5 and HLA-G1 has an additive effect on the inhibition of NK cytotoxicity. It has been shown that the expression of KIR2DL4 on the surface of uterine NK cells was higher in fertile women than among RSA women, indicating that the interaction between membrane-bound HLA-G and KIR2DL4 may favour induction of tolerance at the materno-foetal interface (Yan et al., 2007). HLAG1 may be most important in preeclampsia pathogenesis, however, low levels of its shedded sHLA-G have been found in the blood plasma of pregnant women with severe preeclampsia in late pregnancy (Dahl et al., 2014). Based on what is known today, it is possible that membrane-bound HLA-G interacts with inhibitory immune receptors to induce tolerance of the foetus, and at the same time sHLA-G serves as an activating molecule promoting proinflammatory cytokine secretion University of Ghana http://ugspace.ug.edu.gh 20 allowing trophoblast migration and vascular remodelling (Dahl et al., 2014).In contrast, studies by McCormick et al., (2009) revealed sHLA-G actually inhibited trophoblast invasion, but this may be concentration dependent and tolerance at the materno-foetal interface may be contributed to by several immune cells interacting with HLA-G (Dahl et al., 2014). 2.8 Human Leukocyte Antigen (HLA) -G and Pregnancy The human placenta is made up of several distinct subpopulations of trophoblast cells which originate from the progenitor cells of the trophectoderm layer of the blastocyst. HLA-G is selectively expressed by the trophoblast cells as early as the first trimester and throughout pregnancy. However, there are divergent patterns of expression of HLAG in the various subpopulations of trophoblastic cells (Hviid, 2005). These expressions are as follows; endovascular trophoblast cells express membrane-bound HLA-G (Proll et al.,1999), the syncytiotrophoblast cells express only sHLA-G like all other subpopulations of trophoblast cells (Ishitani et al.,2003;Kovats et al., 1990); moreover, the syncytiotrophblast (ST), which forms the placental interface with the maternal blood and was previously thought to be class I MHC negative, has now been shown to also express message for soluble HLA-G5 and-G6 (Solier et al., 2002), but the invasive extravillous cytotrophoblast (EVT) cells have the strongest expression of membranebound HLA-G. There is a good amount of evidence to support that the extravillous trophoblast cells express membrane-bound HLA-G1, soluble HLAG-5/- G6, and possibly other isoforms, whereas the HLA-G5 and HLA-G2/-G6 expression in villous trophoblast (VT) and syncytiotrophoblast cells have been proposed but are still a matter of controversy (Morales et al., 2003 and 2007; Ishitani et al., 2003). Hence University of Ghana http://ugspace.ug.edu.gh 21 adequate expression of HLA-G at the foetal-maternal interface is important in the acceptance of the semiallogeneic foetus by the pregnant woman’s immune system. HLA-G was found to play an important role in immune tolerance during pregnancy (Tripathi, 2007). It was found to suppress the proliferation of alloreactive CD4+ T- cell, block the effector function of decidual monocyte/macrophage, inhibit cytolysis mediated by NK-cells and T cells, modulate the release of cytokines and shift decidual mononuclear cells toward the T helper (Th) 2 profile (Cai et al., 2012). Of the Th2 cytokines, interleukin (IL)-10 has been shown to induce HLA-G expression, which in return stimulates IL-10 expression (Cai et al., 2012). Cytokines have been shown to influence placental development, growth and invasion (Roth and Fisher, 1999). HLA- G also inhibits CD8+ and CD4+ T cell reactivity (Lila et al., 2001): soluble/membrane bound HLA-G critically regulate CD8+ T cells by eliminating alloreactive or antiparternal T cell by triggering surface expression and secretion of Fas ligands, resulting in apoptosis by the Fas/FasL pathway (Contini et al.,2003; Fournel et al., 2000; Solier et al., 2002) and another way of maternal tolerance is to prevent cytotoxic activity of CD8+ T cells against target cells by using ILT2 to transduce inhibitory signals as well as protecting target cells against T cell lysis (Contini et al.,2003; Shiroishi et al., 2003). T cells have the capacity to produce various cytokines and this is largely done by the expression of surface molecules. A study suggested that CD3+ peripheral blood T cells are at normal levels for non-pregnant recurrent aborters as compared with normal fertile women (Kwak-Kim et al., 2003). Measurement done by Kwak-Kim et al., (2003) revealed significantly low levels of CD3+ peripheral blood T cells in first trimester pregnancy than those who delivered live infants successfully. A decrease in the CD3+ T cell population in women who miscarried may be as a result of proportional increases in CD19+ B cells and or CD 56+ NK cells (Kwak-Kim et al., University of Ghana http://ugspace.ug.edu.gh 22 2003). Endometrial T cells (CD3+, CD8+CD4+TCR) are present in the menstrual cycle and early pregnancy, and significant in the implantation and maintenance of human pregnancies. However, very few T cells were detected in the endometrium. The effect of HLA-G on T cells depends on; numbers of T cells in decidua (Bulmer et al., 1988), and that trophoblastic cells in normal placentas are most unlikely to generate a cytotoxic T lymphocyte (CTL) cell response (Hunt et al., 2004). Two powerful, multifunctional leucocytes also known as antigen-presenting cells (APCs); macrophages and dendritic cells habit the endometrium of the decidua throughout pregnancy (Bulmer et al., 1988; Gardner and Moffet, 2003; Nehemiah et al., 1981). These are in close contact with the invasive trophoblast, uterine blood vessels and may play central roles in uterine and placental homeostasis (Bulmer et al., 1988; Gardner and Moffet, 2003; Nehemiah et al., 1981; Hunt et al., 1989). Decidual macrophages activated by expression of HLA class II and CD86 antigens appear to be programmed for immunosuppression. They function in producing inhibitory molecules that act on lymphocytes, reducing allogeneic and autologous T cell responses in comparison with monocytes and producing prostaglandins as well as spontaneously secreting antiinflammatory cytokines (also immunosuppressive) eg. IL-10 and TGF (Heikkinen et al., 2003; McIntire et al., 2004). Decidual macrophages also express surface markers and altogether cause immune invasion and activation of macrophages. IL-10 according to Moreau et al., (1999) activates HLA-G expression which further enhances IL10 secretion. Decidual dendritic cell (CD83+ type) are involved in immune cell inhibitory profiles by secreting IL-12 and induce T-helper 2 (Th2) cell in cocultures with naïve CD4+ T cells (Hunt et al., 2004). Placental HLA-G induces these APCs into those profiles by recognizing ILT2 and ILT4 receptors on the APCs (Petroff et al., 2002; Samaridis et al., 1997; Colona et al., 1997). University of Ghana http://ugspace.ug.edu.gh 23 The nature of HLA-G and its tissue distribution strongly suggested that it might play a key role in preventing the trophoblast from being recognized as foreign and rejected by the mother’s immune system (Sargent et al., 2005). Indeed, HLA-G can protect foetal trophoblastic cells from maternal NK cells through interaction with their inhibitory receptors. HLA-G expression by embryos seems to be a prerequisite to their implantation. Evidence for a placental contribution comes from the observation that soluble HLA-G levels were found to increase during the early stages of gestation in twin pregnancies (Sargent et al., 2005). The existence of soluble forms of HLA-G extends the potential for its systemic inhibitory action. Soluble HLA-G has been found in the serum of women in early pregnancy. However, the levels was not found to differ from those in preovulatory women (Sargent et al., 2005; Puppo et al.,1999; Rebman et al., 1999; Pfeiffer et al., 2000). Houcai et al., (2011) investigated soluble HLA-G in all trimesters in the serum of normal pregnant women and reported similarities. Soluble HLA-G expression was found to be altered in conditions with poor trophoblast invasion such as miscarriage and preeclampsia (Sargent et al., 2005). Soluble HLA-G in maternal blood may be largely produced by immune competent cells of the mother than the few by the trophoblast cells of the placenta (Steinborn et al., 2003). It has been shown that there is reduced HLA-G expression on the invasive cytotrophoblast in the decidua of women who miscarry (Sargent et al., 2005). Similarly, HLA-G expression by extravillous cytotrophoblast in the implantation sites of term placentas from preeclamptic women was found to be reduced compared to normal pregnancy (Sargent et al., 2005) and there is a corresponding decrease in soluble HLA-G in both the placentas and circulation of pre-eclamptic women (Sargent et al., 2005). University of Ghana http://ugspace.ug.edu.gh 24 Soluble HLA-G levels lower than 9.95ng/ml possesses the relative risk of the development of placental abruption in the further course of pregnancy (Steinborn et al., 2003). Pfeiffer et al., 2000, in a study of 20 women experiencing spontaneous abortion, showed significantly reduced levels of soluble HLA-G in 9 weeks gestation with a sHLA-G measure of 25.9±3.9 SEM ng/ml as compared to 35.9±3.3 SEM ng/ml of 37 women with normal successful pregnancies. HLA-G 010103 and –G0105N alleles are reported by Pfeiffer et al., 2001 to be associated with recurrent spontaneous abortion, and – G0104 and –G0105 alleles with an increased risk of abortion (Aldrich et al., 2001). A great number of women with recurrent spontaneous abortion carried – G 0106 allele compared with women with normal successful pregnancy (Hviid, 2005). Blood levels of HLA-G and 14bp in the 3’UTR may be linked to recurrent spontaneous abortion. However, an interesting development showed that maternal sHLA-G plasma levels are not altered significantly during normal pregnancy and that very similar levels were found between non-pregnant and pregnant women even when different assays are used (Puppo et al., 1999; Rebman et al., 1999; Pfeiffer et al., 2000). O’Brien et al., (2001) and Hylenius et al., (2004) both in independent case control studies reported that a reduced HLA-G mRNA expression in primipara women, as well as those who carry a foetus with +14/+14 HLA-G genotype have the greater risk of preeclampsia. Hylenius et al., (2004) further observed that -14bp polymorphisms HLA-G alleles are inherited more often from the father in heterozygous foetuses in pregnancies not complicated with preeclampsia and the opposite, +14bp HLA-G alleles are more inherited from the mother in foetuses in preeclamptic cases. He also did not find any evidence of HLA-G antigen incompatibility between mother and foetus in preeclampsia. Preeclampsia is a systemic disorder which evolves in the University of Ghana http://ugspace.ug.edu.gh 25 second half of pregnancy in which a pregnant woman develops hypertension, proteinuria and often oedema due to abnormal vascular response and increased systemic vascular resistance, enhanced platelet aggregation, activation of coagulation cascades and endothelial cell dysfunction (Pregnancy, 2000; Hviid, 2006). Preeclampsia leads to intrauterine growth retardation and reduced placental blood flow to the foetus. Pathogenesis of preeclampsia is characterized excessively by maternal inflammatory response to pregnancy (Rebman et al., 1999) and is more of a Th1 response in contrast to normal pregnancy which is a Th2 response (Darmochwal- Kolarz et al., 1999). Preeclampsia affects 2-7% of pregnancies in varying degrees (Hviid et al., 2005). Immune maladaptation is involved in preeclampsia however; it is not clear whether cytokine expressions are a direct aetiological factor. In an attempt to find a link between HLA-G alleles and preeclampsia: HLA-G0105N allele was found to have associations with preeclampsia in the Caucasian population (Hylenius et al., 2004) but not in the African American populations (Aldrich et al., 2000). Combined HLA-G genotype of mother and child predisposes to preeclampsia. Moreover significantly reduced levels of sHLAG in maternal serum had an association with preeclampsia; in a study of 20 preeclampsia women and 14 controls (Yie et al., 2004). Possibly +14/+14 bp HLA-G may predispose one to preeclampsia due to an aberrant and reduced expression in the placenta (O’Brien et al., 2001; Hviid, 2003). Abnormal HLA-G expression in preeclampsia may be associated with general pathology, supporting the pathogenic role of HLA-G in preeclampsia. University of Ghana http://ugspace.ug.edu.gh 26 2.9 Human Leukocyte Antigen (HLA) -G and Diseases HLA-G expression is evident in many different tumours, malignant haematopoietic diseases, inflammatory diseases and transplantations. However, HLA-G is particularly involved in oncology and transplantation (Tiago et al., 2010). HLA-G expression in disease was first described in tumour cells (Paul et al., 1998), since then, many studies have confirmed HLA-G expression in more than a thousand malignancies, where its gene transcription and protein expressions are switched on, and switched off in surrounding normal tissues (Rouas-Freiss et al., 2005). HLA-G expression in tumours favours tumour progression, development and disease as HLA-G interactions with inhibitory receptors of NK cells prevents NK cytolysis of tumour cells (Tiago et al., 2010) hence impairing antitumuor immunity. Another means of escape of immune surveillance by tumours is the use of trogocytosis. Trogocytosis is a cell-to-cell contact-dependent uptake of membrane and associated molecules. It involves transfer of molecules at an area of the membrane as well as molecules not taking part in cell-to-cell crosstalk. Trogocytosis was studied in murine T cells where CD4+ and CD8+ T cells respectively acquired MHC class II and MHC class I molecules in antigen presenting cells (APCs) (Huang et al., 1999; Patel et al., 2001; Hudrisier et al., 1999). This was confirmed in humans as T cells acquired HLA- G1 from APCs and behaved in like manner as murine T cells (Tatari-Calderone et al., 2002; LeMaoult et al., 2007). These T cells acquiring HLA-G1 switch to immunosuppressive modes and those that acquire HLA-DR assume the roles of APCs (Tatari-Calderone et al., 2002). Hence, modulating the role and capabilities of the immune system toward tumours. NK cells may acquire MHC class1 (Sjostrom et al., 2001; Zimmer et al., 2003; Vanherberghen et al., 2004) and viral receptors from their University of Ghana http://ugspace.ug.edu.gh 27 targets (Tabiasco et al., 2003). Activated NK cells may acquire HLA-G1 from tumour cells by trogocytosis, making them HLA-G-negative tumours which escape cytolysis (LeMaoult et al., 2007). Moreover, NK cells that acquire HLA-G1 ceases to proliferate, losses their cytotoxic abilities and become suppressor cells even inhibiting cytotoxic activity of other NK cells (Tiago et al., 2010). Studies reveal HLA-G expression correlates with tumour progression in ovarian and breast carcinomas (Singer et al., 2003) and melanocytic lesions (Ibrahim et al., 2004). HLA-G levels were found to be high in patients with neuroblastoma (Morandi et al., 2007), and produced unfavourable outcomes in chronic lymphocytic leukaemia (Nuckel et al., 2005), gastric and colorectal cancers (Ye et al., 2007). Malignant haematopoietic diseases such as acute myeloid leukaemia, acute lymphoblastic leukaemia also expresses HLA-G molecules (Tiago et al., 2010). Pregnancy-associated malaria (PAM), a peripheral or placental infection by Plasmodium, is a major public health concern due to significant adverse health effects on both mother and child (Moya-Alvarez et al., 2004). This problem is predominant in sub-Saharan Africa example Ghana (WHO, 2012). PAM is associated with increased malaria risk in infancy (Desai et al., 2007; Rachas et al., 2012) and has been associated with congenital malaria, increased malaria episodes, anaemia, and non-malaria episodes and fever episodes in infants (Tonga et al., 2013; Malhotra et al., 1997). The variation in HLA-G 3’UTR have been proposed to be associated with HLA-G gene expression levels and this regulatory region may play a role in the control of response to malaria (Garcia et al., 2013). The transmission of +3178 G allele and of the UTR-1 haplotype carries unique +3178 G transmission in children having lower intensity of the parasite during asymptomatic infection (Garcia et al., 2013). The base, Adenine University of Ghana http://ugspace.ug.edu.gh 28 located at position +3178 has been associated with decrease mRNA stability in vitro and its due to an expansion of an AU-rich motif leading to a less stable mRNA (Yie et al., 2008). The transmission of +3178 G is theoretically associated with increase in HLA-G expression in children. It has also been found that UTR-3 haplotype is associated with an increase in the level of intensity of infection together with mean levels of Plasmodium falciparum density (Garcia et al., 2013). Studies in many populations have reported, the UTR-3 is associated with the coding allele group HLA- G01:04 (Castelli et al., 2001) which is very frequent in the African populations (Donadi et al., 2011). Rebmann et al., (2001) have also reported the coding allele groups are associated with high sHLA-G production. HLA-G is expressed in many inflammatory diseases and seems to shift T helper cells towards a Th2 profiles (Kapasi et al., 2000; Carosella et al., 2001) and may act as a tissue protective molecule in certain inflammatory diseases (Tiago et al., 2010) such as psoriasis, multiple sclerosis and asthma. HLA-G is an attractive molecule for promoting the immune profile characteristic of asthma. This is because airway inflammation in asthma involves a T-helper cell type 2 skewing of molecules similar to pregnancy (Hunt et al., 2005) with the expression of sHLA-G5 (Nicolae et al., 2005). Individuals are genetically predisposed to over-expression of HLA-G in response to specific signals. Once secreted, HLA-G could promote cascade of events relating in worsening inflammation. A coincidence is also seen of HLA-G expression and Th2 phenomenon in allergic rhinitis were higher levels of sHLA-G expression was detected with a strong correlation between sHLA-G levels and clinical severity (Ciprandi et al., 2009a and 2009b). Studies in rheumatoid arthritis reported lower levels of sHLA-G compared to healthy individuals, however, the levels correlates with University of Ghana http://ugspace.ug.edu.gh 29 the presence of disease associated epitopes (Tiago et al., 2010). This may provide a link to genetic factors or a mere consortium with 2695 genotype markers across the MHC in 2321 T1D families which has put HLA-G at the main list of candidate genes susceptible for type -1 diabetics (Tiago et al., 2010). The role of HLA-G in type 1 diabetic disease is not known, however, treatment of dendritic cells with IFN-beta induced monocytes to express HLA-G (Abediankenari et al., 2007). Comparisons of HLA-G expression in 20 normal patients and 20 diabetics showed low expression associated with dendritic cells of the diabetics. Dendritic cells expressing HLA-G mediate inhibition of autologous T cell activation, showing that HLA-G may prevent the immune pathway in diabetic pathogenesis (Abediankenari et al., 2007). HIV infection is characterized by loss of HLA-A and HLA-B, but the expression of HLA-G remains unaffected or at least not decreased (Triphati and Agarwal, 2007). Along with inability of viral Nef to down regulate HLA-G, there could be some changes, particularly increased interleukin 10 indirectly influencing the expression of HLA-G (Navikas et al., 1995). This cytokine, interleukin 10 (IL10) up regulates expression of HLA-G (Triphati et al., 2003). Lozano et al., (2002) demonstrated that after HIV infection expression of HLA-G increased in all monocytes and some T lymphocytes. A contradictory report by Derrien et al., (2004) showed down regulation of HLA-G in HIV infection. Derrien et al., (2004) studied HLA-G expression in acute HIV infection, and their results are similar to other acute viral infections such as human cytomegalovirus and herpes simplex virus. These both decrease cell surface expression of HLA-G1, but the former particularly can increase HLA-G1 expression University of Ghana http://ugspace.ug.edu.gh 30 upon reactivation (Fisher et al., 2000; Onno et al., 2000). Possibly the expression of HLA-G could be enhanced in the natural course of HIV infection so that the situation in chronic infection would be as shown by Lozano et al., (2002). Further, HLA-G polymorphism is also associated with the risk of HIV infection. Extensive study of HLA-G polymorphism in 456 HIV-seropositive and 406 HIV-seronegative African women showed significant association of G*0105N with protection from HIV-1 infection and G*010108 with susceptibility to infection (Matte et al., 2004). HLA- G*0105N is characterized by deletion of cytosine at position 130 of exon 3, which leads to frame shift and introduction of a stop codon in exon 4 (Ober et al., 1998). Hence HLA-G*0105N stops the production of a functional HLA-G molecule. This could be the likely reason for association of G*0105N with protection from HIV infection, would be that this impairs the function of HLA-G and so down regulation by HIV would be absent or decreased (Triphati and Agarwal, 2007). Lajoie et al., (2006) presented more extended and explicit data for HLA-G polymorphism in the same cohort and found that women carrying G*0105N had a 2.2fold decreased risk of HIV- 1 infection compared with women without G*0105N. They also reported an HIV- seronegative woman who was homozygous for G*0105N. 2.10 Enzyme-Linked Immunosorbent Assay (ELISA) Enzyme-linked immunosorbent assay (ELISA) is a common laboratory technique used to measure the concentration of analyte (antibodies or antigens) in solution. It involves separation of specific and non-specific interactions through serial binding to a solid surface, usually a polystyrene multi well plate to achieve quantitative results. ELISA is University of Ghana http://ugspace.ug.edu.gh 31 quick and easy to carry out and its coloured end products correlate to the amount of analyte in the original sample. They are quick to use and designed to rapidly handle large number of samples and are popular choice for research and diagnostic targets. ELISAs can be quite complex, including various intervening steps and the ability to measure protein concentrations in heterogeneous samples such as blood. The most complex step in the process is detection, where multiple layers of antibodies can be used to amplify a signal. There are different ELISA formats; the most appropriate for this study is the sandwich ELISA. Sandwich ELISA has a high specificity, suitable for complex samples and is flexible and sensitive (www.abdserotec.com/an-introductionto- elisa.htm). The human leukocyte antigen (HLA)-G is a HLA class Ib protein, which in contrast to the highly polymorphic classical HLA molecules shows limited polymorphism and restricted tissue distribution and has a unique alternative splice pattern (Ellis et al., 1986; Paul et al., 2000). HLA-G is expressed as several different splice variants including four membrane-bound (HLA-G1 to -G4) and three soluble isoforms (HLA- G5 to -G7). In addition, both membrane-bound β2-microglobulins (β2m)-linked and free dimers, membrane bound β2m-free heavy chains, and possibly soluble β2m-free dimers have been reported (Ishitani et al., 1992; Apps et al., 2007; Morales et al., 2007). According to Hunt, (2000) HLA-G5 which is spread throughout the placenta likely has mainly free heavy chains and lack β2m, however, detecting antibodies in reagents require light chain/heavy chain associate. Approximately 50 HLA-G alleles corresponding to 16 HLA-G proteins have been reported (The IMGT database; Nov. 2013). Investigations of HLA-G genetics in relation to risk of certain pregnancy complications have increased during recent years (Hviid et al., 2001 and 2006; Dahl et al., 2012). University of Ghana http://ugspace.ug.edu.gh 32 In most of the studies, sHLA-G has been determined with a commercially available sHLA-G enzyme-linked immunosorbent assay (ELISA) kit, based on the capture antibody MEMG/9, capturing sHLA-G1/-G5 in association with β2m and a detecting antibody against β2m. Interestingly, the study by Wu et al., (2009) who failed to report an association, used a different ELISA assay with a higher limit of detection. This could account for the differences in results, whereas there is no obvious explanation to the reported lack of association in the study by Zhang et al., (2003) examining children with atopic asthma and positive controls. In a study by Rizzo et al., (2009) sHLA-G1 and HLA-G5 were determined by performing two different ELISA assays: one capturing both sHLA-G1 and HLA-G5 and one capturing only HLA-G5 by the use of the monoclonal antibody (mAb) 5A6G7, which is specific for HLA-G5/-G6. It is interesting to note that low levels of sHLA-G in assays may be due to polymorphisms or undetectable HLA-G5/ sHLA-G1 (Hviid et al., 2004b). ELISA assays measuring sHLA-G in serum detects HLA-G molecules in a β2m-associated form, hence should detect both sHLA-G1 which is shed by membrane-bound HLA-G1 and soluble HLA- G5 isoforms however, shed HLA-G molecules may blur associations between sHLA-G protein expression and even its polymorphisms (Hviid, 2006). Observations by Fournel et al., (2000) showed that the varied differences in ELISA results depend on the specific antibodies used and whether they bind the β2m-associated HLA-G or intron 4-retaining sHLA-G isoforms. University of Ghana http://ugspace.ug.edu.gh 33 CHAPTER THREE 3.0 MATERIALS AND METHODS 3.1 Study Site The study was conducted at the Department of Obstetrics and Gynaecology in the Korle-Bu Teaching Hospital (KBTH). Korle-Bu Teaching Hospital is situated in the nation’s capital, Accra, Ghana. KBTH is the leading teaching hospital and the major referral centre in the country. It also serves as the teaching hospital of the University of Ghana Medical School, (UGMS), in Accra. The Department of Obstetrics and Gynaecology in KBTH is one of the biggest tertiary obstetrics and gynaecology care centre in the country. The Gynaecology Emergency Room (E/R) is a 24-hour OPD emergency clinic, fully equipped with all basic and necessary medical gadgets. There are doctors and nurses around the clock who attend to emergencies and referrals. The E/R receives many cases in a day; approximately thirty cases (not including the night cases) of which about 75% are complications of induced abortion and the rest are spontaneous abortions and other gynaecological emergencies. The E/R serves the needs of the local community who present with emergencies without the notice of referral letters. However, about 70% of the complicated pregnancy cases within the nation are referred to this department. Patients that are referred to this department originate from different social and ethnic groups as well as geographically distinct areas. Thus the demographics of the study participants enrolled in this study were not limited to a specific social group or ethnicity. University of Ghana http://ugspace.ug.edu.gh 34 3.2 Inclusion Criteria Pregnant women who had normal delivery, women diagnosed with spontaneous abortion(s), healthy non-pregnant women and normal healthy males were included in the study. 3.3 Exclusion Criteria Participants with immuno-deficiencies, malignancies, co-morbid factors as well as those on immunosuppressive and immune stimulants therapies were ruled out of the study. 3.4 Study Participants A total of eighty participants were involved in the study. Peripheral blood samples were obtained from thirty two women who attended the Gynaecology Emergency Room for treatment of spontaneous abortion or recurrent spontaneous abortion, twenty eight healthy normal pregnant women who had normal deliveries from the Gynaecology Unit of KBTH, Ghana as well as ten non-pregnant women and ten men drawn from the College of Health Sciences, UGMS. Informed consent was obtained from or given by all participants. Participants were enrolled in the study if they met the following criteria: suffered/suffering from one or two and more miscarriages, had no immune deficiencies, malignancies, co-morbid factors as well as no current history of immunosuppressive and immune modulator University of Ghana http://ugspace.ug.edu.gh 35 therapies. Screening was done for, intrauterine contour showing homogeneous echo pattern of the uterus, clotting profiles, and a semi-structured questionnaire (Appendix VII) was used for the socio-demographic history of both women experiencing spontaneous abortion and those with normal pregnancy and delivery. Non-pregnant women were enrolled based on negative urine pregnancy test (β subunit of human chorionic gonadotrophin) and last menstrual period, and healthy adult men as part of the study. Malaria screening was done for all the participants. 3.5 Collection of Blood Samples Five millimetres of venous blood was obtained from each consented participant using butterfly needles (0.8×19mm×178mm, BD, USA) into heparinised vacuitainers (BD, USA) tubes. Blood samples were transported from the Unit to the laboratory in ice chest within 1 hour of sample collection. 3.6 Separating Plasma from Blood Samples Plasma was separated from heparinised blood samples by centrifugation from whole blood at 2000rpm for 10minutes. After centrifugation the plasma was collected and placed in cryo tubes and stored at -80°C until ready to use. University of Ghana http://ugspace.ug.edu.gh 36 3.7 Preparation of Plasma for ELISA On the day of running the ELISA the frozen plasma samples were allowed to thaw at room temperature (25°C). Plasma was diluted 10 times with dilution buffer. Plasma samples were aliquoted each with a new 100µl pipette tip into clean wells for dilutions. The mixture was vortex to ensure proper missing of plasma and diluent. 3.8 Measuring sHLA-G levels by ELISA The sHLA-G levels were measured by ELISA using the manufacturer’s protocol. In brief, the buffers and solutions used were pre-prepared, and were diluted as outlined in Appendice I-II. Master Calibrators (standards) were reconstituted with distilled water and prepared in dilutions with dilution buffer as outlined in the Appendix III. To the appropriate wells were added 100µl of standards, samples and dilution buffer and incubated at 2-8°C between 16-20 hours without shaking. After the incubation, the plate was washed manually for five times (5x) with 0.35ml of wash solution per well. The microtiter plate was then bloated on paper towels to remove excess wash solution and unbound analytes. This was followed by the addition of 100µl of Conjugate Solution. The microtiter plate was then incubated on an orbital plate shaker with shaking at 300rpm for 1 hour at room temperature (25°C). Wash procedure was repeated and remained the same. To each well, was added 100µl of Substrate Solution. Care was taken not to expose plate to direct sunlight, and plate was covered with University of Ghana http://ugspace.ug.edu.gh 37 aluminium foil. The microtiter plate was then incubated for 25 minute at room temperature (25°C) without shaking. The Conjugate Solution which contains horse radish peroxidase (HRP) is allowed in incubation time to react with the Substrate Solution which is tetramethylbenzidine (TMB). This allows for colour development which is stopped by the addition of 100µl Stop Solution to each well of the microtiter plate (Appendix IV). The absorbance of each well was determined within 5 minutes of addition of Stop Solution. A Micro plate reader with a reference wavelength of 630nm set to 450nm was used to read the microtiter plates. Spectrophotometric readings of the assays were done both at 650nm and 450nm, and the readings were subtracted to obtain the various absorbance. The sensitivity of the assay was 0.6Units/ml. 3.9 Statistical Analysis Data was entered into a database and analyzed using Sigma-Stat Version 3.5. The data was analyzed for significant differences and/or associations between categorical data. Measures of centrality (median) was evaluated with Man-Whitney Rank Sum test, Kruskal-Wallis Rank Sum test and Bonferroni post-hoc test (pairwise). Descriptive statistics; box plot was used in the data presentation. P-values of <0.05 was considered statistically significant. Most analyses were conducted by comparing the sHLA-G levels in categorical data. Regression analysis was used to analyze and correct for possible confounding effects of age and infant birth weight. University of Ghana http://ugspace.ug.edu.gh 38 CHAPTER FOUR 4.0 RESULTS 4.1 Description of Study Participant A total of 80 participants were involved in the study. Thirty two (32) spontaneous abortion (SA) patients represented 40% of the population with mean age of 31.7, (17- 41years). Twenty eight (28) normal pregnant women who had normal delivery (PND) had a mean age of 24.6; (16-35years) and represented 35% of the study population. Ten (10) non-pregnant women (NPW) had a mean age of 22.6; (18-37 years). Ten (10) normal healthy men (NM) used as negative controls had a mean age of 25.6; (22- 33years) and represented 12.5% of the population. Table 1 list all study participants. Table 1: Age Distribution of study participants; Study Group Total Number (%) Age(M±SD) Spontaneous abortion (SA) 32 (40) 31.7 ±3.2 Normal delivered (PND) 28 (35) 24.6 ±2.4 Non- pregnant women (NPW) 10 (12.5) 22.6 ±3.7 Health adult men(NM) 10 (12.5) 25.6 ±5.2 University of Ghana http://ugspace.ug.edu.gh 39 4.2 Levels of sHLA-G among study participants Figure 2 shows sHLA-G levels in women who experienced spontaneous abortion (SA) and pregnant women who had normal delivery (NPD). sHLA-G levels in women who experienced spontaneous abortion (66.5 U/ml) was higher than those pregnant women who had normal delivery (49.35U/ml). This was statistically significant (P<0.05). Groups Figure 2: sHLA-G levels in spontaneous abortion patients (SA) and pregnant women who had normal delivery (NPD). Each box plot represents the median (dark band). P=0.03 1 2 0 5 0 10 0 15 0 20 0 25 0 1= Spontaneous abortion 2= Pregnant women Normal delivery University of Ghana http://ugspace.ug.edu.gh 40 The sHLA-G level in healthy non-pregnant women (58.28U/ml) was higher compared with sHLA-G levels in pregnant women who had normal delivery (49.36U/ml) and lower than women who had spontaneous abortion (66.53 U/ml). Figure 3 shows the differences in the median of the three. Groups Figure 3: sHLA-G levels in spontaneous abortion patients (SA), pregnant women who had normal delivery (NPD), non-pregnant women (NPW). Dark bands show the median sHLA-G levels in the groups. 1 2 3 0 5 0 10 0 15 0 20 0 25 0 1= Spontaneous abortion Pregnant women Normal delivery 2= 3= Non - Pregnant women University of Ghana http://ugspace.ug.edu.gh 41 4.3 Effect of Gender on sHLA-G Levels The sHLA-G level of 79.11U/ml in normal healthy males was higher compared with the sHLA-G levels of 54.04 U/ml in healthy non-pregnant women. The difference was not statistically significant (P=0.18). Figure 4: Comparison of sHLA-G levels in non-pregnant women (NPW) and normal healthy male (NM). Each box plot represents the median (dark band). P=0.18 Groups 1 2 0 20 40 60 80 100 120 140 1= Non - Pregnant women 2= Healthy Adult males University of Ghana http://ugspace.ug.edu.gh 42 4.4 The impact of Gestation on sHLA-G levels From the results, women in the first trimester who had spontaneous abortions (SA) showed a higher sHLA-G level of 66.53 U/ml as compared with pregnant women who had normal delivery (NPD) (sHLA-G of 41.94 U/ml). This was statistically significant (P= 0.04). Women in the second trimester who had spontaneous abortions (SA) showed higher sHLA-G levels of 98.65 U/ml as compared with second trimester of pregnant women who had normal delivery sHLA-G level of 69.01 U/ml. However, this was not statistically significant (P=1.0). There was no statistical significance difference (P=1.0) between first and second trimester sHLA-G levels in women who had spontaneous abortions (SA). Pregnant women who had normal delivery showed a lower sHLA-G level of 39.73 U/ml in the first trimester compared with sHLA-G level of 69.06 U/ml in second trimester of pregnant women who had normal delivery. The difference was not statistical significance (P=0.08). These data are shown in Figure 5. University of Ghana http://ugspace.ug.edu.gh 43 Figure 5: Differences in the levels of sHLA-G of first and second trimesters of normal pregnant women who had normal delivery (PND) and women who had spontaneous abortion. HLA-G in plasma was analyzed by ELISA. Data is presented as box plots with dark band as median. 4.5 Relationship between the levels of sHLA-G, maternal age, infant birth weight and contraception. Results in Table 2 show the relationship between maternal age and sHLA-G levels as well as the sHLA-G levels of spontaneous abortion women and the history of contraception use. Groups 1 2 3 4 0 50 100 150 200 250 1= First Trim ester spontaneous abortion First Trimester Pregnant women Normal delivery 2= 3= Second Trimester Spontaneous abortion 4= Second Trimester Pregnant Women Normal Delivery University of Ghana http://ugspace.ug.edu.gh 44 There is no statistical significance difference (P=0.26) between maternal age and the corresponding sHLA-G levels. Maternal sHLA-G levels have no statistical significant (P=0.38) on infant birth weight. Women who had spontaneous abortion with the history of contraception use (84.68U/ml) was high compared to women who had spontaneous abortion with no history of contraception use (66.24U/ml). The difference was not statistically significant (P=0.62). Table 2: Associations between sHLAG levels and Maternal Age, History of Contraception Use Median r R² P value sHLA-G level(U/ml) MATERNAL 0.083 0.007 0.26 ---- AGE INFANT BIRTH -0.056 0.003 0.38 ---- WEIGHT CONTRACEPTION 0.62 YES (n=7) ---- ----- 84.68 NO (n=25) ---- ----- 66.24 r =correlation coefficient R2 = coefficient of determination University of Ghana http://ugspace.ug.edu.gh 45 CHAPTER FIVE 5.0 DISCUSSION Foetal survival and pregnancy require an evolution of adaptive mechanisms that allows immunologic tolerance of the foetal allograft by the mother and at the same time maintaining immune competence against invading pathogens and HLA-G may represent one of the pathways of mechanisms for protecting the foetus (Ober et al., 2006). Recurrent spontaneous abortion (RSA) of unknown etiology is a frustrating and emotionally charged clinical problem. Spontaneous abortion is one of the least understood pathological processes in spite of being one of the most common pregnancy complications (Meka and Reddy, 2006). Identified causes are uterine malformations and genetic anomalies which account for 20-50% of the cases. Immunologic causes account for the rest of the causes (Vercammen, 2008). HLA-G altered expressions may predispose to RSA and clinical conditions in pregnancy. HLA-G is involved in reproduction; suppressing NK cell cytotoxic activity, regulating cytokines, inducing apoptosis in T cells, inhibit the activation of immune cells and controlling trophoblast invasion and maintenance of successful implantation (Sargent et al., 2005). HLAG expression is restrictive although it has been detected in much different tissues; however, its expression has been repeatedly described only on and by the trophoblast cells of the placenta (Kovats et al., 1990; Lila et al., 2001; Rebman et al., 2003). One of the major findings from this study is that, women who had spontaneous abortion had higher sHLA-G levels of 66.5U/ml compared with pregnant women who University of Ghana http://ugspace.ug.edu.gh 46 had normal delivery (49.35 U/ml) and the difference was statistically significant (P=0.03). This suggests a possible role of sHLA-G in spontaneous abortion. This finding is in agreement with studies by Dahl and colleagues, (2014) who reported that HLA-G may inhibit trophoblast invasion in concentration-dependent manners, and that tolerance at the materno-foetal interface may be contributed to by the immune cells present in the decidua. Again, studies done by Hviid, (2006) have shown that sHLA-G positive preimplantation embryos failured to implant in IVF treatment. It has also been reported by McCormick et al., (2009) that sHLA-G actually inhibited trophoblast invasion of the maternal decidua. Kanai et al., (2001), in in-vitro studies also demonstrated the ability of sHLA-G and/or mHLA-G to control the release of cytokines from human allogeneic peripheral blood mononuclear cells, which could generate allogeneic CTLs response in a concentration-dependent manner. Furthermore, soluble HLA-G levels could be altered in conditions with poor trophoblast invasion such as miscarriage and preeclampsia (Sargent et al., 2005). Again, sHLA-G was detected in both healthy males and non-pregnant women, with the males showing higher sHLA-G level than the females but the difference was not statistically significant (P=0.18). These findings are consistent with Yie et al., 2004 which suggested that sHLA-G can be detected in all plasma from pregnant to non- pregnant women, and men (Rizzo et al., 2007; Fuzzi et al., 2002). HLA-G expression according to Lila et al., (2001); Rebman et al., (2003) and Hviid et al., (2004a) can be detected in serum, and plasma from men and women, but there are controversies in various studies as to whether sHLA-G can be found in the blood of all men and non- pregnant women under normal conditions of health. There again in literature, Ishitani University of Ghana http://ugspace.ug.edu.gh 47 et al., (1999) in a comparative study, had indicated that there is a high of about 83% frequency of an HLA-G allele in Ghanaians. Based on these findings and this result, the current study suggests that high sHLA-G levels in healthy individuals may play a role in immunosurveilance in the Ghanaian populace. This finding is not in line with Vercammen (2008) who stated high HLA-G levels are present in disease conditions. In this study, sHLA-G levels in pregnant women who had normal deliveries and healthy non-pregnant women were similar (sHLA-G levels of 53.13U/ml and 58.28U/ml respectively), showing no substantial differences. The result in this study is consistent with earlier studies which also found similar levels of sHLA-G between non-pregnant women and pregnant women even when different assays are used (Puppo et al., 1999; Rebman et al., 1999; Pfeiffer et al., 2000). This study also looked at the effect of gestation on the levels of sHLA-G. sHLAG levels in first trimester gestation of spontaneous abortions were high compared to the first trimester of pregnant women who had normal delivery (P=0.04). However, substantial differences were not seen in the second trimester between the two groups (P=1.0). Other observations show that women who had spontaneous abortions had no substantial differences in the levels of sHLA-G between the first and second trimester gestations. Further findings in this study, reports that there are marked differences in the sHLA-G plasma levels of first trimester gestation (39.73 U/ml) which was not statistically higher (P=0.08) than the second trimester gestation (69.06 U/ml) of pregnant women who had normal delivery. However, findings by Hviid, (2006), which stated that maternal sHLAG plasma levels are not altered significantly during normal University of Ghana http://ugspace.ug.edu.gh 48 pregnancy. Similar studies by Houcai et al., (2011) also reported similarities in soluble HLA-G levels in all trimesters in the serum of normal pregnant women. Again, sHLA-G levels showed no relationship with age and infant birth weight. Infants born at 38 weeks and above to pregnant women who had normal delivery had birth weight ranging between 2.2kg to 4.7kg but their birth weights had no relationship with sHLA-G levels in maternal blood. However, studies by Hviid, et al., (2004b) and Hviid, (2006) report that HLA-G polymorphism (which influences HLA-G expression) was significantly associated with increased foetal and placental weight. Also in the mouse model, polymorphisms in the mouse Qa-2, a homolog of human HLA-G showed associations with increased foetal and placental weight (Comiskey et al., 2003). However, the current study was conducted in smaller populations. Maternal age also had no effect on sHLA-G levels in women who had spontaneous abortion and pregnant women who had normal delivery. There may be no relationship between maternal age and maternal sHLA-G levels. Finally, women who had spontaneous abortion with a history of contraceptive use had a higher sHLA-G level of 84.68 U/ml compared with women who had spontaneous abortions with no history of contraception but this was not statistically significant (P=0.62). Hence, the history of contraceptive use is not a determinant of maternal sHLA-G levels. University of Ghana http://ugspace.ug.edu.gh 49 CHAPTER SIX 6.0 CONCLUSION AND RECOMMENDATION 6.1 CONCLUSION • Soluble HLA-G levels were higher in the plasma of women who had spontaneous abortions than in normal pregnant women who had normal delivery. Many controversies surround HLA-G functions in pregnancy and its complications. However, the results of this current study are indicative that high levels of sHLA-G in circulation may play a role in spontaneous abortion but reduced sHLA-G levels in circulation may enhance pregnancy survival. • High soluble HLA-G expression was detected in plasma of healthy non- pregnant women and healthy males in normal conditions of health. The current study suggests based on its findings and results that, high HLA-G levels are not present only in disease but high sHLA-G levels in healthy individuals may play a role in immunosurveilance. • Soluble HLA-G levels did not alter substantially during the course of normal pregnancy in pregnant women who had normal delivery. • Maternal age had no corresponding effect on maternal HLA-G levels, which in turn had no effect on infant birth weight. University of Ghana http://ugspace.ug.edu.gh 50 6.2 RECOMMENDATION • There is the need for more research on sHLA-G levels and cytokine profiles in larger population sample among Ghanaians. This may determine the combined effects of sHLA-G levels and cytokines on the outcomes of pregnancy in Ghana. • The in-depth study of cytokines and sHLA-G and how they affect one another in pregnancy may serve as a therapeutic tool, as to how higher sHLA-G levels can be lowered to enhance pregnancy survival. • Soluble HLA-G levels may be used as a diagnostic tool in women who may have spontaneous abortion and/or recurrent spontaneous abortion in Ghana. • There is the need for public education on the effects of stigmatization and the risks of induced abortion, to allow people to freely seek proper medical care and to readily aid diagnosis of spontaneous abortions (miscarriages). University of Ghana http://ugspace.ug.edu.gh 51 REFERENCES Abbas, A., Tripathi, P., Naik, S., and Agrawal, S. (2004). Analysis of human leukocyte antigen (HLA)-G polymorphism in normal women and in women with recurrent spontaneous abortions. Eur J Immunogenet, (6):275-8. Abediankenari, S., Eslami, M. B., Sarrafnejad, A., Mohseni, M., and Laarijani, B. (2007). Dendritic cells bearing HLA-G inhibit T-cell activation in type 1 diabetics. Iran J Allergy Asthma Immunol, 6(1):1-7. Adams, E. J., and Parham, P., (2001). Species-specific Evolution of MHC Class I Genes in the Higher Primates. Immunol Rev, (183):41-64. Åhman E., Dolea C., and Shah I., (2000). The global burden of unsafe abortion in the year 2000. 1-11. Allan D. S., Colonna M., Lanier L. L., Churakova, T. D., Abrams, J. S., Ellis, S. A., McMichael, A. J., and Braud, V. M. (1999). Tetrameric complexes of human histocompatibility leukocyte antigen (HLA)-G binds to peripheral blood myelomonocytic cells. J. Exp. Med, (189): 1149–1156. University of Ghana http://ugspace.ug.edu.gh 52 Aldrich, CL., Stephenson, MD., Karrison, T., Odem, RR., Branch, DW., Scott, JR., Schreiber, JR., and Ober, C. (2001). HLA-G genotypes and pregnancy out- come in couples with unexplained recurrent miscarriage. Mol Hum Reprod, (7): 1167–1172. Aldrich, C., Verp, M. S., Walker, M. A, and Ober, C. (2000). A null mutation in HLAG is not associated with preeclampsia or intrauterine growth retardation. J Reprod Immunol. (47): 41–48. Apps, R., Gardner, L., Sharkey, A. M., Holmes, N., and Moffett, A. (2007). A homodimeric complex of HLA-G on normal trophoblast cells modulates antigenpresenting cells via LILRB1. European Journal of Immunology, 37(7): 1924– 1937. Berrih, S., Arenzana-Seisdedos, F., Cohen, S., Devos, R., Charron, D. and Virelizier, J. L. (1985). Interferon-gamma modulates HLA class II antigen expression on cultured human thymic epithelial cells. J Immunol, 135(2):1165-71 . Bulmer, J. N., Pace, D. and Ritson, A. (1988). Immunoregulatory cells in human decidua: morphology, immunohistochemistry and function. Reprod Nutr Dev, 28(6B):1599-613. University of Ghana http://ugspace.ug.edu.gh 53 Cai, M. B., Han, H. Q., Bei, J. X., Liu, C. C., Lei, J. J., Cui, Q., Feng, Q. S., Wang, H. Y., Zhang J. X., Liang, Y., Chen, L. Z,, Kang, T. B,, Shao. J. Y. and Zeng, Y. X. (2012). Expression of Human Leukocyte Antigen G is associated with Prognosis in Nasopharyngeal Carcinoma. Int J Biol Sci, 8(6):891-900. Carosella S., (2005) Linking Two Immuno-Suppressive Molecules Indoleamine 2,3 Dioxygenase Can Modify HLA-G Cell-Surface Expression. Carosella, E. D., Favier, B., Rouas-freiss, N., Moreau. P. and Lemaoult, J. D. W. (2012). Beyond the increasing complexity of the immunomodulatory HLA-G molecule. Review in translational hematology. 4862–4932. Carosella, E. D, Moreau, P., Aractingi, S. and Rouas-Freiss, N. (2001). HLA-G: a shield against inflammatory aggression. Trends Immunol, 22: 553-5. Castelli, E.C., Mendes-Junior, C.T., Veiga-Castelli, L.C., Roger, M., Moreau, P. and Donadi, E. A. (2011). A comprehensive study of polymorphic sites along the HLA-G gene: implication for gene regulation and evolution. Mol. Biol. Evol. 28: 3069-3086. Ciprandi, G., Contini, P., Murdaca, G., DeAmici, M., Gallina, A. M. and Puppo, F. (2009a). Soluble serum HLA-G and HLA-A, -B, -C molecules in patients with seasonal allergic rhinitis exposed to pollens. Int Immunopharmacol, 9(9): 1058-62. University of Ghana http://ugspace.ug.edu.gh 54 Ciprandi, G., Contini, P., Murdaca, G., Gallina, A. M. and Puppo, F. (2009b). Soluble HLA-G Molecule in Patients with Perennial Allergic Rhinitis. Int Arch Allergy Immunol, 150(3): 278-359. Colona, M., Navarro, F., Bellon, T., Llano, M., Garcia, P., Samaridis, J., Angman, L., Cella, M., and Lopez-Botet, M. (1997). A common inhibitory receptor for major histocompatibility complex classI molecules on human myeloid and myelomonocytic cells. J. Exp. Med, 186: 1809-1818 Comiskey, M., Goldstein, C. Y., De Fazio, S. R., Mammolenti, M., Newmark J. A., and Warner, C. M., (2003). Evidence that HLA-G is the functional homolog of mouse Qa2, the Ped gene product. Hum Immunol, 64(11):999-1004. Comiskey, M., Warner, C. M., and Schust, D. J. (2006). MHC molecules of the preimplantation embryo and trophoblast. Immul of Preg. Contini, P., Ghio, M., Poggi, A., Filaci, G., Indiveri, F., Ferrone, S. and Puppo, F. (2003). Soluble HLA-A,-B,-C and -G molecules induce apoptosis in T and NK CD8 cells and inhibits cytotoxic T cell activity through CD8 ligation. Eur. J. Immunol. 33: 125–134 University of Ghana http://ugspace.ug.edu.gh 55 Dahl, M., Djurisic, S. and Hviid T.V. (2014). The Many Faces of Human Leukocyte Antigen-G: Relevance to the Fate of Pregnancy. J Immunol Res. Dahl, M., and Hviid T.V. (2011). Human leucocyte antigen class Ib molecules in pregnancy success and early pregnancy loss. Hum Reprod Update, 18(1): 92-109 Darmochwal-Kolarz, D., Leszczynska-Gorzelak, B., Rolinski, J. and Oleszczuk, J. (1999). T helper 1- and T helper 2-type cytokine imbalance in pregnant women with pre-eclampsia. Eur J Obstet Gynecol Reprod Biol, 86: 165–170. Davis, D. M., Reyburn, H.T., Pazmany. L., Chiu, I., Mandelboim, O. and Strominger, J. L (1997). Impaired spontaneous endocytosis of HLA-G. Eur J Immunol, 27: 2714– 2719. Desai, M., Kuile, F.O., Nosten, F., Mcgready, R., Asamoa, K., Brabin, B. and Newman, R. D. (2007). Epidemiology and burden of malaria in pregnancy. Lancet Infect Dis, 7(2): 93–104. Derrien, M.1., Pizzato, N., Dolcini, G., Menu, E., Chaouat, G., Lenfant, F., BarréSinoussi, F., Bouteiller, P. L., (2004). Human immunodeficiency virus 1 down University of Ghana http://ugspace.ug.edu.gh 56 regulates cell surface expression of the non-classical major histocompatibility class I molecule HLA-G1. J Gen Virol. 85(7): 1945-1954. Doherty, P. C., and Zinkernagel, R. M. (1975). A biological role for the major histocompatibility antigens. The Lancet, 305(7922): 1406-1409. Donadi, E.A., Castelli, E.C., Arnaiz-Villena, A., Roger, M., Rey, D. and Moreau, P. (2011). Implications of the polymorphism of HLA-G on its function, regulation, evolution and disease association. Cell. Mol. Life Sci. 68: 369–395. Ellis, S. A., Sargent, I. L., Redman, C. W. G. and McMichael A. J. (1986). Evidence for a novel HLA antigen found on human extravillous trophoblast and a choriocarcinoma cell line. Immunology, 59(4): 595–601. Fisher, S., Genbacev, O., Maidji, E. and Pereira, L. (2000). Human cytomegalovirus infection of placental cytotrophoblasts in vitro and in utero: implications for transmission and pathogenesis. J Virol, 74: 6808-6820. Fournel, S., Aguerre-Girr, M., Huc, X., Lenfant, F., Alam, A., Toubert, A., Bensussan, A., Le Bouteiller, P. (2000). Cutting edge: Soluble HLA-G1 triggers CD95/CD 95 University of Ghana http://ugspace.ug.edu.gh 57 ligand-mediated apoptosis in activated CD8+ cells by interacting with CD8. Journal of Immunology, 164(12): 6100-6104. Fuzzi, B.1, Rizzo, R., Criscuoli, L., Noci, I., Melchiorri, L., Scarselli, B., Bencini, E., Menicucci, A. and Baricordi, OR. (2000). HLA-G expression in early embryos is a fundamental prerequisite for the obtainment of pregnancy. European Journal of Immunology, 32(2): 11–315. Garcia, A., Milet, J., Courtin, D., Sabbagh, A, Massaro, J.D., Castelli, E.C., MigotNabias, F., Favier, B., Rouas-Freiss, N., Donadi, E. A and Moreau, P. (2013). Association of HLA-G 3'UTR polymorphisms with response to malaria infection: a first insight. Infect Genet Evol, 16: 263–269 Gardner, L., and Moffet, A. (2003). Dendritic cells in the human decidua. Biol.Reprod. 69(4): 1438-1446 Geraghty, D.E., Koller, B.H., and Orr, H.T., (1987). A human major histocompatibility complex class I gene that encodes a protein with shortened cytoplasmic segment. Proc Natl Acad Sci U S A. 84: 9145–9149. Harrison, G. A., Humphrey, K. E., Jakobsen, I. B., and Cooper, D. W. (1993). A 14 base pair deletion polymorphism in the HLA-G gene. Hum. Mol. Genet, 2:2200. University of Ghana http://ugspace.ug.edu.gh 58 Heikkinen, J., Mottonen, M., Komi, J., Alanen, A., and Lassila, O. (2003) Phenotypic characterization of human decidua macrophages. Clin. Exp. Immunol. 131: 498-505 Heinrichs, H. and Orr, H. T. (1990). HLA non-A, B, C classI genes: their structure and expression. Immunol Res, 9: 265-274. Houcai, Y., Min, B., Wang, J., and Yao Y., (2011) Serum of Normal pregnant Women in different gestational weeks and the level of HLAG. Clin Med Pap. Huang, J.F., Yang, Y., Sepulveda, H., Shi, W., Hwang, I,, Peterson, P.A., Jackson, M.R., Sprent, J. and Cai, Z. (1999). TCR-Mediated internalization of peptide-MHC complexes acquired by T cells. Science. 286(5441):952-4. Hudrisier, D., Riond, J., Mazarguil, H., Gairin, J. E. and Joly, E. (2001). Cutting edge: CTLs rapidly capture membrane fragments from target cells in a TCR signalingdependent manner. J Immunol, 166(6):3645-3654. Hunt, J. S. (1989). Cytokine networks in the uteroplacental unit: macrophages as pivotal regulatory cells. J Reprod Immunol, 16: 1–17 University of Ghana http://ugspace.ug.edu.gh 59 Hunt, J.S., Petroff, M.G. and McIntire, R. H., Ober C.(2005) HLA G and immune tolerance in pregnancy; FASEB J. 19(7):681-93 Hunt, J. S. Jadhav, L., Chu W., Geraghty D. E., and Ober, C. (2000). Soluble HLA-G circulates in maternal blood during pregnancy. The American Journal of Obstetrics and Gynecology, 183(3): 682–688. Hviid, T. V. F., (2005). HLA-G in human reproduction: aspects of genetics, function and pregnancy complications. Hum. Reprod. Update, 12(3):209-32. Hviid T. V. F. (2006). HLA-G in human reproduction: aspects of genetics, function and pregnancy complications. Human Reproduction Update, 12(3): 209–232. Hviid, T. V., Christiansen, O. B., Johansen, J. K., Hviid, U. R., Lundegaad, C., Moller, C., and Morling, N. (2001). Characterisation of a new HLA-G allele encoding a nonconservative amino acid substitution in the alpha3 domain (exon 4) and its relevant to certain complications in pregnancy. Immunogenetics, 53: 48-53. Hviid, T.V., Hylenius, S., Høgh, A.M., Kruse, C. and Christiansen, O.B. (2002). HLAG polymorphisms in couples with recurrent spontaneous abortions. Tissue Antigens, 60:122–132. University of Ghana http://ugspace.ug.edu.gh 60 Hviid, T. V., Hylenius, S, Lindhard, A and Christiansen, O. B. (2004a). Association between human leukocyte antigen-G genotype and success of in vitro fertilization and pregnancy outcome. Tissue Antigens, 64:66–69. Hviid, T. V., Hylenius, S., Rorbye, C., and Nielsen, L. G. (2003). HLA-G allelic variants are associated with differences in the HLA-G mRNA isoform profile and HLA-G mRNA levels. Immu- nogenetics, 55: 63–79 Hviid, T.V., Meldgaard, M., Sørensen, S. and Morling, N. (1997). Polymorphism of exon 3 of the HLA-G gene. J Reprod Immunol, 35:31–42. Hviid, T.V., Rizzo, R., Christiansen, O. B., Melchiorri, L., Lindhard, A., and Baricordi, O. R., (2004b). HLA-G and IL10 in serum in relation to HLA-G genotype and poly morphism. Immunogenetics, 56,135-141. Hylenius, S., Andersen, A. M., Melbye, M., and Hviid, T.V. (2004) Association between HLA-G genotype and risk of pre-eclampsia: a case-control study using family triads. Mol Hum Reprod, 10:237–246. Ibrahim, E. C., Aractingi, S., Allory, Y., Borrini, F., Dupuy, A., Duvillard, P., Carosella, E. D., Avril, M. F. and Paul, P. (2004), Analysis of HLA antigen expression in benign and malignant melanocytic lesions reveals that upregulation of University of Ghana http://ugspace.ug.edu.gh 61 HLA-G expression correlates with malignant transformation, high inflammatory infiltration and HLAA1genotype. Int J Cancer, 108: 243-50. Ishitani, A. and Geraghty, D. E. (1992). Alternative splicing of HLA-G transcripts yields proteins with primary structures resembling both class I and class II antigens. Proc Natl Acad Sci U S A. 89(9): 3947–3951. Ishitani, A., Kishida, M., Sageshima, N., Yashiki, S., Sonoda, S., Hayami, M., Smith, A. G., and Hatake, K. (1999). Re-examintion of HLA-G polymorphism in African Americans. Immunogenetics, 49: 808-811 Ishitani, A., Sageshima, N., Lee N., Dorofeeva, N., Hatake, K., Marquardt H, and Geraghty, D. E. (2003). Protein expression and peptide binding suggest unique and interacting functional roles for HLA-E, F, and G in maternal-placental immune recognition. Journal of Immunology, 171(3): 1376–1384. . Janeway, C. A., Paul, T., Mark, W., and Mark, J. S. (2001), The Immune System in Health and Disease, Immunobiology, 5th ed. University of Ghana http://ugspace.ug.edu.gh 62 Kanai, T., Fujji, T., Kozuma, S., Yamashita, T., Miki, A., Kikuchi, A., and Taketani, Y. (2001). Soluble HLA-G influences the release of cytokines from allogeneic peripheral blood mononuclear cells in culture. Mol Hum Reprod, 7:195-200. Kapasi, K., Abert, S.E., Yie, S., Zavazava, N., and Librach, C. L., (2000). HLA-G has a concentration-dependent effect on the generation of an allo-CTL response. Immunology, 101: 191-200 Kikuchi-Maki, A., Yusa, S., Catina, T. L., and Campbell, K. S., (2003). KIR2DL4 is an IL-2- regulated NK cell receptor that exhibits limited expression in humans but triggers strong IFN-gamma production. J. Immunol, 171: 3415-3425 Kovats, S., Main, E. K., Librach, C., Stubblebine, M., Fisher, S. J., and DeMars, R. (1990). A class I antigen, HLA-G, expressed in human trophoblasts. Science, 248: 220–223. Kwak-Kim, J. Y., Chung-Bang, H.S., Ng S.C., Ntrivalas, E. F., Mangubat, C. P., Beaman, K. D., Beer A.E., Gilman-Sachs, A., (2003). Increased T helper 1 cytokine responses by circulating Tcells are present in women with recurrent pregnancy losses and in infertile women with multiple implantation failures after IVF. Hum Reprod. 18(4): 767-73 University of Ghana http://ugspace.ug.edu.gh 63 Laird, S. M., Tuckerman, E. M., Cork, B. A., Linjawi, S., Blakemore, A. I. F., Li, T. C. (2003). A review of immune cells and molecules in women with recurrent miscarriage. Hum Reprod Update, 9(2):163–74. Lajoie, J., Hargrove, J., Zijenah, L. S, Humphrey, J. H., Ward, B. J., Roger, M. (2006) Genetic variants in nonclassical major histocompatibility complex class I human leukocyte antigen (HLA)-E and HLA-G molecules are associated with susceptibility to heterosexual acquisition of HIV-1. J Infect Dis, 193:298-301. Langat, D. K., Sue, Platt, J., Tawfik, O., Fazleabas, A. T., and Hunt, J. S., (2006). Differential expression of human leukocyte antigen-G (HLA-G) messenger RNAs and proteins in normal human prostate and prostatic adenocarcinoma. Journal of Reproductive Immunology, 71(1) 75–86. Larsen, M. H., Bzorek ,M., Pass, M. B., Larsen, L. G., Nielsen, M. W., Svendsen, S. G., Lindhard, A., Hviid, T. V. (2011). Human leukocyte antigen-G in the male reproductive system and in seminal plasma. Molecular Human Reproduction, 17(12):727–738, 2011. University of Ghana http://ugspace.ug.edu.gh 64 Lee, N., Llano, M., Carretero, M., Ishitano, A., Navarro, F., Lopez-Bote, M. and Geraghty, D. A., (1998). HLA-E is a major ligand for the natural killer inhibitory receptor CD94/NKG2A. Proc Natl Acad Sci USA, 95:5199-5204. Le Friec, G., Gros, F., Sebti, Y., Guilloux, V., Pangault, C., Fauchet R., Amiot, L., (2004). Capacity of myeloid and plasmacytoid dendritic cells especially at mature stage to express and secrete HLA-G molecules. J Leukoc Biol. 76:1125-33. Le Hesran, J. Y., Cot, M., Personne, P., Fievet , N., Dubois, B., Beyeme, M., Boudin, C., Deloron, P., (1997). Maternal placental infection with Plasmodium falciparum and malaria morbidity during the first 2 years of life. Am J Epidemiol, 146:826–831. LeMaoult, J., Caumartin, J., Daouya, M., Favier, B., Le Rond, S., Gonzalez, A., Carosella, E. D. (2007). Immune regulation by pretenders: Cell-to-cell transfers of HLA-G make effector T cells act as regulatory cells. Blood, 109: 2040-2048. Lila, N., Rouas-Freiss, N., Dausset, J., Carpentier, A., Carosella, E. D. (2001). Soluble HLA-G protein secreted by allo-specific CD4+ T cells suppresses the allo-proliferative response: A CD 4+ T cell regulatory mechanism. Proceedings of the National Academy of Science of the United States of America. 98(21):12150-12155. University of Ghana http://ugspace.ug.edu.gh 65 Lozano, J. M., Gonzalez, R., Kindelan, J. M., Rouas-Freiss, N., Caballos, R., Dausset, J, Carosella, E. D., Peña, J. (2002). Monocytes and T lymphocytes in HIV-1-positive patients express HLA-G molecule. AIDS, 16:347-351. Malhotra, I., Dent, A., Mungai, P., Wamachi, A., Ouma, J. H., Narum, D. L., Muchiri, E., Tisch, D. J., and King, C. L. (2009). Can prenatal malaria exposure produce an immune tolerant phenotype? A prospective birth cohort study in Kenya. PLoS Med, 6: 100-116. Martínez, E., Naves, J. F., Parra Cuadrado, A., Pérez Rosado, M., Gómez, D.(2001). Moral Structure and function of "non-classical" HLA class I molecules. Unidad de Inmunología. Facultad de Medicina. Universidad Complutense. Madrid, 20(4):207- 215. Matte, C., Lajoie, J., Lacaille, J., Zijenah, L. S., Ward, B. J., Roger, M. (2004). Functionally active HLA-G polymorphisms are associated with the risk of heterosexual HIV-1 infection in African women. AIDS, 18:427-431. Meka, A., Reddy, B. M., (2006). Recurrent Spontaneous Abortions : An Overview of Genetic and Non-Genetic Backgrounds. 6(2):109–17. University of Ghana http://ugspace.ug.edu.gh 66 McCormick, J., Whitley, G. S. J., Le Bouteiller, P., and Cartwright, J. E., (2009). Soluble HLA-G regulates motility and invasion of the trophoblast-derived cell line SGHPL-4. Human Reproduction, 24(6):1339–1345. McIntire, R. H., Morales, P.J., Petroff, M. G., Colona, M., and Hunt, J. S. (2004) Recombinant HLA-G5 and G6 drive U937 myelomonocytic cell production of TGFbeta1. J. Leukoc. Biol. 76:1220-1228. Meer, A. V., Lukassen, H. G. M., Lierop, M. J. C., Van, Wijnands F, Mosselman, S., Braat, D. D. M., Joosten, I., (2004). Membrane-bound HLA-G activates proliferation and interferon- g production by uterine natural killer cells. Mol Hum Reprod, 10(3):189–95. Morales, P. J., Pace, J. L., Platt, J. S., Langat, D. K., and Hunt, J. S., (2007) Synthesis of β2-microglobulin-free, disulphide-linked HLA-G5 homodimers in human placental villous cytotrophoblast cells. Immunology, 122(2):179–188, 2007. Morandi, F., Levreri, I., Bocca, P., Galleni, B., Raffaghello, L., Ferrone, S., Prigione, I., Pistoia, V. (2007). Human neuroblastoma cellstrigger an immunosuppressive program in monocytes bystimulating soluble HLA-G release. Cancer Res,; 67: 6433- 41 University of Ghana http://ugspace.ug.edu.gh 67 Moreau, P., Adrian-Cabetre, F., and Menier, C., (1999). IL-10 selectively induces HLAG expression in human trophoblasts and monocytes. Int. Immunol, 11:803-811 Moya-Alvarez, V., Abellana, R., and Cot, M., (2014). Pregnancy-associated malaria and malaria in infants: an old problem with present cosequences.. Malar J. 13(1):271. Munz, C., Holmes, N., King, A., Loke, Y. W., Colonna, M., Schild, H., and Rammensee, H. G. (1997). Human histocompatibility leukocyte antigen HLA-G molecules inhibit NKAT3 expressing natural killer cells. J Exp Med, 185(3):385-91. Navikas V, Link J, Persson C, Olsson T, Hojeberg B, Ljungdahl A, Link H, Wahren B. (1995). Increased mRNA expression of IL-6, IL-10, TNF-alpha, and perforin in blood mononuclear cells in human HIV infection. J Acquir Immune Defic Syndr Hum Retroviruses, 9:484-489. Navarro, F., Llano, M., Bellon, T., Colonna, M., Geraghty, D. E., and Lopez-Botet, M. (1999). The ILT2 (LIR1) and CD94/NKG2A NK cell receptors respec- tively recognize HLA-G1 and HLA-E molecules co-expressed on target cells. Eur J Immunol 29:277– 283. University of Ghana http://ugspace.ug.edu.gh 68 Nehemiah, J. L., Schnitzer, J. A., Schulman, H., and Novikoff, A. B., (1981). Human chorionic trophoblast, decidual cells, and macrophages: a histochemical and electron microscopic study. Am. J. Pathhol, 157:159-169 Nicolae, D., Cox, N.J., Lester, L. A., Schneider, D., Tan, Z., Billstrand, C., Kuldanek, S., Donfack, J., Kogut, P., Patel, N. M., Goodenbour, J., Howard, T., Wolf, R., Koppelman, G. H., White, S. R., Parry, R., Postma, D. S., Meyers, D., Bleecker, E. R., Hunt, J. S., Solway, J., Ober, C. (2005). Fine mapping and positional candidatestudies identify HLA-G as an asthma susceptibility gene on chromosome 6p21. Am J Hum Genet, 76(2):349-57. Nuckel, H., Rebmann, V., Durig, J., Duhrsen, U., and Grosse-Wilde, H.(2005) HLA-G expression is associated with an unfavorable outcome andimmunodeficiency in chronic lymphocytic leukemia. Blood, 105:1694-8. Ober, C., Aldrich, C., Rosinsky, B., Robertson, A., Walker, M. A., Willadsen, S., Verp, M. S., Geraghty, D. E., Hunt, J. S. (1998). HLA-G1 protein expression is not essential for fetal survival. Placent,a 19:127-132. Ober, C., Aldrich, C. L., Chervoneva, I., Billstrand, C., Rahimov, F., Gray, H. L., and Hyslop, T. (2003). Variation in the HLA-G promoter region influences mis- carriage rates. Am J Hum Genet ,72:1425–1435. University of Ghana http://ugspace.ug.edu.gh 69 Ober C, Billstrand C, Kuldanek S, Tan Z. (2006). The miscarriage-associated HLA-G 725G allele influences transcription rates in JEG-3 cells. Human reproduction, 21(7): 1743-1752 O'Brien, M., McCarthy, T., Jenkins, D., Paul, P., Dausset, J., Carosella, E. D., and Moreau, P. (2001). Altered HLA-G transcription in pre-eclampsia is associated with allele specific inheritance: possible role of the HLA-G gene in susceptibility to the disease. Cell. Mol. Life Sci, 58. Odlind, V. (1997).Induced abortion—a global health problem. Acta Obstet Gynecol Scand Suppl, 164: 43 – 45. Onno, M., Le Friec, G., Pangault, C., Amiot, L., Guilloux, V., Drenou. B., CauletMaugendre, S., Andre, P., and Fauchet, R., (2000a). Modulation of HLA-G antigens in myelomonocytic cells. Hun.Immunol. 61:1086-1094. Onno, M., Pangault, C., Le Friec, G., Guilloux, V., Andre P, and Fauchet, R. (2000b) Modulation of HLA-G antigens expression by human cytomegalovirus: specific induction in activated macrophages harboring human cytomegalovirus infection. J Immunol; 164:6426-6434. University of Ghana http://ugspace.ug.edu.gh 70 Park G. M., Lee S., Park B., Kim E., Shin J., Cho K., and Ahn, K. (2004) Soluble HLAG generated by proteolytic shedding inhibits NK-mediated cell lysis. Biochem Biophys Res Commun, 313,606-611. Patel, D. M., Arnold, P.Y., White, G. A., Nardella, J. P. and Mannie, M.D. (1999). ClassII MHC/peptide complexes are released from APC and are acquired by T cell responders during specific antigen recognition. J Immunol, 63: 5201-5211. Paul P., Cabestre, F. A., Ibrahim, E. C., Lefebvre, S., Khalil-Daher, I., Vazeux, G., Quiles, R. M., Bermond, F., Dausset, J. and Carosella, E.D. ( 2000). Identification of HLA-G7 as a new splice variant of the HLA-G mRNA and expression of soluble HLAG5, -G6, and -G7 transcripts in human transfected cells. Human Immunology, 61(11): 1138–1149. Paul, P., Rouas-Freiss, N., Khalil-Daher, I., Moreau, P., Riteau, B., Le Gal, F. A., Avril MF, Dausset J, Guillet JG, Carosella,. E. D. (1998). HLA-G expression in melanoma: a way for tumor cells to escape from immune surveillance. Proc Natl Acad Sci USA; 95: 4510-5. Pende, D., Sivori, S., Accame, L., Pareti, L., Falco, M., Geraghty, G., Le Bouteiller, P., Moretta, L., and Moretta, A.(1997) HLA –G recognition by natural killer cells; University of Ghana http://ugspace.ug.edu.gh 71 Involvement of CD94 as inhibitory and activating receptor complex. Eur. J. Immunol. 27, 1875-1880 Petroff, M. G., Sedlmayr, P., Azzola, D., and Hunt, J. S.(2002). Decidual macrophages are potentially susceptible to inhibition by class Ia and class Ib HLA molecules. J.Reprod. Immunol.. 56, 3-17 Perez-Villar, J.J., Melero, I., Navarro, F., Carretero, M., Bellon, T., Llano, M., Colonna,M., Geraghty, D. E., and Lopez-Botet, M., (1997). The CD94/NKG2-A inhibitory receptor complex is involved in natural killer cell mediated recognition of cells expressing HLA G1. J. Immuno, 158:5736-5743 Pfeiffer, K. A., Rebmann, V., and van der Ven, K. (2000). Soluble histocompatibility antigen levels in early pregnancy after IVF. Hum Immunol 61:559–564. Pfeiffer, K. A., Fimmers, R., Engels, G., van der Ven, H., and van der Ven, K., (2001) The HLA-G genotype is potentially associated with idiopathic recurrent spontaneous abortion. Mol Hum Reprod, 7:373–378. Pregnancy WGRoHBPi. (2000). Report of the National high Blood Pressure Education Program. Am J Obstet Gynecol, 6:88-95 University of Ghana http://ugspace.ug.edu.gh 72 Ponte, M., Cantoni, C., and Biassoni, R. (1999). Inhibitory receptors sensing HLA-G1 molecules in pregnancy: decidua-associated natural killer cells express LIR-1 and CD94/NKG2A and acquire p49, an HLA-G1-specific receptor. Proceedings of the National Academy of Sciences of the United States of America, 96(10):5674–5679,. Population Council. (2000). Is it possible to reduce the abortion rate by increasing contraceptive use? Annual report. Pröll, J., Blaschitz, A., Hutter, H. and Dohr, G. (1990). First trimester human endovascular trophoblast cells express both HLA-C and HLA-G. Am J Reprod Immunol. 42(1):30-6 Puppo, F., Costa, M., Contini, P., Brenci, S., Cevasco, E., Ghio, M., Norelli, R., Bensussan, A., Capitanio, G. L., and Indiveri, F. (1999). Determination of solu- ble HLA-G and HLA-A-B, and -C molecules in pregnancy. Transplant Proc 31:1841– 1843. Rachas, A., Le Port, A., Cottrell, G., Guerra, J., Choudat, I., Bouscaillou, J., Massougbodji, A.,Garcia, A. (2012). Placental malaria is associated with increased risk of nonmalaria infection during the first 18 months of life in a Beninese population. Clin Infect Dis, 55:672–678. University of Ghana http://ugspace.ug.edu.gh 73 Rajagopalan, S., Fu, J., and Long, E. O. (2001). Cutting edge: induction of IFN- gamma production but not cytotoxicity by the killer cell Ig-like receptor KIR2DL4 (CD158d) in resting NK cells. J. Immunol., 167:1877–1881 Rebman, V., Busemann, A., Lindermann, M., Grosse-Wilde, H. (2003). Detection of HLA-G5 secreting cells. Hum Immunol, 64:1017-1024. Rebmann, V., Pfeiffer, K., Passler, M., Ferrone, S., Maier, S., Weiss, E., and Grosse- Wilde, H. (1999). Detection of soluble HLA-G molecules in plasma and amniotic fluid.Tissue Antigens, 53:14–22. Rebmann, V., Switala, M., Eue, I., and Grosse-Wilde., H., (2010). Soluble HLA-G is an independent factor for the prediction of pregnancy outcome after ART: a German multicentre study. Human Reproduction, 25(7):1691–1698. Rebmann, V., van der Ven, K., Passler, M., Pfeiffer, K., Krebs, D., Grosse-Wilde, H., (2001). Association of soluble HLA-G plasma levels with HLA-G alleles. Tissue Antigens 57:15–21. University of Ghana http://ugspace.ug.edu.gh 74 Ribic, A. (2005). Immune Privilege Revisited : The Roles of Neuronal MHC Class I Molecules in Brain Development and Plasticity. http://cdn.intechopen.com/pdfs-wm/36522.pdf 12 -12- 2013 Rizzo, R., Fuzzi, B., Stignani, M., Criscuoli, L., Melchiorri, L., Dabizzi, S., Campioni, D., Lanza, F., Marzola, A., Branconi, F., Noci,I., Baricordi, O. R. (2007). Soluble HLA-G molecules in follicular fluid: a tool for oocyte selection in IVF? Journal of Reproductive Immunology, 74(1-2) :133–142. Roth, I., and Fisher, S. J. (1999) IL-10 is an autocrine inhibitor of placental cytotrophoblast MMP-9 production and invasion. Dev Biol 205:194-204 Rouas-Freiss, N., Moreau, P., Ferrone, S., Carosella, E. D. (2005). HLA-G proteins in cancer: do they provide tumor cells with an escapemechanism? Cancer Res, 65:10139-10144 Rouas-Freiss, N., Naji, A., Durrbach, A., Carosella E. D. (2007). Tolerogenic functions of human leukocyte antigen G: from pregnancy to organ and cell transplantation. Transplantation, 84(1):21-25. University of Ghana http://ugspace.ug.edu.gh 75 Rousseau, P., Le Discorde, M., Mouillot, G., Marcou, C., Carosella, E. D., and Moreau, P. (2003) The 14 bp deletion-insertion polymorphism in the 3’ UT region of the HLA-G gene influences HLA-G mRNA stability. Hum Immunol, 64:1005–1010. Ryan, K. J., Berkowits, R. S., Barbieri, R. L., and Dunaif, A. (1999). Kistner's Gynecology and Women's Health. 7th ed. St. Louis: Mosby; 15 – 18. Sadki, K., Bakri, Y., Tijane, M. H., and Amzazi S. (2011). MHC Polymorphism and Tuberculosis. Disease. 14 http://cdn.intechopen.com/pdfs-wm/29409.pdf 8-11-2013 Samaridis, J., and Colona, M. (1997) Cloning of novel immunoglobulin superfamily receptors expressed on human myeloid and lymphoid cells: structural evidence for new stimulatory and inhibitory pathways. Eur.J. Immunol. 2:660-665. Sargent IL. (2005). Does “soluble” HLA-G really exist? Another twist to the tale. Mol Hum Reprod 11(10):695–8. Schwandt, H. M., Creanga, A. A., Danso, K. A., Adanu, R. M. K., Agbenyega, T., Hindin, M. J. A. (2010). Comparison of women with induced abortion, spontaneous abortion and ectopic pregnancy in Ghana ☆. Contraception [Internet]. Elsevier B.V.; http://dx.doi.org/10.1016/j.contraception.2010.10.011 Accessed 20/01/2014 University of Ghana http://ugspace.ug.edu.gh 76 Singer, G., Rebmann, V., Chen, Y. C., Liu, H. T., Ali, S. Z., Reinsberg, J., McMaster, M. T., Pfeiffer, K., Chan, D. W., Wardelmann, E., Grosse-Wilde, H., Cheng, C. C., Kurman, R. J., and Shih, I. M. (2003) HLA-G is a potential tumor marker in malignant ascites.Clin Cancer Res. 9(12):4460-464. Sivori, S., Parolini, S., Marcenaro, E., Millo, R., Bottino, C., and Moretta, A. (2000) Triggering receptors involved in natural killer cell-mediated cytotoxicity against choriocarcinoma cell lines. Hum. Immunol. 61:1055–1058 Shankarkumar U., (2004). The Human Leukocyte Antigen (HLA) System. 2004;4(2):91–103. Sher, Y. P., Chou, C. C., Chou, R. H., Wu, H. M., Wayne Chang, W. S., Chen, C. H., Yang, P. C., Wu, C. W., Yu, C. L., Peck, K., (2006). Human kallikrein 8 protease confers a favorable clinical outcome in non-small cell lung cancer by suppressing tumor cell invasiveness. Cancer Res 66:11763-11770. Shiroishi, M., Tsumoto, K., Amano, K., Shirakihara, Y., Colonna, M., Braud, V. M., Allan, D. S., Makadzange, A., Rowland- Jones, S., Willcox, B., Jones, E. Y., van der Merwe, P. A., Kumagai, I. and Maenaka, K. (2003). Human inhibitory receptors Ig- University of Ghana http://ugspace.ug.edu.gh 77 like transcript 2 (ILT2) and ILT4 compete with CD8 for MHC class I binding and bind preferentially to HLA-G. Proc. Natl. Acad. Sci. USA 100:8856–8861 Sjöström, A., Eriksson, M., Cerboni, C., Johansson, M. H., Sentman, C. L., Kärre, K., Höglund, P., (2001). Acquisition of external major histocompatibility complex class I molecules by natural killer cells expressing inhibitory Ly49 receptors. J Exp Med 194:1519-30. Soderstrom, K., Corliss, B., Lanier, L. L., and Phillips, J.H., (1997). CD 94/NKG2 is the predominant inhibitory receptor involved in recognition of HLA –G by decidual and peripheral blood NK cells. J. Immunol. 156:1072-1075 Sridhar Rao P.N., Major MHC (major histocompatibility complex). http://www.microrao.com/micronotes/pg/MHC.pdf Accessed 20/01/2014 Steinborn, A., Rebmann, V., Scharf, A., Sohn, C., and Grosse-Wilde, H. (2003). Pla- cental abruption is associated with decreased maternal plasma levels of soluble HLAG. J Clin Immunl, 23:307–314. Suarez, M. B., Morales, P., Castro, M. J., Fernandez, V., Varela, P., Alvarez, M., Martinez-Laso, J., and Arnaiz-Villena, A. (1997). A new HLA-G allele University of Ghana http://ugspace.ug.edu.gh 78 (HLAG*0105N) and its distribution in the Spanish population. Immuno-genetics, 45:464–465. Tabiasco, J., Vercellone, A., Meggetto, F., Hudrisier, D., Brousset, P., and Fournie, J. J. (2003). Acquisition of viral receptor by NK cells through immunological synapse. J Immunol, 170:5993–5998. Tamaki, J., Arimura, Y., Koda, T., Fujimoto, S., Fujino, T., Wakisaka, A., and Kakinuma, M. (1993). Heterogeneity of HLA-G genes identified by polymerase chain reaction/single strand conformational polymorphism (PCR/SSCP). Micro-biol Immunol 37:633–640. Tatari-Calderone, Z., Semnani, R., T., Nutman, T., B., Schlom, J., Sabzevari, H. (2002). Acquisition of CD80 by human T Cells at early stages of activation: functional involvement of CD80 acquisition in T cell to T cell interaction. J Immunol, 69:6162- 6169. The MGT/HLA Database, 2013 http://www.ebi.ac.uk/ipd/imgt/hla/docs/version_r3150.html accessed 10-09-2013 University of Ghana http://ugspace.ug.edu.gh 79 The MHC Sequencing Consortium. (1999). Complete sequence and gene map of a human major histocompatibility complex. Nature 401: 921-923. Tiago, D. V., Vianna, P., Chies, J. A. B., (2010) HLA-G- From fetal tolerance to a Regulatory Molecule. Current Immunology Reviews, 6, 000-000. Tonga, C., Kimbi, H. K., Anchang-Kimbi, J. K., Nyabeyeu, H. N., Bissemou, Z. B., Lehman, L. G. (2013). Malaria risk factors in women on intermittent preventive treatment at delivery and their effects on pregnancy outcome in Sanaga-Maritime, Cameroon. PLoS One,8:65876. Tripathi, P., Abbas, A., Naik, S., and Agrawal, S., (2004). Role of 14-bp deletion in the HLA-G gene in the maintenance of pregnancy. Tissue Antigens, 64,706–710. Tripathi, P., and Agrawal, S. (2007a). Editorial Review: The Role of Human Leukocyte Antigen E and G in HIV Infection. AIDS, 21(11):1395-1404. Tripathi, P., Naik, S., and Agrawal, S. (2006). HLA-E and immunobiology of pregnancy. Tissue Antigens, 67:207-213. University of Ghana http://ugspace.ug.edu.gh 80 Tripathi, P., Naik, S., and Agrawal, S., (2007b). Role of HLA-G, HLA-E and KIR2DL4 in Pregnancy. Tissue Antigen, 7(3):219–33. Trowsdale, J., and Moffett, A. (2008). NK receptor interactions with MHC class I molecules in pregnancy. Seminars in Immunology, 20( 6):317–320 . Trundley, A., and Moffett, A., (2004). Human uterine leukocytes and pregnancy. Tissue Antigens, 63, 1–12. Vanherberghen, B., Andersson, K., Carlin, L. M., Nolte-'t, Hoen, E. N., Williams, G. S., Höglund, P., Davis, D. M., (2004). Human and murine inhibitory natural killer cell receptors transfer from natural killer cells to target cells. Proc Natl Acad Sci USA; 101:16873-8. van Lierop, M. J., Wijnands, F., Loke, Y. W., Emmer, P. M., Lukassen, H. G., van der Braat, D. D. M. A., Mosselman, S., and Joosten, I. (2002). Detection of HLA-G by a specific sandwich ELISA using monoclonal antibodies. Mol Hum Reprod. 8,776-784. University of Ghana http://ugspace.ug.edu.gh 81 Vercammen, M. J., Verloes, A., Velde, H., Van De, Haentjens, P., (2008). Accuracy of soluble human leukocyte antigen-G for predicting pregnancy among women undergoing infertility treatment . meta-analysis, 14(3):209–18. Warner, C. M., Brownell, M. S., Rothschild, M. F. (1991). Analysis of litter size and weight in mice differing in Ped genephenotype and the Q region of the H-2 complex. J Reprod Immunol, 19(3):303-313. White, S. R., Loisel, D. A., McConville, J. F., Stern, R., Tu, Y., Marroquin, B. A., Noth, I., and Ober, C. (2010). Levels of soluble human leukocyte antigen-G are increased in asthmatic airways. Eur Respir J. 35(4):925-927. WHO: World Malaria Report. Geneva: World Health Org. 2012 Wu, L., Feng H., and Warner C., M., (1999). Identification of two major histocompatibility complex class Ib genes, Q7 and Q9, as the Ped gene in the mouse. Biol Reprod, 60(5), 1114-1119. University of Ghana http://ugspace.ug.edu.gh 82 Wu, F. X., Wu, L. J., Luo, X. Y., Tang, Z., Yang, M. H., Xie, C. M., Liu, N. T., Zhou, J. G., Guan, J. L., Yuan, G. H., (2009). Lack of association between HLA-G 14-bp polymorphism and systemic lupus erythematosus in a Han Chinese population. Lupus, 18(14), 1259–1266. www.abdserotec.com/an-introduction-to-elisa.htm accessed 07-05-2014 www.copewithcytokines.de/cope.cgi?key=HLA-G accessed 15-07-2014 www.medscape.com/viewarticle/559456_6 accessed 15-07 -2014 Xu, Y., Jin, P., and Warner, C. M. (1993). Modulation of preimplantation embryonic development by antisense oligonucleotides to major histocompatibility complex genes. Biol Reprod; 48(5), 1042-1046. Yan, W. H., Lin, A., Chen, B. G., Zhou, M. Y., Dai, M. Z., Chen, X. J., Gan, L. H., Zhu, M., Shi, W. W., Li, B. L., (2007). Possible roles of KIR2DL4 expression on uNK cells in human pregnancy. The American Journal of Reproductive Immunology, 57(4)233–242 University of Ghana http://ugspace.ug.edu.gh 83 Ye, S. R., Yang, H., Li, K., Dong, D. D., Lin, X. M., and Yie, S. M. (2007). Humanleukocyte antigen G expression: as a significant prognostic indicator for patients with colorectal cancer. Mod Pathol 20,375-83. Yie, S. M., Li, L. H., Li, Y. M., and Librach C. (2004). HLA-G protein concentrations in maternal serum and placental tissue are decreased in preeclampsia. Am J Obstet Gynecol 191,525–529. Yie, S. M., Li, L. H., Xiao, R., and Librach, C. L. (2008). A single base-pair mutation in the 3 untranslated region of HLA-G mRNA is associated with pre-eclampsia. Mol. Hum. Reprod. 14, 649–653. Yu, Y.-R., Tian, X.-H., Tian, Y., Wang, and Feng, M. F., (2006). Rapid production of human KIR2DL4 extracellular domain and verification of its interaction with HLAG. Biochemistry, 71(1): 60–64 Zhang, W. Q., Xu, D. P., Liu, D., L, Y. Y., Ruan, Y. Y., Lin, A., and Yan, W. H. (2014). HLA-G1 and HLA-G5 isoforms have an additive effect on NK cytolysis. Human Immunology, 75(2), 182–189 University of Ghana http://ugspace.ug.edu.gh 84 Zimmer, J., Ioannidis, V., Held, W., (2001) H-2D ligand expression byLy49A+ natural killer (NK) cells precludes ligand uptake from environmental cells: implications for NK cell function. J Exp Med; 194,1531-1539 University of Ghana http://ugspace.ug.edu.gh 85 APPENDICES Appendix I: Conjugate Solution 100x (Dilution) Volume: 13ml Components Amount Lot no.: Amount used Check steps done ( ) Conjugate Solution 0.13ml K21-033 Conjugate Diluent 13ml K00-600 PROCEDURE 1. Pipette 0.13ml of Conjugate Solution into flask. 2. Add 13ml of Conjugate Diluent and mix. University of Ghana http://ugspace.ug.edu.gh 86 Appendix II: Wash Solution 10x (Dilution) Volume: 500ml Working solution: 1x Components Amount Lot no.: Amount used Check steps done ( ) Wash Solution 100ml K00-579 Distilled Water 50 ml n/a PROCEDURE 1. Pipette 50ml of wash solution into a measuring cylinder. 2. Add 450mlof distilled water and mix. University of Ghana http://ugspace.ug.edu.gh 87 Appendix III: Master Calibrator stock solution Volume: 500ml Components Amount Lot no.: Amount used Check steps done ( ) Master Calibrator 2 vials K21-030S3 Dilution Buffer 13ml K00-595 Distilled Water 0.8ml N/A PROCEDURE 1. Pipette 0.8ml of distilled water into vial to obtain 625U/ml concentration of Reconstituted master calibrator (stock). 2 Aliquot 100µl of the stock into new a vial and add 400µl of the dilution buffer. 3. Aliquot 100µl into appropriate well. 4. Add 250µl of dilution buffer and pipette 100µl appropriate well 5. Repeat step 4 five times, to obtain specific dilutions for appropriate wells. University of Ghana http://ugspace.ug.edu.gh 88 Appendix IV: sHLA-G ELISA Plate sHLA-G ELISA Plate 1 2 3 4 5 6 7 8 9 10 11 12 A Calibrator 125.00 Calibrator 125.00 SA0 1 SA0 9 SA1 7 SA2 6 AMC0 01 AMC0 09 AFC0 07 DP0 45 DP1 76 DP2 23 B Calibrator 62.50 Calibrator 62.50 SA0 2 SA1 0 SA1 8 SA2 7 AMC0 02 AMC0 10 AFC0 08 DP0 88 DP1 94 DP2 28 C Calibrator 31.25 Calibrator 31.25 SA0 3 SA1 1 SA1 9 SA2 8 AMC0 03 AFC00 1 AFC0 09 DP1 25 DP1 97 DP2 43 D Calibrator 15.63 Calibrator 15.63 SA0 4 SA1 2 SA2 0 SA2 9 AMC0 04 AFC00 2 AFC0 10 DP1 37 DP1 98 DP2 77 E Calibrator 7.81 Calibrator 7.81 SA0 5 SA1 3 SA2 1 SA3 0 AMC0 05 AFC00 3 DP00 7 DP1 42 DP2 02 DP2 83 F Calibrator 3.91 Calibrator 3.91 SA0 6 SA1 4 SA2 2 SA3 1 AMC0 06 AFC00 4 DP01 0 DP1 55 DP2 04 DP2 85 G Blank Blank SA0 7 SA1 5 SA2 3 SA3 2 AMC0 07 AFC00 5 DP04 2 DP1 58 DP2 08 DP2 63 H Blank Blank SA0 8 SA1 6 SA2 4 SA3 3 AMC0 08 AFC00 6 DP04 3 DP1 75 DP2 22 DP2 91 University of Ghana http://ugspace.ug.edu.gh 89 Appendix V. Median sHLA-G levels (U/ml) and P value for Categories Compared Study Participants Median sHLA-G levels (U/ml) P value Spontaneous Abortion 66.52 0.03 Pregnancy with Normal Delivery 49.35 1st Trimester of Spontaneous Abortion 66.53 0.04 1st Trimester of Pregnancy with Normal Delivery 41.94 2nd Trimester of Spontaneous Abortion 98.65 1.0 2nd Trimester of Pregnancy with Normal Delivery 69.05 1st Trimester of Spontaneous Abortion 66.53 1.0 2nd Trimester of Spontaneous Abortion 74.08 1st Trimester of Pregnancy with Normal Delivery 39.73 0.08 2nd Trimester of Pregnancy with Normal Delivery 69.06 Non Pregnant Women 54.04 0.18 Healthy Adult Males 79.11 Krukal-Wallis Rank Sum Test (Trimesters) P=0.015 University of Ghana http://ugspace.ug.edu.gh 90 Appendix VI The characteristics of study participants Sample ID Age/Yrs. Category Contrace ptive Usage Gestation/ months Infant Weight/ Kg Units/ ml SA01 41 Spontaneous Abortion No 1 n/a 11.7 SA02 41 Spontaneous Abortion No 5 n/a 186.5 SA03 46 Spontaneous Abortion Yes 3 n/a 64.02 SA04 30 Spontaneous Abortion Yes 4 n/a 140.7 SA05 39 Spontaneous Abortion Yes 3 n/a 28.3 SA06 35 Spontaneous Abortion No 1 n/a 141.8 SA07 30 Spontaneous Abortion Yes 3 n/a 46.3 SA08 27 Spontaneous Abortion No 1 n/a 112.3 SA09 23 Spontaneous Abortion No 3 n/a 23.4 SA10 31 Spontaneous Abortion No 4 n/a 63.5 SA11 28 Spontaneous Abortion Yes 1 n/a 88.6 SA12 33 Spontaneous Abortion No 3 n/a 206 SA13 29 Spontaneous Abortion No 1 n/a 90.3 SA14 30 Spontaneous Abortion No 2 n/a 27.2 SA15 22 Spontaneous Abortion No 3 n/a 113.4 SA16 41 Spontaneous Abortion No 2 n/a 66.2 SA17 25 Spontaneous Abortion No 1 n/a 135.5 SA18 34 Spontaneous Abortion Yes 4 n/a 84.7 SA19 17 Spontaneous Abortion No 3 n/a 119.1 SA20 29 Spontaneous Abortion No 2 n/a 68.5 SA21 41 Spontaneous Abortion No 3 n/a 25.6 SA22 29 Spontaneous Abortion No 4 n/a 49.1 SA23 40 Spontaneous Abortion No 1 n/a 51.3 University of Ghana http://ugspace.ug.edu.gh 91 SA24 35 Spontaneous Abortion No 1 n/a 29.4 SA26 33 Spontaneous Abortion No 3 n/a 69 SA27 24 Spontaneous Abortion No 2 n/a 62.9 SA28 37 Spontaneous Abortion No 5 n/a 171.5 SA29 21 Spontaneous Abortion No 4 n/a 58.5 SA30 25 Spontaneous Abortion Yes 3 n/a 150.4 SA31 38 Spontaneous Abortion No 3 n/a 66.8 SA32 25 Spontaneous Abortion No 2 n/a 21.3 SA33 34 Spontaneous Abortion No 4 n/a 34.8 AMC001 n/a Normal Male n/a 0 n/a 128.2 AMC002 n/a Normal Male n/a 0 n/a 41.4 AMC003 n/a Normal Male n/a 0 n/a 123 AMC004 n/a Normal Male n/a 0 n/a 96.5 AMC005 n/a Normal Male n/a 0 n/a 91.4 AMC006 n/a Normal Male n/a 0 n/a 66.8 AMC007 n/a Normal Male n/a 0 n/a 666.6 AMC008 n/a Normal Male n/a 0 n/a 52.9 AMC009 n/a Normal Male n/a 0 n/a 13.8 AMC010 n/a Normal Male n/a 0 n/a 54.6 AFC001 n/a Non Pregnant Female n/a 0 n/a 52.4 AFC002 n/a Non Pregnant Female n/a 0 n/a 32.1 AFC003 n/a Non Pregnant Female n/a 0 n/a 112.8 AFC004 n/a Non Pregnant Female n/a 0 n/a 55.7 AFC005 n/a Non Pregnant Female n/a 0 n/a 61.8 AFC006 n/a Non Pregnant Female n/a 0 n/a 31.6 AFC007 n/a Non Pregnant Female n/a 0 n/a 37.6 AFC008 n/a Non Pregnant Female n/a 0 n/a 63.5 AFC009 n/a Non Pregnant Female n/a 0 n/a 102.1 AFC010 n/a Non Pregnant Female n/a 0 n/a 33.2 University of Ghana http://ugspace.ug.edu.gh 92 DP007 28 Pregnancy with normal delivery n/a 2 3.6 25.1 DP010 30 Pregnancy with normal delivery n/a 2 3 10.1 DP042 30 Pregnancy with normal delivery n/a 3 3 21.8 DP043 20 Pregnancy with normal delivery n/a 5 3.5 82.4 DP045 30 Pregnancy with normal delivery n/a 5 2.5 29.4 DP088 28 Pregnancy with normal delivery n/a 3 3.6 22.9 DP125 28 Pregnancy with normal delivery n/a 2 2.9 48.5 DP137 28 Pregnancy with normal delivery n/a 3 3.4 33.7 DP142 25 Pregnancy with normal delivery n/a 1 3.2 71.8 DP155 18 Pregnancy with normal delivery n/a 2 2.7 56.3 DP158 20 Pregnancy with normal delivery n/a 3 3.8 41.9 DP175 25 Pregnancy with normal delivery n/a 2 2.2 16.5 DP176 26 Pregnancy with normal delivery n/a 9 3.2 36.5 DP194 19 Pregnancy with normal delivery n/a 6 3 98.7 DP197 21 Pregnancy with normal delivery n/a 4 3.8 41.4 DP198 23 Pregnancy with normal delivery n/a 4 3.3 60.7 DP202 25 Pregnancy with normal delivery n/a 6 3.9 49.6 DP204 26 Pregnancy with normal delivery n/a 3 3.8 60.7 DP208 31 Pregnancy with normal delivery n/a 6 3 71.3 DP222 23 Pregnancy with normal delivery n/a 4 3.9 61.8 DP223 19 Pregnancy with normal delivery n/a 8 2.5 37 DP228 20 Pregnancy with normal delivery n/a 6 2.6 135.5 DP243 17 Pregnancy with normal delivery n/a 4 2.9 95.9 DP277 27 Pregnancy with normal delivery n/a 3 3.1 57.9 DP283 16 Pregnancy with normal delivery n/a 6 3 81.3 DP285 24 Pregnancy with normal delivery n/a 2 2.3 49.1 University of Ghana http://ugspace.ug.edu.gh 93 DP263 35 Pregnancy with normal delivery n/a 4 4.7 63.5 DP291 27 Pregnancy with normal delivery n/a 4 3.9 26.1 University of Ghana http://ugspace.ug.edu.gh 94 Appendix VII QUESTIONNAIRE Identification number ……… ………… Date…………….. Age………..Duration of Pregnancy:………………..weeks Marital Status: a. Single b. Married c. Widowed d. Separated e. Divorced Do you work? ………………… If yes, what do you do? …………………………... During the pregnancy, did you have any exposure to X-rays? No Yes, If yes how many times……………………………………… Herbal Drugs No Yes, Alcohol No Yes, Medications No Yes, If yes please specify………………………………………………. Cigarettes No Yes, During the pregnancy, did you have any viral infections/Rashes No Yes, If yes please specify ………………………………………………………………………………………… ………………………………………………………………………………………… ………………………… Were there any complications during the pregnancy? No Yes, if yes please specify ………………………………………………………………………………………….. Do you have a history of infertility? No Yes, Was the pregnancy the result of infertility treatment? No Yes, How many pregnancies have you had, including any miscarriages, abortions, and this one: …………………………………………………………………………………… Have you had recurrent pregnancy loss or a stillbirth? No Yes, How many miscarriages have you had?......................................................................... How many abortion have you had? …………………………………………………… University of Ghana http://ugspace.ug.edu.gh 95 ………………………………………………………………… Were you on birth control pills before conception? No Yes, If yes please specify ………………………………………………………………………………………… ………………………………………………………………………………………… ……………….. Are you Diabetes? No Yes Hypertensive? No Yes Have you had a history of trauma or domestic violence? No Yes; if yes, please explain: ………………………………………………………………………………………….. ………………………………………………………………………………………….. Have you ever had a blood transfusion? No Yes; if yes date………………………. Do you have a history of a sexually transmitted disease? No Yes, If yes which of the following: gonorrhoea, chlamydia, HPV syphilis University of Ghana http://ugspace.ug.edu.gh