MATERNAL INFECTIONS AND DEVELOPMENT OF PREECLAMPSIA: A SYSTEMATIC REVIEW OF THE EPIDEMIOLOGICAL LITERATURE By Abdul Wajid A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Epidemiology Master of Science 2015 ABSTRACT MATERNAL INFECTIONS AND DEVELOPMENT OF PREECLAMPSIA: A SYSTEMATIC REVIEW OF THE EPIDEMIOLOGICAL LITERATURE By Abdul Wajid We systematically reviewed the associations between H. pylori (HP) , cytomegalovirus (CMV) and C. pneumoniae (CP) infection in pregnancy and preeclampsia (PE), a disorder found in 5 - 8% of pregnancies and a leading cause of maternal and perinatal mortality. We used Preferred Reporting Items for Systematic Reviews and Meta - Analyses (PRISMA) Guidelines and searched PubMed, EMBASE and Web of Science. We also assessed the studies for risk of bias by utilizing a modified version of A Cochrane Risk of Bias Asse ssment Tool for Non - Randomized Studies of Interventions (ACROBAT - NRSI). After exclusions based on criteria from these tools, 16 studies were reviewed in detail, of 1,031 initially identified by our search algorithm. Evidence of infection was based on ser ology (12 studies), Polymerase Chain Reaction (2) or both (2). All four studies of the association between HP and PE found significant odds ratios ranging from 2.7 9.2. Two of four studies of CMV and PE found significantly elevated odds ratios (1.9 and 2 .7), while only three of ten studies of CP found significant odds ratios, ranging from 3.1 to 4.1. Not all studies controlled fully for confounding, and ten studies were at serious risk of bias. The available literature provides partial support for the association between these infectious agents and PE, especially for HP, but more rigorous studies are needed in this area, because more than half of the studies examined were at high risk of bias. iii ACKNOWLEDGMENTS I am thankful to my supervisor, Dr. Nigel Paneth , for his guidance and support that helped significantly in giving shape to this thesis. I am grateful to him for his valuable suggestions that helped clear many of issues and concepts related to the subject matter and epidemiology. I am indebted to Dr. Mat Reeves, for his patience and continuous support that contributed a lot to the fabric of this document. I am gratified to Dr. Mahdi Saeed for his support and encouragem ent. His suggestions in understanding the nomenclature of the microorganisms played an important role. My special thanks to the authors of the studies which were included in this review without which this research work would not have been possible. I sinc erely wish to express my gratitude to the entire faculty of the Master of Science Program in Epidemiology and Biostatistics and my colleagues for their extensive support during the preparation of this thesis. I am highly obliged to my wife, Sabahat, and my children who stood by me during the challenging time of completing my thesis requirements. iv TABLE OF CONTENTS LIST OF TABLES ................................ ................................ ................................ ....................... vi LIST OF FIGURES ................................ ................................ ................................ ..................... vii KEY TO ABBREVIAT IONS ................................ ................................ ................................ ........ viii INTRODUCTION/BACKGROUND ................................ ................................ ............................... 1 Descriptive epidemiology of preeclampsia ................................ ................................ ....................... 2 Infection during pregnancy ................................ ................................ ................................ .............. 3 Risk Factors for the Development of an Atherosclerotic Plaque ................................ ................................ ...... 4 Clinical Manifestation of Atherosclerosis ................................ ................................ ................................ ......... 4 Pathophysiology of Preeclampsia ................................ ................................ ................................ ..... 5 Me chanisms for Development of Preeclampsia ................................ ................................ ................... 5 Lack of remodeling of spiral arteries ................................ ................................ ................................ ................ 5 Maternal endothelial response and coagulopathy ................................ ................................ ........................... 6 Th1 and Th2 imbalance ................................ ................................ ................................ ................................ .... 6 Placental fa ctors ................................ ................................ ................................ ................................ ............... 7 Infection and atherosclerosis ................................ ................................ ................................ ................ 8 OBJECTIVES ................................ ................................ ................................ ........................... 12 METHODS ................................ ................................ ................................ .............................. 13 Data Abstraction Process ................................ ................................ ................................ ............... 14 Methodology for Quality Assessment ................................ ................................ ............................. 14 RESULTS ................................ ................................ ................................ ................................ 16 Descriptive Analysis of Review ................................ ................................ ................................ ....... 16 Sources of the Studies ................................ ................................ ................................ ......................... 16 Preeclamps ia Definitions and Diagnosis ................................ ................................ ......................... 17 Association between Micro - organisms and Preeclampsia ................................ ............................... 21 Serological Tests ................................ ................................ ................................ ................................ .. 21 Tests for the detection of DNA ................................ ................................ ................................ ........... 22 He licobacter pylori (H. pylori) and Preeclampsia ................................ ................................ ................ 23 Cytomegalovirus (CMV) and Preeclampsia ................................ ................................ ......................... 23 Chlamydophila pneumoniae (C. pneumoniae) and Preeclampsia ................................ ...................... 24 Assessment of Risk of Bias ................................ ................................ ................................ ............. 29 DISCUS SION ................................ ................................ ................................ ........................... 30 LIMITATIONS ................................ ................................ ................................ ......................... 33 CONCLUSIONS ................................ ................................ ................................ ....................... 34 APPENDICES ................................ ................................ ................................ .......................... 35 v Appendix A: Structured Summary ................................ ................................ ................................ .. 36 Appendix B: Main terms searched ................................ ................................ ................................ . 39 Appendix C: Terminology used for PUBMED search engine ................................ ............................. 40 Appendix D: Risk of Bias assessment ................................ ................................ .............................. 41 Appendix E: Search and selection process ................................ ................................ ...................... 42 Appendix F: Antigens of H. pylori ................................ ................................ ................................ ... 43 Appendix G: PRISMA checklist ................................ ................................ ................................ ........ 44 REFERENCES ................................ ................................ ................................ .......................... 47 vi LIST OF TABLES Table 1: Recommendation of ACOG for the diagnosis of Preeclampsia ............................................ 18 Table 2: Recommendations of ISSHP for the diagnosis of Preeclampsia ........................................... 19 Table 3 : Studies retrieved and reviewed systematically, arranged by the organism investigated using serological t ests ............................................................................................................................. . 26 Table 4: Studies retrieved and reviewed systematically, arranged by the organism investigated detecting DNA ............................................................................................................................. .... 28 Table 5: Modified version o f the ACROBAT - N RSI ............................................................................. 41 Table 6: Types of Antigens of H. pylori...................................................................................... ......43 Ta ble 7: PRISMA Checklist of items to include when reporting a systematic review (with or without meta - analysis) ............................................................................................................................. ..44 vii LIST OF FIGURES Figure 1: Trends in preeclampsia incidence across high income countries. ................................ ........ 3 Figure 2: Pathological changes in preeclampsia with inflammatory cells. Error! Bookmark not defined. Figure 3: Search and selection process of s tudies viii KEY TO ABBREVIATIONS ACOG The American College of Obstetrics & Gynecology ACROBAT - NRSI A Cochrane Risk of Bias Assessment Tool for Non - Randomized Studies of Interventions ACS Acute Coronary Syndrome adjOR Adjusted Odds Ratio BMI Basal Metabolic Rate BP Blood Pressure CagA Cytotoxin - associated protein CC Case control CDC T he Center for Disease Control and Prevention CMV Cytomegalovirus CP Chlamydophila pneumoniae (C. pneumoniae ) DNA Deoxyribonucleic Acid ELISA The enzyme - linked immunosorbent assay EMBASE Excerpta Medica dataBASE EVT Extra - villous Tissue HLA Human Leukocyte Antigen HP Helicobacter pylori (H. pylori) Hsp Heat Shock Proteins IgA/IgG/IGM Immunoglobulin A/ Immunoglobulin G/Immunoglobulin M IL Interleukins INF Interferon ISSHP The International Society for the Study of Hypertension in Pregnancy MCH Major Histocompatibility Complex ix MeSH Medical Subject Headings MIF Micro - immunofluorescent mm Hg Millimeter of Mercury mmol Milli mole NHBPEP The National High Blood Pressure Education P rogram NK Natural Killer NS Non - significant OR Odds Ratio PCR Polymerase Chain Reaction PE Preeclampsia PlGF Placental Growth Factor PRISMA The Preferred Reporting Items for Systematic Reviews and Meta - Analyses RoB Risk of Bias RR Risk Ratio sEng soluble Endoglin sFlt - 1 soluble fms - like tyrosine kinase 1 STROBE Strengthening the reporting of observational studies in epidemiology Th1/Th2 T Helper cell 1/ T Helper cell 2 TNF Tissue Necrotic Factor UreC Urease subunit C of H. py lori UreE Urease subunit E of H. pylori VacA Vacuolating cytotoxin protein VEGF Vascular Endothelial Growth Factor VT Villous Tissue WGO The World Gastroenterology Organization x WHO THE World Health Organization WoS Web of Science 1 INTRODUCTION/BACKGROUND Preeclampsia (PE) is one of the leading causes of perinatal morbidity and mortality in the world ( 1) . About 10 - 15 % of direct maternal deaths in both developing and developed countries are attributed to PE or its complications ( 2 - 5 ). This suggests that once the chain of events related to PE starts it becomes difficult to stop and to reverse the pathologic cycle, especially in the more severe categories of disease. Maternal deaths are not the only fatal sequel of PE; a quarter of stillbirths worldwide have also been found to be related to PE ( 6 ). In addition to effects on mortality, there is substantial morbidity from PE. Some women may develop seizures, which signal progression to ecl ampsia. Other acute consequences can include stroke, renal and hepatic failure and coagulopathy. These conditions may require intensive care. Apart from these immediate effects, even when the woman apparently recovers uneventfully, there may be both shor t and long term health consequences. Women may be at an elevated risk for hypertension and cardiovascular disease in later life ( 7 ), and newborns may suffer growth retardation and have higher risks of cerebral palsy and other neurodevelopment disorders la ter ( 8 ) . Preeclampsia is most commonly diagnosed when elevated blood pressure (140/90 mmHg or higher, measured on more than one occasion) occurs after 20 weeks of gestation and is accompanied by proteinuria (300 mg or more of protein in a 24 - hour urine sa mple). In unusual cases, the diagnosis can be made in the absence of prote i nuria, when hypertension is accompanied by one or more of the following conditions: thrombocytopenia, renal or hepatic failure, pulmonary edema and cerebral or visual symptoms ( 9 ). 2 Research on the origins of preeclampsia, primarily from developed countries, has explored genetic, dietary, metabolic, environmental and cardiovascular risk factors (10 - 12). However, no cause has been identified yet. The World Health Organization (WHO) Global Survey on Maternal and Perinatal Health has identified some risk factors which include: older age (more than 30 years), low literacy, high body mass index, nulliparity, chronic hypertension, diabetes, cardiac and renal diseases (13). Over the past few decade s , efforts have been made to explore the possible role of infections in the development of preeclampsia. However, the scientific and health communities have yet to produce a strong evidence to establish a clear link between infections and the d evelopment of preeclampsia. Descriptive epidemiology of preeclampsia The burden of preeclampsia varies across different regions. The World health Organization (WHO) estimates the incidence of PE around the world as between 2 - 10 % of all pregnancies (14). The frequency is almost always low in high income countries as compared to low income countries. Again, the prevalence fluctuates from 1.4 - 4.0 % across high income countries as shown in the (15) . The low income countries exhibit a wider range. I n Africa, PE incidence ranges from 1.8 - 7.8 % of pregnancies excluding Nigeria where preeclampsia presents with the highest frequency, 1.8 - 16.7 % (16). A WHO report found that PE incidence was 7 times higher in low income compared to high income countries ( 14). 3 Figure 1 : Trends in preeclampsia incidence across high income countries. Source: Roberts et al, 2011 (15). Evidence is lacking on the trends of preeclampsia especially in low income countries. Information is also not available for all high - income countries. Figure 1 provides some information on time trends for PE incidence for a selective group of countries. This figure shows that over the previous two decades, the frequency of preeclampsia remained almost constant i n Sweden, Denmark and Western Austra lia but it fluctuated for other s. Since 2003, the rates have been different in different countries. Rates went down in Scotland, minimally increased in Alberta, Canada, while rates significantly increased in Massachuse tts, USA from 2.5 % in 1987 to 3.2 % in 2004 (17). Infection during pregnancy Evidence of infection during pregnancy varies across the countries as well as for different organisms. Moreover, status of infection is also not similar for all organisms; the re may be primary episode or chronic infection with the development of immune status. According to the CDC, 1 - 4 % of the pregnant women get CMV primary infection in the US (18). However, 4 higher estimates are found for primary CMV infection in other count ries , especially low - income countries such as Nigeria where the overall seroprevalence was very high with prevalence levels (based on IgM) reaching to 92% or higher (19,20). H. pylori has also been found to be present in pregnant women with high prevalenc e; the burden ranges from 20 - 80 % across the globe (21). Risk Factors for the Development of an Atherosclerotic Plaque Major Factors Unhealthy blood cholesterol, hypertension, smoking, insulin resistance, diabetes, obesity and low physical activity Minor Factors Sleep apnea, stress and alcohol. Clinical Manifestation of Atherosclerosis Atherosclerosis can affect any vessel in the body; however, the three m ain categories are significant . These are coronary heart disease, cerebrovascular disease and perip heral vascular disease. The coronary heart disease is the result of atherosclerosis in the coronary arteries, while carotid arteries atherosclerosis results in cerebral or neural manifestation. The peripheral vascular disease may result in the involveme nt of a variety of organs and systems such as liver, kidney and limbs (22). 5 Pathophysiology of P reeclampsia Preeclampsia (PE) is a disorder involving multiple organ systems, and its possible causative factors have been explored from various perspectives, including genetic, dietary and metabolic, environmental, infectious and cardiovascular factors (10 - 12). But the central facto r for the development of PE is the placenta, and after placental delivery, the condition of the woman with PE improves (23). The initiating event in PE is thought to be placental hypoperfusion resulting in placental ischemia. Among several different expl anations for the development of PE, three mechanisms have been cited most frequently. These are: lack of remodeling of spiral arteries; maternal endothelial response and coagulopathy and placental factors. Mechanisms for Development of Preeclampsia Lac k o f remodeling of spiral arteries In normal pregnancies, cytotrophoblasts are responsible for widening of the lumen of spiral arteries by a process which replaces the endothelial layer after they invade these vessels. This allows a marked increase of bloo d flow to the placenta and to the growing fetus. In PE, however, cytotrophoblasts do not invade spiral arteries deeply enough to convert these arteries to the dilated vessels which are necessary for the maintenance of uninterrupted blood supply to the fetu s. This results in placental and fetal hypoxia and under - nutrition. However, whether hypoxia is the result or the cause of the superficial invasion of spiral arteries is still not entirely clear. But in any case, the narrowing of the vessels supplying t he placenta cannot maintain the needed blood supply to the fetus, setting the stage for the next steps leading to the development of PE. These processes can begin before the end of the first trimester, and are therefore especially invoked as the mechanism behind early onset PE (24). 6 Maternal endoth elial response and coagulopathy Possibly in response to the changes described above, PE is characterized by an important maternal endothelial response. As far as the clinical features of PE are concern ed, it is the response of maternal endothelium which corresponds with these features. The endothelial reaction results in increased vascular permeability which explains the proteinuria and edema. The change in vascular tone can underly the development of hypertension. Another feature of PE is the production of pro - coagulants which promote hyper - coagulopathy (25). Several markers of endothelial injury are found during this phase. These markers are normally found in pregnant women (26). However, when PE develops, an abnormality in the concentrations of these factors appears. The factors which are favorable for nor mal pregnancy, like prostacyclin, are reduced in concentrations while the factors which promote vasoconstriction or hyper - coagulation, such as endothelin - 1, von Willebrand factor, circulating cellular fibronectin, and thromboxane are increased. Moreover, the responsiveness of preeclamptic women to vasopressors is increased as compared to women with normal pregnancy (27). Also, these features show impairment in the endothelial - derived relaxation of vessels (28, 29). The maternal endothelial response indu ces hypertension due to high levels of vasoconstrictors and low levels of vasodilators. The probability of coagulopathy is increased as a result of enhanced concentrations of clotting factors. In more severe situations, a higher consumption of platelets in the process of coagulation may result in thrombocytopenia (30). Th1 and Th2 imbalance Two variants of the T cell, Th1 and Th2, are involved as immune mediators in pregnancy. In normal pregnancy, Th2 predominates, and is responsible for the product ion of different types of interleukins (ILs) such as, IL - 4, IL - 5, IL - 6, IL - 10, IL - 13 and antibodies. Th2 and the interleukins produced promote normal pregnancy. In contrast, Th1 cells, which produce IL - 2, 7 tissue necrotic factor (TNF) - - situation in women with PE (31). Figure 2 : Pathological changes in preeclampsia with inflammatory cells. Up - regulation of Th1 results in cytotoxic activity due to stimulation of natural killer (NK) cells and CD - 8 positive T cells (31). Experimental evidence suggests that cultures of peripheral blood mononuclear cells in the blood of pre eclamptic women resulted in high concentrations of TNF - - 2 and INF - factors. This experiment supports the association between PE and these factors (32). Additionally, Th1 has been found to be associated with an increase in endothelin - 1 and reduct ion in plasminogen activator inhibitor - 2 (33, 34). These factors are both important mediators of different phases of PE, especially hypertension. Placental factors Apart from issues related to the development of spiral arteries and endothelial inju ry, some other biomarkers have also been extensively studied and are hypothesized to play important roles in the causation of PE. These biomarkers are derived from the placenta. These factors too are normally present in pregnancy. However, there is a re versal in the concentration of these factors. 8 Factors which are in high concentration in normal pregnancy, such as placental - like growth factor (PlGF) and vascular endothelial growth factor (VEGF), are reduced in preeclamptic women. These factors keep blo od flow and placental condition within normal limits (23, 27). On the other hand, factors such as soluble fms - like tyrosine kinase 1 (sFlt - 1) and soluble Endoglin (sEng) are also placental in origin but their concentration corresponds to the existence and severity of the PE. These are potent antagonists of PlGF and VEGF. These factors bind to PlGF and VEGF and thus reduce the concentration of free PlGF and VEGF circulating in the blood. This induces endothelial dysfunction. Endothelial dysfunction resul ts in reductions in prostacyclin and nitric oxide concentrations leading to the vasoconstriction and eventually hypertension. At the same time, there is increased production of endothelin, cellular fibronectin, thrombomodulin and von Willebrand factors (2 7). The imbalance between two types of markers - angiogenic (PlGF, VEGF) and antiangiogenic (sFlt - 1) - as a result of endothelial dysfunction seems to be part of the development of PE; sFlt - 1 production takes place about 5 - 6 weeks before the onset of PE (23). Infection and atherosclerosis Some of the biomarkers discussed in the previous section such as, IL - 6 and IL - 10 are considered indicators of inflammatory responses including infections (35). Based on such evidence, the role of different infections agents including Chlamydophila pneumoniae (C. pneumoniae), Helicobacter pylori (H. pylori) and cytomegalovirus (CMV), in the development of atherosclerosis has been explored by the research community (36 - 44). Several factors contributed to the selection of these three microorganisms for conducting this review: 1) High frequency and chronic infection or re - infection in the population: 9 A review conducted by Grayston concluded that the presence of C. pneumoniae IgG antibodies remains low (less than 10%) up to the age of 10 years followed by an abrupt increase to attain a level of 50% and then it increases steadily over the rest of life reaching up to 80% (45). According to the World Gastroenterology Organization (WGO) prevalence of H. pylori is higher in developing countries where it reaches up to 85 - 90 % while across the globe it is 50% (46). According to the Center for Disease Control and Prevention (CDC) Atlanta, more than 50% of the US population is infected by CMV by the time they reach to 40 years of age; this prevalence may go up to 80% (47). 2) Potential role of these micro - organisms in the development of atherosclerosis (as discussed below); 3) Different studies conducted to assess if any association exist b etween these micro - organisms independently or in combination with the development of PE. The findings of these studies were mixed and in - conclusive; some showed a significant association (21, 48, 49) others no association (50, 51). A large body of lit erature is available on the role of infections caused by these micro - organisms and atherosclerosis. In one study of 40 subjects explored the coronary artery specimen and found 22 specimens with acute coronary syndrome (ACS) where C. pneumoniae was found i n significantly higher concentrations per millimeter of the specimen as compared to 18 non - ACS specimens (36). Another study investigated the presence of C. pneumoniae in the atherosclerotic plaques of 76 patients who presented with unstable angina and 75 % of the plaques revealed presence of C. pneumoniae DNA (37). A nested - case control study compared the presence of antibodies to C. pneumoniae in the serum sample with the presence of C. 10 pneumoniae DNA in the coronary artery atherosclerotic plaque (38). Higher proportions of plaques showed the evidence of C. pneumoniae. With regards to H. pylori , one study looked for an association between inflammatory markers such as IL - 8 which are involved in the process of atherosclerosis development and H. pylor i (39). The findings showed a significant association between IL - 8 and H. pylori infection. The subjects with carotid intima - media thickness (IMT) had significantly higher levels of IL - 8 than those without IMT. A follow up study with H. pylori infected and non - infected individuals found a significant association between infection and carotid artery plaque formation as compared to the non - infected subjects (40). In addition, a meta - analysis of 13 studies reported a significantly higher risk of ischemic s troke in H. pylori infected subjects as compared to non - infected ones (41). The role of CMV in the development of atherosclerosis has been supported by the animal model as well as in humans. Research into an animal model found that a higher proportion o f CMV - infected mice showed an evidence of atherosclerotic plaque presence as compared to the control group (42). Another study conducted in Iran reported that the subjects with a higher prevalence of coronary artery syndrome had a significant association with CMV and the virus was found in the atherosclerotic plaque (43). Similarly, a study conducted in India found significant association between CMV - infected young patients and the development of coronary artery disease (44). Based on the findings fr om available literature on the role of infections in the development of atherosclerosis, we propose an analogy between the atherosclerotic process of preeclampsia and the atherosclerosis in coronary and cerebral blood vessels because both pathologic proces ses include evidence of an inflammatory response to endothelial injury. After 11 injury to the endothelium of vessels in the placenta, such as occurs in preeclampsia, a series of events ensue, including fibrinoid necrosis of the vessel walls, movement of mon onuclear cells to the surrounding tissue of the vessel, and accumulation of lipid laden macrophages and presence of lipoproteins in more than normal concentration (52). Using the association of infection and atherosclerosis as a basic model, a relations hip between infection in pregnancy or earlier in life and the subsequent development of preeclampsia has already been explored by the research community. A literature search retrieved two systematic reviews looking at the role of maternal infections and P E (53, 54). T hese reviews provided opposing results and since then more research work has been published, so we planned to carry out a systematic review focusing on these three infectious agents and PE. Conde et al conducted the review in 2007 to explore the association between maternal infections and preeclampsia (53). Their review included 49 studies and found a significant association between urinary tract infections as well as periodontal disease and the development of preeclampsia. They did not find association between C. pneumoniae , CMV, H. pylori , and treated or untreated HIV. In contrast, Rustveld et al reviewed 16 studies to investigate relationship between maternal infections and PE. Their findings showed that some studies were significantly associated while others were not as far as viral and bacterial infections and PE is concerned, including C. pneumoniae (54). 12 OBJECTIVES 1. To identify all observational studies on the association between Chlamydophila pneumoniae, Helicobacter pylori and cytomegalovirus (CMV) and preeclampsia; and 2. To carry out a systematic review to determine whether there is an association between these microorganisms 1 and development of PE. 1 Microorganisms are represented by either detection of their DNA or antibodies against their proteins/antigens. 13 METHODS We followed the Preferred Reporting Items for Systematic Reviews and Meta - Analyses (PRISMA) Guidelines to carry out this systematic review (55). This review explored the scientific literature to investigate the role of three infectious agents, H. pylori , CMV and C. pneumoniae, in the causation of preeclampsia in singleton pregnancies. The studies were identified by searching the electronic databases (discussed below), contacting the authors and manually searching the reference lists of the selected studie s. For this purpose, we searched PubMed, EMBASE and Web of Science (WoS), limited to papers in the English language and involving human subjects without any restrictions related to regions of the world, where a study was conducted, or time period. Th e main terms searched are enlisted as A ppendix B . These terms were combined with a broad set of organisms and related infectious diseases including: all bacteria, all viruses, bacterial infections, or virus diseases. The Appendix C provides search terminology used for Pubmed search engines. The agents of specific interest - Helicobacter pylori, Chlamydophila pneumoniae and cytomegalovirus were included in the br oader organism searches of bacteria and viruses, and were also searched as title words to ensure that nothing of interest was missed. We included all the studies which used cross - sectional, case control or cohort stud y designs that examined the associat ion between three microorganisms described above and preeclampsia. Eligible studies included those with 1) original data; 2 ) which used an appropriate and acceptable definition of preeclampsia in a way that it was in compliance with the recognized defini tion of preeclampsia that has been recommended by relevant organizations or societies such as the American College of Obstetrics and Gynecology (ACOG) , the International Society 14 for the Study of Hypertension in Pregnancy (ISSHP), and the National High Bl ood Pressure Education Program (NHBPEP) and 3 ) that described the techniques of detection of infectious agents or antibodies, such as using PCR technique for the identification of DNA or serological testing to determine the presence of antibodies and 4 ) a nd finally, those us ed at least one of the three microorganisms as an exposure and described the methodology of their detection. The following literature was excluded: case reports, case series, studies with only abstract, commentaries, letters to editor s, studies in language other than English. There was no restriction of time period applied on for searching the studies. Data Abstraction Process We developed a data abstraction form to collect the required information from the selected studies. This f orm was refined after the pilot - testing of 5 randomly selected studies not the part of the final study sample. The information obtained through these forms focused on the criteria for study eligibility, methods used, exposure and outcome assessment, confo unders and results. Apart from obtaining the information on the variables mentioned above, we also extracted the information on association between the microorganism (s) and PE. For this purpose, we extracted both the crude and adjusted Odds Ratio (OR); w hen we found only crude OR we took only that value. We calculated OR for the studies which did not calculate it. Methodology for Quality Assessment We could not use the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) Statement (56). This statement provides guidelines for reporting observational studies and at the same time makes emphasis that this resource is not appropriate to be used for the quality assessment of observational studies. Alternatively, w e as sessed the risk of bias (RoB) for the studies included in the review as a proxy measure for the assessment of quality of the studies. For the assessment of RoB, we modify A Cochrane Risk of Bias Assessment Tool: for Non - Randomized Studies of Information 15 ( ACROBAT - NRSI) (5 7 ). This tool is for studies on interventions but our corresponding variable was the The guidelines in ACROBAT - NRSI suggest that the tool should be followed right from the design stage. Unfortunately, this tool was published after we had already finished our analysis. However, we still had the opportunity to assess the RoB by re - analyzing the related items. In the next stage, as rec ommended by this tool, the review of each individual study is provided. This review was conducted utilizing the following seven domains to identify any bias which may exist: 1. Bias due to confounding 2. Bias in the selection of participants into the study 3. Bias in measurement of interventions/exposure 4. Bias due to departures from the intended interventions/exposure 5. Bias due to missing data 6. Bias in measurement of outcome 7. Bias in selection of the reported results. Each domain has certain questions with options of responses as yes, probably yes, no, probably no and no information. Based on the responses to the questions each domain may receive one of the four levels of bias: low risk, moderate risk, serous risk and critical risk of bias. For each study, the overall risk of bias was assessed by the cumulative assessment for all the seven domains. The final RoB for each study is also assessed at the same scale as for each domain. The tables/checklist to show these domains with analysis for each study is attached as A ppendix D . 16 RESULTS Descriptive Analysis of Review The initial attempt with Pubmed, Embase and Web of Science search terms retrieved 1,031 articles. Further analysis of the titles and abstracts of th e articles excluded 1,014 search results based on exclusion criteria as shown in figure 1. After reviewing the reference lists of the remaining 14 articles, two more were added to achieve a final number of 16 articles which resulted in obtaining informati on about 5,614 women. These studies looked for the association between at least one of the three microorganisms, C. pneumoniae, H. pylori and cytomegalovirus (CMV), and development of preeclampsia (PE). The search and selection process of studies is pro vided in a figure as A ppendix E . Sources of the Studies Eight countries contributed to the data in this review. Three studies each were conducted in Canada, Turkey and the USA while two studies each were from Italy and the UK. One study each was conducted in Finland, Norway and Venezuela. All but one study was carried out in North America and Europe; one was done in South America. All but two studies (one nested case - control and the other a cohort study) used the case control design. Fiftee n studies used the case - control (CC) design and one was carried out as cohort study. Including the cohort studies, three were population based while the rest of them (13) were conducted in hospital setting. The controls were pregnant women who did not ha ve the features of preeclampsia. The hospital studies included the women who were visiting hospitals for antennal care or were admitted either to get services for an emergency condition or to deliver in the hospital. 17 Nine of the sixteen studies provid ed information on controlling for confounding variables. Three studies controlled for confounding variables at design phase by using a matched design while six controlled confounding variables at analysis phase by constructing a logistic regression model. Most of the studies controlled for parity and maternal age while using matching or adjusted analysis . Eight articles exclusively looked at the role of C. pneumoniae. Six of these studies assessed the evidence of C. pneumoniae by looking at antibodies, on e looked for C. pneumoniae using PCR, and one study used both techniques . Three articles each explored an association between PE and H. pylori and CMV. The researchers of these studies utilized different methods to assess the presence of these organisms, such as: antibodies against the microorganism or carrying out PCR or extracting the DNA. One study each assessed the presence of CMV or H. pylori through PCR/DNA and the remaining two studies each assessed evidence by using antibodies. Two articles stud ied two microorganisms as exposures for the development of PE; one looked at C. pneumoniae and CMV, while the other explored the role of C. pneumoniae and H. pylori. Preeclampsia Definitions and Diagnosis All studies except one (5 8 ) provided information on how the blood pressure (BP) of pregnant women was assessed in making the diagnosis of preeclampsia. Thirteen studies checked BP twice for making a diagnosis of hypertension. Six studies measured BP twice six hours apart, and fou r measured BP twice four hours apart. The remaining three studies did not provide information on the interval between BP assessments taken at two point s . Four studies divided preeclampsia into mild and severe categories based on both BP readings and protein levels in the 18 urine; two studies made sub - groups of early and late onset of preeclampsia based on the timing of development of the clinical features. Table 1 : Recommendation of ACOG for the diagnosis of Preeclampsia . Blood Pressure Greater than or equal to 140 mm Hg systolic or greater than or equal to 90 mm Hg diastolic on two occasions at least 4 hours apart after 20 weeks gestation in a woman with previous normal blood pressure. Greater than or equal to 160 mm Hg systolic or greater than or equal to 110 mm Hg diastolic, hypertension can be conformed within a short interval (minutes) to facilitate timely hypertensive therap y. And Protein Greater than 300 mg/24 hours urine collected (or this amount extrapolated from a timed collection). Or Protein/creatinine ratio greater than 0.3 (both measured in mg/dl). Dipstick recording of 1+ (useful only if other quantitative methods are not available). Or in the absence of proteinuria, new - onset hypertension with the new onset of any of the following Thrombocytopenia Platelet count less than 100,000/ml Renal Insufficiency Serum creatinine concentration more than 1.1 mg/d l or a doubling of the serum creatinine concentration in the absence of other renal disease. Impaired Liver Function Elevated blood concentration of liver transaminases to twice normal concentration. Pulmonary Edema Cerebral/Visual symptoms All but one study used a cut off value of 140/90 mm Hg for diagnosing mild hypertension while 160/110 mm Hg was taken as the cut off value for severe hypertension which correspond to mild preeclampsia and severe preeclampsia respectively. Four studies exp licitly mentioned following the American College of Obstetrics and Gynecology (ACOG) guidelines as summarized in table 1 (5 9 ), while one study each mentioned following guidelines of the International Society for the Study of Hypertension in Pregnancy (ISSHP) , narrated in table 2 ( 60 ) and National High Blood Pressure Education Program (NHBPEP). One study took only diastolic BP as the criterion for diagnosing preeclampsia ( 61 ). One study did not provide any criterion for diagnosing PE, but stated that th ey enrolled preeclamptic women as cases ( 58 ). 19 Proteinuria was assessed by two methods, either measuring proteins in 24 hours - collected urine or using urinary dipstick findings. All studies that obtained 24 - hour urine samples used the same level of protein s in urine (>300 mg/24 hours) for mild preeclampsia. Studies that subdivided their sample into mild and severe PE also agreed upon one value of >500mg /24 hours. However, for dipstick methods, studies differed. For mild PE, dipstick 1+ and 2+ were used; in comparison either dipstick 3+ or 4+ was used for severe PE by different studies. Table 2 : Recommendation s of ISSHP for the diagnosis of Preeclampsia . Blood Pressure De Novo hypertension after gestational week 20 And the new o nset of one or more of the following Protein or Renal Insufficiency Neurological Problems Convulsions (eclampsia), hyper - reflexia with clonus, se vere headache with hyper - reflexi a, persistent visual disturbances (scotomoa). Hematological Disturbances Thrombocytopenia, Disseminated Intravascular Coagulation (DIC) and Hemolysis. Fetal Growth Restriction The timing of physical examination and biological information collection was similar in almost all studies. Except for four studies, the rest did not provide information on the semester during which the biosamples were collected. The d iagnosis of preecl ampsia was based on the findings of hypertension and proteinuria assessed after the 20 th weeks of gestation. Six studies explicitly mentioned that they followed some guidelines in the diagnosis of PE; four followed ACOG, one each followed ISSHP and NHBPEP . The risk of bias (RoB) analysis found that more than six studies were at the lowest level of risk, low risk (21, 49, 50, 6 1 - 6 3 ) and one study was at moderate risk of bias (6 4 ). More than 20 half of the studies reviewed were either at serious risk (48, 6 5 - 6 8 ) or critical risk of bias (51, 5 8, 69 , 70 ). 21 Association between Micro - organisms and Preeclampsia To determine the evidence of an infectious agent, usually two types of tests are used: serological tests which show the antigen - antibody reaction and extraction of DNA of an infectious agent in body tissues. All studies we reviewed used serological tests but four also used tests for the detection of microorganism DNA from the placental tissue. All studies explored the association between PE and thes e micro - organisms either for all or at least for one. Five studies did not calculate the OR so we calculated it based on the provided data. Maternal age was the most commonly used matching variable. Serological Tests These tests are based on antigen - antibody reactions. The serological tests have been named according to the type of antigen - antibody reaction/mechanism, of complex formation (7 1 ). Different types of testing strategies come under this main domain. However, be low is a brief description of the two tests utilized in the studies which are investigated in this review. The Enzyme - Linked Immunosorbent Assay (ELISA) is one of the techniques which follow the principle of antigen - antobody reactions. In this test, th e antibodies change color when a substance reacts with them. Antigens from the serum of patients are attached to a surface to which a specific antibody is added which binds to the antigen. The added antibody is linked to an enzyme; the substance which co ntains the substrate for that enzyme is added as a final step. The reaction usually changes the color of the substrate. Another test which utilizes the serological techniques is the Micro - immuno Fluorescence (MIF) Test. It is a specific type of serological tests utilized for some micro - organisms including C. pneumoniae. This test uses an indirect fluorescent antibody (FA) technique which helps in observing the binding of antigen antibody. This is facilitated by anti - globulin wh ich is 22 fluorescein - conjugated and corresponds to specific antibody molecules. The antigen used in this test comes from whole elementary body C hlapmydophila organisms. These elementary bodies are grown in cell culture, purified and then treated with 0.05% formalin. This preparation of antigens can be stored in refrigerator for many years (72 ). Tests for the detection of DNA For the detection of DNA from placental tissue, a Polymerase Chain Reaction (PCR) test is applied. The PCR test is a laboratory techn ique through which not only the DNA is detected but it is amplified by making millions of copies from a single strand or a few copies of DNA (7 3 ). To carry out a PCR test, a nucleic acid target source (micro - organism) is required along with small DNA prim ers for the amplification of the 5` and 3` ends, the enzyme polymerase and an appropriate instrument to regulate the temperature during different phases of the test. One study utilized the PCR test to detect CMV DNA by using the genetic information pre sent in the Major Histocompatibility Complex (MCH). The MCH is a collection of molecules on the cell surface which is coded by a large gene family; this controls the major part of the immune system (7 4 ). Its gene family has been divided into three subcla sses, class I, class II and class III. The class I and class II are also called Human Leukocyte Antigens (HLA). There are nine HLA genes which have been studied the most. The studies explored in this review investigated for DRB1, DQA1 and DQB1 and DR7. CMV DNA was amplified by using PCR. Apart from indicating the evidence of an infection, the level of anti - body titer also provides information on the status of infection. Immunoglobulin A (IgA) is more frequently found in the elderly and the individuals who have chronic infections and IgM is the first antibody to appear in the serum in response to infection due to bacteria, viruses or toxins. Usually, IgG appears in the body when IgM levels start decreasing and then it persists for a longer duration. It also appears as a response to chronic infection (7 5 ). 23 Helicobacter pylori (H. pylori) and Preeclampsia Most studies focused on using serological methods to identify various types of antigenic structures found in H. pylori ( A ppendix F ). The literature discusses a long list of these antigenic structures, however, the most commonly used for diagnostic purpose are: Cytotoxin - associated protein (CagA), the vacuolating cytotoxin protein (VacA), urease subunits, flagellin subunits and heat - shock proteins (HspA and HspB) (21). As shown in tables 3 and 4, four studies (21, 51, 6 4 , 6 5 ) looked at the role of H. pylori in the development of preeclampsia. One study explored antibodies produced in response to different antigens of H. pylori by u sing serological methods as well as evidence of H. pylori DNA using the PCR test (64 ) while the remaining three studies expl ored only antibodies (21, 51, 65 ). Of these three latter studies, one looked at antibodies produced in response to both H. pylori a nd C. pneumoniae (51) while the other two for H. pylori only (21, 6 4 ). All the studies which looked at the association between antibodies and PE found a statistically significant association: [Adj OR=9.2 (2.8 - 30.0)] (21), [OR=3.8 (1.2 - 11. 8)] (53), [OR=2.9 (1.1 - 7.8)] (65 ) and [OR (CagA) 26.04 (8.19 - 82.73)] but placenta test for DNA extraction was found to be negative (6 4 ). Cytomegalovirus (CMV) and Preeclampsia Four studies examined the relationship between CMV and PE each used a case - control design (4 8, 50, 63 , 6 6 ). Two of the studies looked at the association between anti - CMV antibodies, IgA, IgG and IgM and PE (48, 50). Another study looked at the association of PE with antibodies against both C. pneumoniae and CMV (6 3 ) while the fourth explored th e association between the presence of CMV DNA in peripheral blood and PE (6 6 ). The study that investigated an association of PE with C. pneumoniae and CMV simultaneously made a distinction between early onset PE (diagnosed during 20 - 34 weeks 24 gestation) and late onset PE (diagnosed after 34 weeks gestation) (6 3 ). Both the micro - organisms were found as risk factors for the early onset PE [OR CMV =2.1(0.3 - 17.1); OR CP =1.8(0.4 - 7.4); OR BOTH =2.9(0.7 - 12.2)]. In contrast, the two microorganisms were protective for late onset PE [OR CMV = 0.6 (0.2 - 1.5); OR CP = 0.5(0.2 - 1.3); OR BOTH = 0.6(0.2 - 1.4)]. The remaining two studies found a significant association between the evidence of infection with CMV and PE (48, 6 6 ). One investigated CMV in peripheral blood assessed by using PCR (6 6 ). As compared to the controls, preeclamptic women had a risk of 7.15 of detecting DR7 (p<.01); similarly the risk was also higher among preeclamptic mothers than the controls for DQA1*0201 [RR: 4.9 (p<.02) and for DR7 and/or DR06 [RR=8.53 (p<.0003)]. The other study explored the association by using serological tests [OR for IgG: 1.9 (1.4 - 2.7)] (48). Chlamydophila pneumoniae (C. pneumoniae) and Preeclampsia Ten studies explored the evidence of infection with C. pneumoniae and developmen t of PE (49, 51, 5 8 , 6 1 - 63 , 6 7 - 70 ). Five of them found a significant association between microorganisms and PE (49, 5 8 , 6 2 , 6 7 , 6 8 ). Eight of the ten studies attempted to find only the role of C. pneumoniae infection in the development of PE. Of the remai ning two studies, one examined the association of C. pneumoniae along with CMV and PE (6 3 ) and the other study investigated C. pneumoniae along with H. pylori and PE (53). Similarly, nine studies used serological methods to find out this evidence while on e study utilized DNA extraction and one study used both methods to establish evidence of this relationship. All studies were conducted using a case - control design except one, which used a cohort design. The eight studies which used only serologic al tests varied in their choice of antibodies against C. pneumoniae . Six of them looked for all three antibodies - IgA, IgG and IgM (58 , 6 1 , 6 2 , 6 8 - 70 ) while one study investigated IgG and IgM (51) and two othe rs explored for IgG only (49, 63 ). Out of th e two studies which looked for IgG only, just one study found a significant 25 association between IgG and PE [OR: 5.3(1.4 - 2.0)] this was carried out by using the cohort study design (49). The other study assessed early (diagnosis of PE between 20 - 34 weeks g estation) and late onset PE (diagnosis of PE after 34 weeks of gestation) but the association was not found to be significant [OR EARLY : 1.8 (0.4 - 7.4); OR LATE: 0.5 (0.2 - 1.3)] (6 3 ). The study which looked for IgM and IgG by using the serological test did not find a significant association for both the immunoglobulins [OR IgM: 1.1 (0.4 - 3); OR IgG: 1.3 (0.5 - 3.1) (51). For the rest of the six studies which looked for three immunoglobulins, IgA, IgM and IgG by using the sero logical tests, two studies showed an association for IgG only; one was sig nificant [OR: 3.1 (1.8 - 7.9)] (68 ) and the other was not [OR: 1.1(0.8 - 1.6)] (6 2 ). The other two were at a borderline for a significant association (58 , 70 ). One of them reported est imates for IgG only [OR: 1.6 (1.0 - 2.6)] (70 ) while the other reported estimates for all three immuno - globulins. However, there was almost no difference among these estimates for all three immuno - globulins [OR:1.2 (1.0 - 4.5)] (58 ). One of the remaining fou r studies did not report the estimates association (6 9 ), another was found to be non - significant with protecti ve effects [OR: 0.6(0.2 - 1.6) (61 ). The two s tudies which detected C. pneumoniae by identifying DNA both found supportive evidence. One study tested the correlation between gDNA copy numbers and anti - C. pneumoniae which was found to be strongly correlated (p<.0001) (6 2 ), the other study explored C. pneumoniae in placental tissue by using PCR. This study explored both the villous tissue (VT) and extra - villous tissue (EVT); a significant association was found for the combination of VT and EVT with PE [OR: 4.10(1.07 - 15.62)] but not for EVT only (6 7 ). 26 Table 3 : Studies retrieved and reviewed systematically, arranged by the organism investigated using serological tests. SNo Author, Year, Country, Ref Study Design Final Study Size, No. Cases with PE, No. Assessment of Exposure Timing of collection of Biosamples Assessment of Outcome OR 95 % CI Matching and Adjustment Variables Quality Assessment (weighted score) Studies of Helicobacter Pylori antibodies 1 Cardaropoli et al, 2011, Italy (21) Case - control 66 17 Serum serology for specific Antibodies against HP antigens: CagA, VacA, Urease C, Flagellin, Urease H, Urease A, Urease E. Before delivery Records/ clinical H. pylori : 9.2 CagA: 17.7 VacA: 4.9 UreC: 2.8 UreE: 4.4 2.8 - 30.0 5.3 - 59.5 1.6 - 14.7 1.0 - 7.8 1.6 - 12.3 Adjusted for: m aternal age, pre - pregnancy BMI, parity, maternal and family risk factors 49 2 Aksoy et al, 2009, Turkey (65 ) Case - control 83 53 ELISA for anti - H. pylori IgG Antenatal period Records/ clinical 2.9 1.1 - 7.8 No information on adjusted variables. 55 Studies of Cytomegalovirus antibodies 3 Strand et al, 2012, Norway (50) Nested case control 2,461 1,470 Serum samples for anti - CMV IgA, IgG and IgM Antenatal period Records / Clinical IgG: 0.9 IgM: 1.1 0.7 - 1.1 0.5 - 2.4 Adjusted for: m at age, parity , smoking in pregnancy 66 4 Xie et al, 2010, Canada (48) Case - control 187 78 Serum samples for anti - CMV IgA, IgG and IgM Antenatal period Records / Clinical IgG: 1.9 1.4 - 2.7 No information on adjusted variables. 55 Studies of Chlamydophi l a pneumoniae antibodies 5 Chrisoulidou et al, 2011, UK (61 ) Case - control 60 30 Fasting blood samples for CP antibodies IgA, IgG, IgM. No information provided Records/ clinical 0.6* 0.2 - 1.6 Matched on: age, parity , w ee k of g estation 50 6 Karinen et al, 2008, Finland (49) Cohort 1556 77 MIF^ for anti - CP IgG 1 st trimester; 10.4 weeks Records/ clinical IgG: 5.3 1.4 - 20 Adjusted for: BMI, smoking, family history of PE. 63 7 Aral et al, 2006, Turkey (58 ) Case - control 116 69 ELISA: anti - C. pneumoniae IgA, IgG and IgM No information provided Records/ clinical IgA:1.9* IgG:2.1* IgM:2.2* 0.8 - 4.3 1.0 - 4.5 0.9 - 5.1 Adjusted for none, crude analysis . 27 8 Goulis et al, 2005, UK (6 9 ) Case - control Multiparous 20 Primiparous 33 Multiparous 9 Primiparous 6 Blood specimens for Enzyme Immunoassay for anti - C. pneumoniae IgA, IgG and IgM. 16 - 22 weeks; 28 - 40 weeks Records/ Clinical (ISSHP*** guidelines) Multiparous IgA IgG IgM Primiparous IgA IgG IgM NS NS NS NS NS NS No information on adjusted variables. 48 27 9 Raynor et al, 2004, USA (70) Case - control 287 81 ELISA: anti - C. pneumoniae IgA, IgG and IgM Records/ Clinical (ACOG** guidelines) IgG:1.6 0.9 - 2.6 Adjusted for: age, parity, seropositivity 60 10 Heine et al, 2003, USA (68) Case - control 74 37 MIF^ for anti - C. pneumoniae IgA, IgG, IgM At delivery Records/ clinical IgA* : 1 IgG*: 3.1 IgM*: 1 NS 1.8 - 7.9 NS Adjusted for: age, gestational age. 50 Studies using antibodies of more than one microorganisms 11 Ustun et al, 2010, Turkey (51) Case - control 80 40 C. pneumoniae IgG, IgM H. pylori IgA Antennal period, fasting sample ACOG guidelines C. pneumoniae * IgG: 1.3 IgM: 1.1 H. pylori * 3.8 0.5 - 3.1 0.4 - 3.0 1.2 - 11.8 No information on adjusted variables. 45 12 Von Dadelszen et al, 2003, Canada (63) Nested case - control Early onset=122; Late onset=142 Early onset=9; Late onset 29 Serology for CMV antibodies and C. pneumoniae antibodies Antenatal period Records/ Clinical. NHBPEP criteria Early onset* CMV: 2.1 CP: 1.8 Both: 2.9 Late onset* CMV: 0.6 CP: 0.5 Both: 0.6 0.3 - 17.1 0.4 - 7.4 0.7 - 12.2 0.2 - 1.5 0.2 - 1.3 0.2 - 1.4 Matched om: maternal age (5 years) and parity (0,1,>=2) 53 CP= C. pneumoniae 28 Table 4 : Studies retrieved and reviewed systematically, arranged by the organism investigated detecting DNA. SNo. Author, Year, Country, Ref Study Design Final Study Size, No. Cases with PE, No. Assessment of Exposure Timing of collection of Biosamples Assessment of Outcome OR 95 % CI Matching and Adjustment Variables Quality Assessment (weighted score) Studies of Helicobacter Pylori DNA 1 Ponzetto, 2006, Italy (6 4 ) Case - control 86 For PCR Placenta:20 43 For PCR Placentae:10 Serum serology for anti - H. pylori IgG and for CagA proteins. PCR for placenta DNA. 5 ml of blood before delivery Records/ Clinical (ACOG** guidelines) H. pylori : 2.67 CagA : 26.04 Placenta test for DNA extraction found NEGATIVE. 1.08 - 6.57 8.19 - 82.73 Matched on: parity 50 Studies of Cytomegalovirus DNA 2 Carreiras et al, 2002, Venezuela (6 6 ) Case - control 56 27 PCR amplification for CMV detection NA Records/ clinical DR7: 7.15 DQA1*0201:4 .9 P=0.01 P=0.02) No information on adjusted variables. 50 Studies of Chlamydophila pneumoniae DNA 3 Xie et al, 2010, Canada (62 ) Case - control 107 50 ELISA: anti - C. pneumoniae IgA, IgG and IgM Venous blood for DNA Antenatal Records/ clinical IgG: 1.14 0.84 - 1.55 Matched on: maternal age, parity, gestational age at sampling. 51 4 Gomez et al, 2009, USA (67 ) Case - control 78 48 Placental tissue for detection of C. pneumoniae by using PCR. 1 st trimester Records/ Clinical (ACOG** guidelines) EVT" & VT'=4.10 EVT"=3.23 1.07 - 15.62 0.65 - 16.12 No information on adjusted variables. 49 *OR calculated by reviewer/author; **ACOG=American College of Obstetricians and Gynecologists; ISSHP=the International Society for the Study of Hypertension in p regnancy; MIF^=Microimmunofluorescence; EVT" = Extravillous Trophoblast Cells; VT'=Villous Trophoblast Cells. NHB PEP=National High Blood Pressure Education Program. 29 Assessment of Risk of Bias Six of the 16 studies were at low risk (21, 49, 50, 6 1 - 6 3 ) and one study had moderate level risk (6 4 ). Out of the remaining nine studies, five had serious risk (48, 6 5 - 6 8 ) and four (51, 5 8 , 6 9 , 70 ) were assessed as at the level of critical risk of bias. By organisms, out of the four studies which explored H. pylori one each was at a risk of the level low (21), moderate (64), serious (65 ) and critical (51). In comparison, out of the four studies which investigated the role of CMV, two each had a risk of the level low (50, 6 3 ) and serious (48, 6 6 ). Out of the ten studies on C. pneumoniae , two showed serious risk (6 3, 67 ) and four each had low (49, 6 1 - 6 3 ) as wel l as critical (51, 5 8 , 6 9 , 70 ) risk of bias. The most common problem with the quality of study was bias due to confounding. 30 D ISCUSSION Of the sixteen research articles reviewed in this study three of the ten showed a positive association between C. pneumoniae and PE. Four of them investigated association between H. pylori and PE, and all were found significantly associated. Of the four studies which explored the relationship between CMV and PE, two reflected a significant association between CMV and PE. Our investigation added nine more studies to the review in compariso n to the previous two meta - analyses (53, 54); still the overall evidence is not sufficient to provide a clear conclusion except for H. pylori and CMV to some extent. To our knowledge, of the previously conducted meta - analyses; one showed an association be tween infection and PE (54) while the other did not (53). But these two reviews focused on infection almost everywhere in the body while our review is exclusively on three micro - organisms which have been found associated in the development of atherosclero sis (21, 48, 49). With regards to the studies focused on H. pylori , all four studies utilized serological tests for the evidence of H. pylori infection (21, 51 , 64 , 6) and found a significant association between the microorganism and PE; one study addi tionally looked at H. pylori DNA by using PCR in placental tissue but could not establish an association (6 4 ). This may be due to the smaller sample size for the PCR study (n=20) as compared to the serological analysis (n=86). All four studies had a stron g association but only one of them was at a low risk of bias (21), the risk of bias (ROB) ranged from moderate (6 4 ) to critical or serious (51, 6 5 ). The main factor for the increased level of ROB was that these studies either did not provide information o n the confounders or did not control all of the potential confounders. Usually, a weak association could be a result of uncontrolled confounders; however caution should be taken before applying these results. 31 Out of the four studies on CMV, two found a significant association (48, 6 6 ). One had a moderate association (48) while the other study did not report a level of association (6 6 ). The latter study used DNA extraction method to determine the evidence of infection. However these both studies were at a serious ROB. On the other hand, none of the other two studies was significantly associated (50, 6 3 ). One of the latter studies assessed the outcome as early and late onset PE (6 3 ). For the early onset PE, the sample size for PE cases was small (n= 9) and the late onset it was a bit higher (n=29). For the early onset PE, CMV was reflective of a risk factor (RF) [OR: 2.1(0.3 - 17.1)] while the late onset PE indicated CMV as a preventive factor [OR: 0.6(0.2 - 1.5)]. This article used C. pneumonia along w ith CMV as an exposure and the trend of its association with PE was similar to CMV ( C. pneumoniae as an RF for early onset while preventive for late onset PE). It may suggest that infection was of severe in nature for the early onset PE however it could n ot reach to a significant level; in contrast the late onset PE was of mild in nature and acted as a preventive factor. As far as C. pneumoniae is concerned, out of the ten studies, three found a significant association between C. pneumoniae and PE (49, 6 7 , 6 8 ). All three showed a strong association. One of them used PCR techniques to look for the evidence of C. pneumoniae DNA in the placental tissue at two levels, extra - villous (EVT) and villous tissue (VT). The association was significant when the sam ples were assessed for both the tissues as compared to only EVT. In the former case (EVT and VT combined), the number of PE cases was 15 as compared to the latter case (EVT only) where nine cases of PE were found. The low number of PE cases in EVT only s cenario might have resulted in no association. However, two of these three studies were found to be at a serious risk of bias, the main factor being no information on controlling for the confounders (6 7 , 6 8 ). 32 The rest of the studies showed an assoc iation between C. pneumoniae and PE where C. pneumoniae was found as a RF (OR>1) (51, 5 8 , 6 1 , 6 2 , 6 9 , 70 ) except for two studies (6 1 , 6 3 ) where C. pneumoniae acted as a protective factor. Nine of the ten studies utilized serological tests and all of them looked for IgG; in all of them C. pneumoniae was found as a RF (49, 51, 5 8 , 6 2 - 6 3 , 6 8 - 70 ) except one (6 1 ). As IgG usually indicates chronic infection, it may point out here as well that these studies had an infectious episode during early weeks of pregnancy. However, two studies also found an association with IgM along with IgG (51, 5 8 ) which may suggest a recent ep isode during pregnancy in addition to previous episode(s). Nine out of the 16 total studies found a statistically significant association between infectious agents and PE; only three of them were of moderate to high quality studies as assessed by the ex istence of risk of bias. The current body of evidence is too small to reach any clear conclusion to determine the role of infections in the development of PE. Moreover, this review did not find any study from low income countries, and the generalizabilit y of the current findings is also very much limited. The findings of this review are not strong enough to determine the definite association of infection caused by H. pylori , CMV or C. pneumoniae with that of PE. However, this review has uncovered the nee d for more research with a robust methodology 33 LIMITATIONS Our review had several limitations. Our search was limited to studies published only in the English language; this may have reduced our sample size due to missing non - English language studies. In future, researchers can get the required information from the authors of the articles in languages other than English by personally contacting them. The quality of the studies in this review varied significantly. More than half of the studies were either at critical bias or serious bias and this made the results questionable. Two of the studies cited in different reviews and found in the reference lis ts of some articles could not be accessed. There was not a single study from low income countries. The number of studies was also not sufficiently large; when considered separately, there were not enough studies for as the recommednde number of studies for categorical outcome is 4 for each group (Cochrane handbook) . Therefore, we could not pool the results or carry out meta - analysis as the number of studies for a particular infectious agent was low, or these studies used different methods of analysis (s erological tests, PCR) and provided different measures of association. 34 CONCLUSIONS This review attempted to investigate an association between C. pneumoniae, H. pylori and cytomegalovirus (CMV) and PE and found that just above half of the studies (9 out of 16 found a significant association with at least one of these microorganisms; most of the studies come from high and middle income countries. Since the two previous meta - analyses were conducted, nine more studies on this topic have been added still the available literature does not strongly indicate an association between these infectious agents and PE except for H. pylori. Overall the quality of studies, as assessed by the existence of risk of bias, was not considered to be using a robust methodology. These were at the risk of serious or even more severe bias. Most of the studies were found at a high risk of bias due to in ability to report or control for the confounders, the findings of this review guide us for the need of more research on this topic with high quality studies. 35 APPENDICES 36 Appendix A: Structured S ummary Background : Preeclampsia (PE) affects 5 - 8 % of pregnancies and is one of the leading causes of perinatal morbidity and mortality in the world. Presently, no causative factor has been identified for PE. Over the last decade efforts have been made to explore the pos sible role of maternal infections as a risk? factor for the development of PE. Objectives : We carried out a systematic review to determine if an association exists between PE and infection with H. pylori (HP) , cytomegalovirus (CMV) or C. pneumoniae (CP). Data Sources: We systematically searched PubMed, EMBASE and Web of Science for articles in the English language. Additionally, studies were added after manual searches by reviewing the reference lists of the selected articles. Search terms included infe ctions, bacterial infections, viral infections, H. pylori (HP) , cytomegalovirus (CMV) C. pneumoniae (CP), preeclampsia, eclampsia, pregnancy induced hypertension and gestational hypertension. Study quality was assessed by applying a modified version of the Cochrane Risk of Bias Assessment Tool (ACROBAT - NRSI). Studies were grouped corresponding to the infectious agents evaluated and a synthesis of results was carried out accordingly. Findings were reported using the Preferred Reporting Items for Systemat ic Reviews and Meta - Analyses (PRISMA) Guidelines. Study Selection: Articles included cross - sectional, case control and cohort studies designs. Eligible studies included those with 1) original data, 2) that used a recognized definition of preeclampsia that followed guidelines of relevant organizations or societies (i.e. the American College of Obstetrics and Gynecology (ACOG), the International Society for the Study of Hypertension in Pregnancy (ISSHP), and the National High Blood Pressure Education Program 37 (NHBPEP), and 3) which described the techniques of detection of infectious agents or antibodies, and 4) included at least one of the three microorganisms of interest . Data Extraction : A data abstraction form was used to extract and document the required i nformation from the selected articles including the year of publication, the country of publication, type of study design with population base used (hospital or population), the sample size and the adjustment for confounding, whether by matching at design phase or at analysis. give some basic variables here?. Data Synthesis : We identified 1031 hits after searching the three databases; after reviewing the titles and abstracts we found 15 relevant articles that underwent full text review . All 15 studies me t our final inclusion criteria. After review of the reference lists we found 3 additional articles, 1 of which met our final inclusion criteria after full text review , Thus 16 studies underwent data abstraction and were included in the final review . F ifteen studies utilized the case - control design, one was a cohort. Three of them, including the cohort study, were population based studies and the rest were (13 studies) were hospital - based CCS. All studies used either serological tests, Polymerase Chai n Reaction (PCR) tests, or both to determine the presence of infection. Four studies described the exact timing of collection of the samples (2 samples were drawn in the 1 st trimester, 1 in the 2 nd trimester and 1 at the time of delivery); for the rest of the studies, no information was provided for the timing of collection of samples. The diagnosis for PE was made based on the assessments made after the 20 th week of gestation. Four studies explicitly mentioned following ACOG guidelines while one study followed the guidelines of ISSHP and the other NHBPEP. All studies explored the association between PE and at least one of the three microorganisms. 38 Four studies explored the association between HP and PE and each found a significant association [Adj OR: 9.2 (2.8 - 30)], [OR: 2.9 (1.1 - 7.8)], [OR: 3.8: (1.2 - 11.8)], and [OR: 2.7 (1.1 - 6.6)]. Of the four studies investigating the relationship between CMV and PE, two showed significant association [OR: 1.9(1.4 - 2.7)] and [OR: 7.2 (p.02)] while three out of ten st udies found a significant association between CP and PE [OR: 5.3 (1.4 - 20)], [OR: 3.1(1.8 - 7.9)] and [OR: 4.1(1.1 - 15.6)]. Nine of the sixteen studies provided information on controlling for confounding variables; three studies used matched case - control des ign and six controlled confounding variables at analysis. T he variables most commonly matched or controlled at analysis were parity and maternal age. The review also looked at the quality of studies by assessing the risk of bias (ROB) utilizing a modified version of A Cochrane Risk of Bias Assessment Tool: for Non - Randomized Studies (ACROBAT - NRSI). The ROB assessment found six studies at low ROB, nine at serious or critical and one was found to be at a moderate ROB; bias due to confounding was the most common serious flaw. Limitations: This review did not include articles from languages other than the English. The final number of articles included was low and meta - analysis could not be carried out. Additionally, about 2/3 of the studies were found to be at a high risk of bias. Conclus ions: A small body of evidence exists on the relationship between PE and HP , CMV and CP. The available literature does not indicate an association between these infectious agents and PE except for HP; more than half of the studies were at a high risk of bias. Literature using a more rigorous methodology is needed in this subject area. 39 Appendix B : Main terms searched 1. Preeclampsia 2. Eclampsia 3. Pregnancy induced hypertension 4. Gestational hypertension 5. Chronic hypertension 6. Seizures in pregnancy 7. Infection s 8. Bacterial infections 9. Viral infections 10. Chlamydophila pneumoniae 11. Helicobacter pylori 12. Cytomegalovirus 40 Appendix C : Terminology used for PUBMED search engine Preecl ampsia[majr] OR eclampsia OR pregnancy induced hypertension OR Gestational hypertension AND (Bacteria[mh] OR bacterial infections[mh] OR viruses[mh] OR virus diseases[mh] OR Helicobacter pylori[ti] OR chlamydophila pneumonia[ti] OR cytomegalovirus*[ti] OR Infection[ti] OR infectio ns[ti] OR infectious[ti] OR infect[ti] OR infected[ti]) 41 Appendix D : Risk of Bias assessment Table 5: Modified version of the ACROBAT - NRSI. SNo. Domain s Assessment of Risk of Bias Comments Author, Year, Country, Ref Bias due to confounding Bias in selection of participants Bias in measurement of exposure Bias due to departures from intended exposure Bias due to missing data Bias in measurem ent of outcome Bias in selection of reported result 1 Cardaropoli et al, 2011, Italy (26) Low risk Low risk Low risk Low risk Low risk Low risk Low risk Low risk Controlled for variables 2 Aksoy et al, 2009, Turkey (37) Serious risk Low risk Low risk Low risk Low risk Low risk Low risk Serious risk 3 Strand et al, 2012, Norway (29) Low risk Low risk Low risk Low risk Low risk Low risk Low risk Low risk 4 Xie et al, 2010, Canada (27) Serious risk Low risk Low risk Low risk Low risk Low risk Low risk Serious risk 5 Chrisoulidou et al, 2011, UK (36) Low risk Low risk Low risk Low risk Low risk Low risk Low risk Low risk Matching on variables 6 Karinen et al, 2008, Finland (28) Low risk Low risk Low risk Low risk Low risk Low risk Low risk Low risk Controlled for variables 7 Aral et al, 2006, Turkey (33) Critical risk Moderate risk Low risk Low risk Low risk Moderate risk Low risk Critical risk 8 Goulis et al, 2005, UK (42) Critical risk Low risk Low risk Low risk Low risk Low risk Low risk Critical risk 9 Raynor et al, 2004, USA (43) Critical risk Low risk Low risk Low risk Low risk Low risk Low risk Critical risk 10 Heine et al, 2003, USA (44) Serious risk Low risk Low risk Low risk Low risk Low risk Low risk Serious risk 11 Ustun et al, 2010, Turkey (30) Critical risk Low risk Low risk Low risk Low risk Low risk Low risk Critical risk 12 Von Dadelszen et al, 2003, Canada (45) Low risk Low risk Low risk Low risk Low risk Low risk Low risk Low risk Matching on variables 13 Ponzetto, 2006, Italy (38) Moderate risk Low risk Low risk Low risk Low risk Low risk Low risk Moderate risk Matching on 2 variables 14 Carreiras et al, 2002, Venezuela (39) Serious risk Low risk Low risk Low risk Low risk Low risk Low risk Serious risk 15 Xie et al, 2010, Canada (40) Low risk Low risk Low risk Low risk Low risk Low risk Low risk Low risk Matching on variables 16 Gomez et al, 2009, USA (41) Serious risk Low risk Low risk Low risk Low risk Low risk Low risk Serious risk ACROBAT - NRSI= A Cochrane Risk of Bias Assessment Tool for Non - Randomized Studies of Interventions. 42 Appendix E : Search and selection process Figure 3: Search and selection process of studies Final sample of articles reviewed for systematic review, n=16 Excluded, n=1 The article could not be found in the search engine, contacted author but received no response preventive treatment and did not provide the required information Articles after addition, n=17 Found article, n=3 Articles included, n=14 Searched the articles by review of reference list Excluded, n=1 The article was on preventive treatment and did not provide the required information Full text review with application of inclusion criteria Selected for further review n=15 Excluded, n=1014 Titles and abstract with no relevant information Articles screened on the basis of title and abstract Search results n = 1031 Databases searched: Pubmed, EMBASE and Web of Science (WoS) Identification Screening Eligibility I nclusion 43 Appendix F : Antigens of H. pylori Table 6: Types of Antigens of H. pylori Cag Cytotoxin - associated protein Vac Vacuolating cytotoxin protein UreA Urease subunits UreB UreC HspA Heat - shock protein HspB Flagellin subunits Catalase lipopolysaccharide 44 Appendix G : PRISMA checklist Table 7: PRISMA Checklist of items to include when reporting a systematic review (with or without meta - analysis). Section/Topic S . No . Checklist item Reported on page # TITLE Title 1 Identify the report as a systematic review, meta - analysis, or both. Title page, i ABSTRACT Structured summary 2 Provide a structured summary including, as applicable: background; objectives; data sources; study eligibility criteria, participants, and interventions; study appraisal and synthesis methods; results; limitations; conclusions and implications of key findi ngs; systematic review registration number. 2 - 4 INTRODUCTION Rationale 3 Describe the rationale for the review in the context of what is already known. 12 Objectives 4 Provide an explicit statement of questions being addressed with reference to participants, interventions, comparisons, outcomes, and study design (PICOS). 14 METHODS Protocol and registration 5 Indicate if a review protocol exists, if and where it can be accessed (e.g., Web address), and, if available, provide registration information including registration number. The protocol was not registered Eligibility criteria 6 Specify study characteristics (e.g., PICOS, length of follow - up) and report characteristics (e.g., years considered, language, publication status) used as criteria for eligibility, giving rationale. 1 5 Information sources 7 Describe all information sour ces (e.g., databases with dates of coverage, contact with study authors to identify additional studies) in the search and date last searched. 1 5 Search 8 Present full electronic search strategy for at least one database, including any limits used, such that it could be repeated. 15 & 37 Study selection 9 State the process for selecting studies (i.e., screening, eligibility, included in systematic review, and, if applicable, included in the meta - analysis). 15 & 3 9 Data collection process 10 Describe method of data extraction from reports (e.g., piloted forms, independently, in duplicate) and any processes for obtaining and confirming data from investigators. 1 5 - 16 Data items 11 List and define all variables for which data were sought (e.g. , PICOS, funding sources) and any assumptions and simplifications made. 37 45 Risk of bias in individual studies 12 Describe methods used for assessing risk of bias of individual studies (including specification of whether this was done at the study or outcome level), and how this information is to be used in any data synthesis. 27 & 38 Summary measures 13 State the principal summary measures (e.g., risk ratio, difference in means). 24 - 26 Synthesis of results 14 Describe the methods of handling data and combining results of studies, if done, including measures of consistency (e.g., I 2 ) for each meta - analysis. Pooling of results was not done Risk of bias across studies 15 Specify any assessment of risk of bias that may affect the cumulative evidence (e.g., publication bias, selective reporting within studies). Pooling of results was not done Additional analyses 16 Describe methods of additional analyses (e.g., sensitivity or subgroup analyses, meta - regression), if done, indicating which were pre - specified. Pooling of results was not done RESULTS Study selection 17 Give numbers of studies screened, assessed for eligibility, and included in the review, with reasons for exclusions at each stage, ideally with a flow diagram. 17 & 39 Study characteristics 18 For each study, present characteristics for which data were extracted (e.g., study size, PICOS, follow - up period) and provide the citations. 17 - 19 Risk of bias within studies 19 Present data on risk of bias of each study and, if available, any outcome l evel assessment (see item 12). 27 & 38 Results of individual studies 20 For all outcomes considered (benefits or harms), present, for each study: (a) simple summary data for each intervention group (b) effect estimates and confidence intervals, ideally with a forest plot. 21 - 27 Synthesis of results 21 Present results of eac h meta - analysis done, including confidence intervals and measures of consistency. 28 - 29 Meta - analysis was not done. Risk of bias across studies 22 Present results of any assessment of risk of bias across studies (see Item 15). Pooling of results was no t done Additional analysis 23 Give results of additional analyses, if done (e.g., sensitivity or subgroup analyses, meta - regression [see Item 16]). Not done. DISCUSSION Summary of evidence 24 Summarize the main findings including the strength of evidence for each main outcome; consider their relevance to key groups (e.g., healthcare providers, users, and policy makers). 28 - 29 Limitations 25 Discuss limitations at study and outcome level (e.g ., risk of bias), and at review - level (e.g., incomplete retrieval of identified research, reporting bias). 30 Conclusions 26 Provide a general interpretation of the results in the context of other evidence, and implications for future research. 30 46 FUNDING Funding 27 Describe sources of funding for the systematic review and other support (e.g., supply of data); role of funders for the systematic review. 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