3.3. V . :0: ...f.,\. r l-”" ~53“: MICHIGAN STATEU I II III IIIIIIIIIIIIIIIIIIIIIIIIIIIIIII 913 4754 II This is to certify that the thesis entitled THE ASSOCIATION OF BACTERIAL VAGINOSIS WITH SPERM CERVICAL MUCUS PENETRATION TEST OUTCOME presented by Penny L. Waltman has been accepted towards fulfillment of the requirements for Master Of Science degree in Nursing 4M ;/44«4W Major professor Date January 7, 1992 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution .--~ sun“ I 4 '1"; ‘Iv ' ' ' I I Laemnv I Michigan 3mm I University! PLACE IN RETURN BOX to remove this checkout from your record. TO AVOID FINES return on or before date due. DATE DUE DATE DUE DATE DUE I MSU Is An Affirmative ActlorVEqual Opportunity Institution ouch-Imam»! — g...— THE ASSOCIATION OF BACTERIAL VAGINOSIS WITH SPERM CERVICAL MUCUS PENETRATION TEST OUTCOME BY Penny L. Waltman A THESIS ‘Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE IN NURSING College of Nursing 1991 THE ASE ,w l \n h." I. I" hi’y' M) if" Walk ~ a." I ABSTRACT THE ASSOCIATION OF BACTERIAL VAGINOSIS WITH SPERM CERVICAL MUCUS PENETRATION TEST OUTCOME BY Penny L. Waltman A retrospective non-experimental descriptive study was done to examine whether infertile couples infected with bacterial vaginosis and/or Ureaplasma urealyticum were more likely to have adverse sperm-cervical mucus penetration test (SCMPT) outcome than infertile couples not infected. The sample consisted of 14 infertile couples divided into four groups according to their bacterial status. Data were analyzed by analysis of variance with SCMPT score as the dependent variable, and the presence and absence of bacterial vaginosis and Ureaplasma urealyticum used as the grouping variables. There were significant differences in mean SCMPT score between couples with combined bacterial vaginosis and Ureaplasma urealyticum profile and couples with bacterial vaginosis and Ureaplasma urealyticum alone. Therefore, in this study a combined presence of bacterial vaginosis and Ureaplasma urealyticum in infertile couples was associated with adverse SCMPT score outcome. The study. implications include attention to SCMPT score outcome for infertile couples with combined infections of bacterial vaginosis and Ureaplasma urealyticum for possible treatment. Copyright by PENNY L. WALTMAN 1991 T° my hush, shall neve To my husband Michael Waltman and my four daughters Rachel, Kristen, Lauren, and Sarah who's sacrifice for my sake I shall never forget. iv A spec Millie Oma preparing ACKNOWLEDGMENTS A special thanks to Rachel Schiffman, Manfred Stommel, Millie Omar, and Linda Beth Tiedje for their assistance in preparing this thesis. List of List of CHAPTER Introdul Stateme Definit Assumpt Limitat Summary Overvie CHAPTER Concept ROY’S A AppliCa Summary CHAPTER Literat Support Mechani Ureapla TABLE OF CONTENTS List of Tables . . . . . . . . . . . . . . . . . . . . viii List of Figures . . . . . . . . . . . . . . . . . . . . . ix CHAPTER I Introduction . . . . . . . . . . . . . . . . . . . . . . 1 Statement of the Problem . . . . . . . . . . . . . . . . 3 Definition of Concepts . . . . . . . . . . . . . . . . . 6 Assumptions . . . . . . . . . . . . . . . . . . . . . . . 13 Limitations . . . . . . . . . . . . . . . . . . . . . . . 14 Summary . . . . . . . . . . . . . . . . . . . . . . . . . 14 Overview of Remaining Chapters . . . . . . . . . . . . . 15 CHAPTER II Conceptual Framework . . . . . . . . . . . . . . . . . . 17 Roy's Adaptation Model . . . . . . . . . . . . . . . . . 17 Application of Conceptual Framework to the Study . . . . 20 Summary . . . . . . . . . . . . . . . . . . . . . . . . . 24 CHAPTER III Literature Review . . . . . . . . . . . . . . . . . . . . 26 Support for Bacterial Vaginosis as a Pathogen . . . . . . 27 Mechanisms by Which Bacterial Vaginosis may Affect Sperm Mucus Interaction . . . . . . . . . . . . . . . . . . 32 Ureaplasma Urealyticum . . . . . . . . . . . . . . . . . 34 Critique/Implications . .'. . . . . . . . . . . . . . . . 40 Summary CHAPTER Methods Backgro Methods Researc Human 8 Summary CHAPTEF Results Descri; Hypothe Summary CRAFTS; sunflar} Summary Discus: Implic Cone 1U List 0 APPEnd D°Cume ApPEnc‘ Appent Summary . . . . . . . . . CHAPTER IV Methods and Procedures . Background . . . . . . . Methods . . . . . . . . . Research Hypotheses . . . Human Subjects . . . . . Summary . . . . . . . . . CHAPTER V Results . . . . . . . . . Descriptive Data . . . . Hypothesis Testing . . . Summary . . . . . . . . . CHAPTER VI Summary and Conclusions . Summary of the Study . . Discussion . . . . . . . Implications for Nursing Conclusions . . . . . . . List of References . . . Appendix A Protection of Documentation . . . . . . Rights and Human Subjects Appendix B Semen Parameter Standards . . . . . . . Appendix C Cervical Mucus Scoring . . . . . . . . vii 42 44 44 45 49 60 60 61 61 66 69 71 71 74 80 85 89 101 104 105 ’5 Table 1 Criteri 2 Sample I 3 Prevalei 4 Compari: 5 Mean SCI Vaginosis Ureap LIST OF TABLES 1:11.13 £49.: 1 Criteria for Scoring SCMPT . . . . . . . . . . . . . . 48 2 Sample Attrition . . . . . . . . . . . . . . . . . . . 64 3 Prevalence of Bacteria in the Initial and Final Sample 64 4 Comparison of SCMPT with Prostate Evaluation . . . . . 68 5 Mean SCMPT Scores by Presence/Absence of Bacterial Vaginosis and Ureaplasma Urealyticum . . . . . . . . . . . . . . . 68 viii 'PD _‘,_. .. Fm e 1 The Ass Cervical Mucus P LIST OF FIGURES timers Page 1 The Association of Bacterial Vaginosis with Sperm Cervical Mucus Penetration Test Outcome Conceptual Model . . . 21 ix Mil States c gynecolc health ; estimate of ever) In all, suffer 1 Multiple infertil tlechnolc Current] polymicr Riff, 5 f91313185 Shared l repreSei relates Br< The “01‘! potentia aSSOCiaI with the s'liepm‘c£ ls to e} CHAPTER I Introduction Millions of dollars are spent annually in the United States on infertility health care. Despite improved gynecological and infertility care, infertility remains a health problem for many couples. Infertility has been estimated to affect 15% of the total population or one out of every six couples of childbearing age (Andrews, 1984). In all, about seven million couples in the United States suffer from compromised fertility (Mosher, 1988). Multiple factors are known to contribute to this dilemma of infertility. Despite recent advances in knowledge and new technology many issues remain unexplored. The syndrome currently known as bacterial vaginosis, because of its polymicrobial nature (Thomason, Schreckenberger, Spellacy, Riff, & LeBeau, 1984), its incidence among sexually active females (Eschenbach, 1984), and its likelihood of being shared by a sexual partner (Gardner & Dukes, 1959), may represent yet another unexplored link to infertility as it relates to sperm-cervical mucus interaction responses. Broadly stated this study will investigate infertility. The more narrowly defined research focus is to analyze the potential virulence or biological maladaptation which may be associated with bacterial vaginosis alone or in combination with the organism Ureaplasma urealyticum, with respect to sperm-cervical mucus interaction. The purpose of this study is to expand the body of knowledge that exists regarding the 1 .. t" potentia associat infertil that bac ascensic childbea DeRouen, Zabrans) Thomasor Lehtiner Sperling Broekhuj KrOhn, I‘ to date bacteria Cervical Ade aSSeSSec mobilit} than 10 m°tility currentl interact Interact 2 potential virulence or biological maladaptation that may be associated with bacterial vaginosis and therefore infertility. There is increasing evidence in the literature that bacterial vaginosis is pathogenic, is capable of ascension into the cervix, and is prevalent in couples of childbearing age (Eschenbach, Hillier, Critchlow, Stevens, DeRouen, & Holmes, 1988; Josephson, Thomason, Sturino, Zabransky, & Williams, 1988; Mead, Eschenbach, Ledger, & .Thomason, 1989; Paavonen, Teisala, Heinonen, Aine, Laine, Lehtinen, Miettinen, Punnonen, & Gronroos, 1987; Silver, Sperling, St. Clair, & Giggs, 1989; Thomason, Gelbart, & Broekhuizen, 1989; Watts, Eschenbach, & Kenny, 1989; Watts, Krohn, Hillier, & Eschenbach, 1990). In addition, no study to date has investigated whether couples with a diagnosis of bacterial vaginosis, are at risk for maladaptive sperm- cervical mucus interactions. Adequate sperm-cervical mucus interaction is generally assessed by the Huhner's test which identifies sperm mobility and quantity. According to the Huhner’s test, less than 10 sperm per high power field and/or reduced sperm motility, without an increased number of white blood cells, currently suggests inadequate sperm-cervical mucus interaction. This inadequate sperm cervical-mucus interaction is currently thought to be related to cervical mucus hostility, sperm allergy or reduced semen parameters on the part of the male. Sperm allergy, cervical mucus hostility, and poor semen parameters therefore become necessa: This in possibl reduced evaluat vaginos cervica penetra investi for qua In addj Ureaplg BU85chj Eschem concur] 3 necessary competing clinical diagnoses for the provider.~ This investigation, however, will investigate another possible explanation for reduced sperm numbers and/or reduced sperm mobility that may be found on routine huhner’s evaluation; that is, whether the presence of bacterial vaginosis has any association with this maladaptive sperm- cervical mucus interaction. The sperm-cervical mucus penetration test will be the unit of measure for this investigation since it does, unlike the huhner's test, allow for quantitative analysis of sperm penetration and motility. In addition, the coexistence of an anaerobic organism, Ureaplasma urealyticum, with bacterial vaginosis (Bump & Buesching, 1988; Gravett, Nelson, DeRouen, Critchlow, Eschenbach, & Holmes, 1986) will be assessed for its concurrent incidence with bacterial vaginosis and with maladaptive sperm-cervical mucus interaction. statement of the Problem Information about sperm-cervical mucus interaction responses in infertile couples will have an impact on clinical decision making and delivery of infertility health care by the primary care provider. The Clinical Nurse Specialist, as a primary care provider, in infertility health care has a responsibility to promote human responses that lead to adaptation (Andrews & Roy, 1986). Adaptation, as applied to infertile couples, is a response to the environment that promotes the couple's general goal of reproduction. The nurse, therefore, has a responsibility, as do at potentia potentia The not void referenc 20 years known fc inferti] nurse 5; eXDahdec infertij meeting nurse 5] Drastic, nursing Scepe 0 and 815 alOng ‘ It or the or Pot. R°Y (l! mu'Sinr °f mar 4 as do other health care providers, to investigate potentially causative stimuli that may alter the client's potential to achieve conception. The specialty of infertility despite its complexity is not void of nursing expertise. Many of the psychosocial references found in the infertility literature for the past 20 years are nursing based. Menning, for example, is well known for her work on the emotional issues related to infertility (1977, 1980, 1984). Today, however, clinical nurse specialists providing infertility care are called to expanded role performance. Clinical nurse specialists in infertility practice are more often primary care providers, meeting more diverse health care needs of couples. Clinical nurse specialists must strive to infuse infertility care and practice, with a holistic framework that is unique to nursing. That is to say, nurses must begin to broaden their scope of practice in the field of infertility health care and elsewhere to include biophysical and spiritual care, along with the predominately psychosocial care they deliver. Thereby, this thesis will focus on the first two levels of the nursing process, i.e. the assessment of behavior and of potentially causative stimuli, according to Andrews and Roy (1986), of infertility. These first two levels of the nursing process, as applied to this study are the assessment of maladaptive sperm-cervical mucus responses and the assessment of bacterial vaginosis as an associated stimuli. I" The as the i cervical variable bacteria various normal I and the: cervix A the res urealyt of its (Bump & Therefc °r9anis analyze °°exist Women ‘ Tl infertj Ureapla cemici infert; 5 The major variables will be bacterial vaginosis (BV), as the independent variable, and the outcome of the sperm cervical mucus penetration test (SCMPT), as the dependent variable. This investigation rests on theory that with bacterial vaginosis there are increased concentrations of various micro-organisms which may be more virulent than the normal lactobacillus dominated flora of the normal vagina and thereby, potentially more hostile to the sperm in the cervix (Eschenbach, 1984; Tan, Scammell, & Houang, 1987). . A possible antecedent variable that is of interest to the researcher is the presence or absence of Ureaplasma urealyticum. This organism is included in the study because of its established association with bacterial vaginosis (Bump & Buesching, 1988; Gravett, Nelson et al., 1986). Therefore, any confounding effect explained by this organism, with or without bacterial vaginosis may be analyzed. The likelihood for Ureaplasma urealyticum to coexist with bacterial vaginosis, and its prevalence in women without bacterial vaginosis will also be established. The problem of interest in this study is: Are infertile couples infected with bacterial vaginosis and/or Ureaplasma urealyticum more likely to have adverse sperm- cervical mucus penetration test (SCMPT) outcome than those infertile couples not infected? Mor been ide cou II. Are ma Ini unProtec Kase, 15 an arral Partner that Su: than a 1 inferti StUdy, : for the Status maladap‘ % Ba. Et al., nonspec Gardnerr 6 More specifically the following research questions have been identified: I. Are couples with bacterial vaginosis more likely, than couples without bacterial vaginosis, to have lower SCMPT scores? II. Are SCMPT scores more likely to be lower in couples with bacterial vaginosis, when one or both spouses are also positive for Ureaplasma urealyticum? Definition of Concepts Infertility Infertility is generically defined as one year of unprotected coitus without conception (Speroff, Glass, & Kase, 1983). Implicit in this definition is the couple with an array of potential problems that can not affect one partner without affecting the other partner. The couple that suffers from infertility, therefore, possesses less than a maximum potential for reproduction. Therefore, infertility will conceptually, for the purposes of this study, be defined as a state of compromised fertility status for the couple. More specifically a compromised fertility status found in study subjects will be defined as maladaptive sperm-cervical mucus interaction responses. W Bacterial vaginosis is the term more recently (Thomason et al., 1989) applied to the condition previously known as, nonspecific vaginitis; Hemophilus, Corynebacterium or Gardnerella vaginitis; anaerobic vaginitis; or anaerobic vaginosi that eff females with vul others h bacteria in priva reported Kaufman, approxir Vaginos; in cert.- Gel SeXuallj Spiegel some de 7 vaginosis. Bacterial vaginosis (BV) is a vaginal condition that effects approximately 15% of young sexually active females and accounts for 45% of the symptomatic females seen with vulvovaginal complaints (Eschenbach, 1984). Still others have reported similar incidence of 10-15% for bacterial vaginosis among married women seen by physicians in private practice. Sexually active unmarried women were reported to have as high as 50% incidence (Gardner & Kaufman, 1980). Brown, Kaufman, and Gardner (1984) reported approximately a 58% relative frequency for bacterial vaginosis in nonpregnant patients and up to a 60% occurrence in certain population groups, such as prostitutes. Generally bacterial vaginosis is considered to be a sexually transmitted disease (Brown et al., 1984; Holmes, Spiegel, Amsel, Eschenbach, Chen, & Totten, 1981); however, some debate still exists about whether bacterial vaginosis is exclusively sexually transmitted or can be present as part of the normal genital flora (Bump & Buesching, 1988). Josey and Lambe (1976) also reported that bacterial vaginosis frequently was associated with other sexually transmitted diseases and found this association to be highly significant. A major problem associated with the condition is that bacterial vaginosis rarely creates sufficient symptomatology to cause suspicion of infection for the client. The subtleness of the condition's signs and symptoms also results in a potential lack of recognition by the health care p! the prc unlike of any with be dischar vaginal standar of bact overloc malada; Fe haVe 31 norm is Vaginit Charact a gray IeXCept lateral “Win with a intercO new, T In Possibl gardner. bacteri; 8 care provider as well. One reason for the client's and/or the provider's lack of suspicion for infection is that, unlike other vaginal infections, bacterial vaginosis is void of any inflammatory response. Therefore, females infected with bacterial vaginosis lack any purulent vaginal discharge, burning, or itching typically created by other vaginal pathogens and utilized by client and provider as standard cues to action. This overall lack of recognition of bacterial vaginosis certainly would make it easily overlooked as a potentially causative factor when maladaptive sperm-cervical mucus responses were observed. Females infected with bacterial vaginosis do, however, have altered vaginal discharge. But the alteration from the norm is less obvious than the changes found with other vaginitis conditions. Typically this vaginosis is characterized by increased epithelial shedding which creates a gray to white vaginal discharge that is thin, nonfrothy (except for 10-15% of the cases), and adherent to the lateral vaginal walls. Sometimes the discharge is copious enough to pool at the vaginal introitus and is associated with a "fishy" malodor that is most noticeable after intercourse or at the time of menses (Brown et al., 1984; Mead, Thomason, Ledger, & Eschenbach, 1986). In recent years, many investigators have pondered the possible pathogenic significance of the coccobacillus, gardnerella Vaginalis, as well as a variety of other bacteria, all of which are recognized as causative for ”I bacterial L I I et al., III Eschenbacf Eschenbacl' Eschenbacl Cummings, Morales, .1 et al., 1: 1984; war incidence 9 bacterial vaginosis (Gardner, 1983; Gravett, Hummel, Eschenbach, & Holmes, 1986; Gravett, & Nelson et al., 1986; Eschenbach et al., 1988; Fingold, & Sbarra, 1987; Josephson et al., 1988; Martius, Krohn, Hillier, Stamm, Holmes & Eschenbach, 1988; Minkoff, Grunebaum, Schwarz, Feldman, Cummings, Crombleholme, Clark, Pringle, & McCormack, 1984; Morales, Angel, O’Brien, Knuppel, & Finazzo, 1988; Paavonen et al., 1987; Spiegel, 1987; Wahbeh, Hill, Eden, & Gall, 1984; Watts et al., 1989; Watts et al., 1990). Given the incidence of bacterial vaginosis, the 100-1000 fold increase ' of abnormal bacteria present with this vaginosis (Spiegel, Amsel, Bschenbach, Schoenknecht, & Holmes, 1980), and the recent associations of this vaginosis with such pathologic conditions as endometritis, premature labor, preterm premature rupture of the membranes (PROM), and amniotic fluid infection, it is not surprising that the pathogenicity of bacterial vaginosis has been investigated (Gravett, Hummel et al., 1986; Gravett, Nelson et al., 1986; Martius et al., 1988; Morales et al., 1988; Spiegel, 1987; Wahbeh et al., 1984). A variety of other bacteria have been found to frequently coexist in the vagina when bacterial vaginosis is diagnosed. These organisms include: Bacteroides bivius, B. disiens, B. melaninogenicus, Peptostreptococcus, Peptococcus, Eubacterium, and two varieties of curved rods recently placed in the species Mobiluncus (Blackwell, Fox, Phillips, and Barlow, 1983; Spiegel et al., 1980; Spiegel, 1987; Tayl Whether or collective bacterial Rome] component, and higher vaginosis process. endocervi; the organ which is third sta. of the ba Vaginosis with adve rupture O produc'Cio (Romero e with bact (GraVett al., 1988 bacterial IEVQI' Wj 10 1987; Taylor, Blackwell, Barlow, and Phillips, 1982). Whether or not these bacteria are individually, or collectively associated with the suspected pathogenicity of bacterial vaginosis is unknown. Romero, Mazor and Oyarzun (1988) have outlined how the component organisms of bacterial vaginosis invade the cervix and higher structures, describing the syndrome of bacterial vaginosis as the first stage of an ascending infectious process. The vaginal infection leads to infection of the endocervix with eventual progression into the decidua where the organisms can invade the amnion and chorionic membranes, which is labeled stage II of the ascension process. The third stage of the infectious process occurs with cross over of the bacteria into the amniotic cavity. Bacterial vaginosis in this purposed view is thought to be associated with adversities (i.e., chorioamnionitis and premature rupture of the membranes) secondary to bacteria protease production and/or a direct host inflammatory response (Romero et al., 1988). Despite cited evidence that the bacteria associated with bacterial vaginosis innervate higher structures (Gravett, Nelson et al., 1984; Wahbeh et al., 1986; Watts et al., 1989) there have been few investigations addressing bacterial vaginosis' potential effects at the cervical level, with respect to sperm survival. A few researchers have begun to investigate whether or not anaerobic bacteria associated with bacterial vaginosis are involved in any Pf disruptio: when thosr Gerhard, I 1987; Trev These stu: specificai positive I Vaginosis current cj SP’ECified There will be Cc (Thomason (Gardner , female par II‘m-thel‘moi Vaginal ir cemix am: FOr the me urethral j and highej Bacterial inducing a in the re U W I organ 131113 11 disruption of sperm migration through the cervical mucus when those anaerobes are clinically evident (Eggert-Kruse, Gerhard, Hofmann, Runnebaum, & Petzoldt, 1987; Tan et al., 1987; Tredway, Wortham, Condon-Mahony, Baker 8 Shane, 1985). These studies did not however study bacterial vaginosis specifically, but instead studied subjects who cultured positive for anaerobes commonly associated with bacterial vaginosis. Whether or not the subjects, according to current clinical criteria, had bacterial vaginosis was not specified. Therefore, the condition known as bacterial vaginosis will be conceptually defined as a polymicrobial infection (Thomason et al., 1984) that is shared by sexual consorts (Gardner & Dukes, 1959), but typically personified by the female partner as an abnormal vaginal syndrome. Furthermore, bacterial vaginosis will be identified as a vaginal infection that is capable of ascension into the cervix and higher pelvic structures (Romero et al., 1987). For the male, bacterial vaginosis will be defined as a urethral infection capable of ascension into the prostate and higher reproductive structures (Gardner & Dukes, 1959). Bacterial vaginosis will also be considered capable of inducing a hostile environment to the sperm once established in the reproductive track of either the female or the male. MW Ureaplasma urealyticum is one of two human mycoplasma organisms that have been isolated in the human genital 12 tract, since 1937 (McCormack, Braun, Lee, Klein, & Kass, 1973). McCormack and associates (1973) also point out that these organisms have for many years been studied for their' possible link to disorders of the human genital tract. More recently evidence has appeared linking these organisms to bacterial vaginosis however, only Ureaplasma urealyticum will be assessed in this study (Bump & Buesching, 1988; Gravett, Nelson et al., 1986). Prior to the Bump and Buesching (1988) study that found adolescent girls with bacterial vaginosis to be more likely to have concurrent vaginal isolation of Ureaplasma urealyticum than those without bacterial vaginosis, the existing evidence suggested that Ureaplasma urealyticum (T- Mycoplasmas) was as prevalent in bacterial vaginosis victims as controls (McCormack et al., 1973). McCormack and colleagues (1973) reviewed several studies of patients with vaginitis and found Ureaplasma urealyticum to be present in 67.3% of women with nonspecific vaginitis (BV), and also in 61.7% of the controls. According to Taylor-Robinson and McCormack (1980) research evidence available in 1980 made it apparent that of the two mycoplasmas, Ureaplasma urealyticum was more often found in "normal" women and men who were sexually active, especially those women with multiple sexual partners. It is not known however how many of these sexually active ”normal" women and men studied may or may not have had bacterial vaginosis using the diagnostic criteria established today. 13 Ureaplasma urealyticum therefore will be defined here as a mycoplasma organism capable of existing in the reproductive tract of sexually active men and women and more likely to exist in couples known to have bacterial vaginosis. Furthermore, it will be considered a pathogenic organism for either the female or the male when it is found to be present in either member of the couple. Research is needed in the area of infertility and bacterial vaginosis to expand the body of knowledge about the potential virulence or biological maladaptation responses that may be associated with bacterial vaginosis, with respect to sperm-cervical mucus interaction. No study to date has investigated whether couples with bacterial vaginosis are at risk for any maladaptive sperm-cervical mucus interaction, using strict clinical diagnostic criteria for the diagnosis of bacterial vaginosis. Assumptions For the purposes of this research, the following assumptions were made: 1. Bacterial vaginosis exists and can be measured. 2. Increased concentration of those micro-organisms associated with bacterial vaginosis are present in cervical mucus. 3. The SCMPT is a good indicator of sperm mucus integrity and therefore of fertility status. 14 4. Bacterial vaginosis is sexually transmitted and therefore is present in the male cohort if present in the female. Limitations The following limitations are identified in this study: 1. This study will not identify if bacterial vaginosis is causal to adverse SCMPT outcome. 2. This study will use a convenience sample whose biological responses may differ from a randomized sample. Thus, the naturally occurring groups in this convenience sample may possess some systematic bias that may explain the effect found on the dependent variable. 3. The gram stain provides limited proof of the confirmation of the presence of bacterial vaginosis. 4. The chance of infection existing in any subject because of extramarital partners is not controlled for in this study. Sullsry Bacterial vaginosis is a subtle, prevalent condition whose potential pathogenic significance has been of great interest to researchers (Eschenbach et al., 1988; Fingold, 1987; Gardner, 1983; Gravett, Hummel et al., 1986; Gravett, Nelson et al., 1986; Josephson et al., 1988; Martins et al., 1988; Minkoff et al., 1984; Morales et al., 1988; Paavonen et al., 1987; Spiegel, 1987; Wahbeh et al., 1984; Watts et al., 1989). Bacterial vaginosis and anaerobes commonly 15 associated with the condition however have not been adequately assessed for their potential role as cervical pathogens (Eggert-Kruse et al., 1987; Tan et al., 1987; Tredway et al., 1985). Therefore, the focus of this study will be to investigate whether or not couples with bacterial vaginosis are involved in ineffective biological responses that affect fertility status. More specifically, this study will investigate whether or not the presence of bacterial vaginosis adversely affects a woman's ability to maintain adequate sperm-mucus interaction. It too will be the focus of this study to determine whether or not Ureaplasma urealyticum, when present by culture, alters SCMPT outcome for women who also have bacterial vaginosis, according to established clinical criteria and gram stain. Thirdly, this investigation will examine how often women seeking routine infertility care are culture positive for Ureaplasma urealyticum. Overview of Remaining Chapters This thesis is organized into six chapters. In Chapter I the introduction, background of the problem, purpose of the study, a statement of the problem, research questions, concept definitions, assumptions and limitations of the study are included. In Chapter II the theoretical framework within which the relevant concepts and problem are to be analyzed is discussed. The relationship of these concepts within the 16 adopted theoretical framework is presented in a conceptual model. Chapter III includes a review of the literature pertinent to the study. In addition, a discussion of the strengths and limitations of past research is provided. Research methods, including a description of the study design, assumptions, setting, study subjects and their selection, are discussed in Chapter IV. Furthermore, Chapter IV will review limitations of the study and describe data collection and analysis techniques. In Chapter V, the results are presented, analyzed, and discussed in relation to the research questions posed. The summary, conclusions, and implications of the findings in relation to theory and nursing practice comprise Chapter VI. CHAPTER II Conceptual Framework 9112:1132! This chapter describes the theoretical framework from which the problem will be analyzed. The theorist, her formulated theoretical framework, and the concepts inherent in the framework will be introduced. Rationale for the adoption of this theory in the analysis of the stated problem is also offered. Furthermore, a model is provided that shows the conceptual framework of the study. The relationship between the model and the theoretical framework adopted is then discussed. Roy's Adaptation Model Sister Callista Roy introduced her conceptual nursing framework in 1970. Roy's conceptual framework is representative of nursing science's view of person, environment, nursing, and health, which have been identified by some as the major concepts of the metaparadigm of nursing science. Roy's model has led to a more systematic development of nursing knowledge (Chinn & Jacobs, 1987). The major concepts of the model introduced by Roy include person, environment, nursing, and health. Persons, according to Roy, are those individuals or persons in groups cared for by nurses. Based in general systems theory the person is described by Roy as an adaptive system, open and taking in input, and processing it to produce a response called output. Behaviors, according to Roy, could be viewed 17 18 as the output of the person as an adaptive system. Behaviors are influenced by the environment, which is seen as the world within and around the person, and by the person's abilities to deal with that world (Andrews 8 Roy, 1986; Chinn 8 Jacobs, 1987). The environmental input for the person is seen as stimuli and is classified in three categories: 1) focal; 2) contextual; and 3) residual. Persons and their mechanisms to cope with stimuli are viewed broadly as regulator and cognator subsystems. The regulator subsystems coping mechanisms are automatic and occur through neural, chemical, and endocrine coping processes, which are personified primarily through the physiological response mode. More specifically, the regulator subsystem reacts to stimuli from the internal and external environment and channels an automatic, unconscious response so that physiological responses occur. However, the regulator subsystem can also initiate responses through the other adaptive modes by the same process (Andrews 8 Roy, 1986). The cognator subsystems coping mechanisms, on the other hand, occur through cognitive-emotive channels and represent the thinking and emotional responses of the person to internal and external stimuli. These cognitive-emotive channels unique to this subsystem include: perceptual/information processing, learning, judgment, and emotion. Included in the perceptual/information processing channel are: attention, coding, and memory. Through the 19 person's emotions, defenses are used to seek relief from anxiety and to make affective appraisal, and attachments (Andrews 8 Roy, 1986). The coping mechanisms that are processed through the two subsystems are not directly observable. They are, however, eventually observed through one of four adaptive modes: 1) physiologic, 2) self-concept, 3) role function, or 4) interdependence. These adaptive modes are the channels through which the responses of the broader subsystems' mechanisms can be observed or manifested in the person. According to Roy (Andrews 8 Roy, 1986) it is through these four major categories that responses are carried out and through which the person's adaptation level can be observed and assessed. For this study only the physiological mode will be addressed. The physiological mode according to Roy (Andrews 8 Roy, 1986) is defined as the manner in which a person manifests her/his physiological activity. The basic underlying need of the physiological mode is physiological integrity. Integrity is defined by Roy as the degree of wholeness achieved by adapting to change (stimuli) or changing needs. Adaptive mode responses are dichotomous, that is to say, they are either adaptive or ineffective (maladaptive). According to Roy, those responses that are adaptive contribute to growth, survival, reproduction, and self- mastery. Ineffective or maladaptive responses, on the other hand, do nothing to promote these on going purposeful 20 functions. The nurse using this framework and the nursing process, assists the client by promoting adaptive responses to facilitate health and wellness (Chinn 8 Jacobs, 1987). This framework, therefore, was chosen to analyze the stated problem in this study because it offered a framework by which reproductive physiologic integrity could be assessed. Application Of Conceptual Framework To The Study Using Roy’s conceptual framework the problem as stated will be analyzed» and the concepts inherent in the problem defined according to Roy's conceptual framework. A model of the conceptual framework of the study is provided in Figure 1 which shows the relationship of the study concepts to Roy's conceptual framework. Those concepts of the study to be defined using Roy’s framework include: persons, environment, health, considered to be reproductive physiologic integrity, and the nurse. Persons for the purposes of this study will be couples, (in their reproductive years) with perceived maladaptation in their reproductive integrity, that are seen as adaptive systems as described by Roy (1984). The concept of persons (couples) as adaptive systems will be utilized in this study to more adequately investigate couples and their adaptation to threats to their reproductive physiologic integrity from stimuli in the environment. The environment, according to Andrews and Roy (1986, p. 7), is viewed as "the world within and around the person" or 21 Figure 1 Conceptual Model 6‘ “M- d“. g. b". I 9 s, 8”” rs“ see-as; 1‘.“ g. .Q “0"“: 0:.‘ng a. .Q 8"“ .09. 257.235 9.300 sun... 01> ass... on: mac: =35..m EceEceafcu I 22 more broadly defined, as "all conditions, circumstances, and influences surrounding and affecting the development and behavior of persons or groups" (Roy, 1984, p. 13). In this study, the internal environment consists of the presence or absence of bacterial vaginosis, and other co-infections in couples trying to achieve a pregnancy. Bacterial vaginosis is an internal environmental condition that develops in the couple as a result of exposure to certain external environmental focal stimuli. These stimuli are those organisms thought to be causative to the condition known as bacterial vaginosis. In the same regard, Ureaplasma urealyticum is a condition or state that exists as a result of the couple's exposure to the external focal stimuli (UU) present in the couple's environment. This is seen in Figure 1 as the environmental focal stimuli labeled bacterial vaginosis and Ureaplasma urealyticum. The adaptation level as the couples reproductive physiologic integrity is consistent with Roy’s (1984) definition of the term: "The condition of the person relative to adaptation, which helps to determine whether the response to a changing environment will be adaptive or :maladaptive (p. 4)". The focal stimulus is the degree of bacterial exposure that allows for a diagnosis consistent with bacterial vaginosis via gram stain and/or to a positive Ureaplasma urealyticum culture. Adaptive and maladaptive functioning is measured by sPerimcervical mucus penetration test (SCMPT) outcome. This 23 is seen in Figure 1 as either an adaptive diagnosis with reproductive physiologic integrity intact or as a maladaptive diagnosis with reproductive physiologic disintegration. Adaptive reproductive physiologic functioning results in excellent SCMPT scores, which would be consistent with maintaining reproductive physiologic integrity. If physiologic integrity is maintained by the couple, then normal fertility and conception can occur. Maladaptive reproductive physiologic functioning is also measured by SCMPT outcome. Maladaptive reproductive physiologic functioning is defined as a poor or fair score on the SCMPT. A poor or fair score on the SCMPT is thought to represent a potential for infertility. The nurse, according to Roy’s conceptual framework as adopted in this study, employs the nursing process to promote the couple's adaptation so as to maintain reproductive physiologic integrity. The first step of the nursing process, as described by Roy’s Adaptation Model, is the assessment of behavior. The behavior to be assessed in this study is the couple's perceived infertility, as evidenced by their failure to conceive after at least one year of unprotected coitus. This is seen in Figure 1 as the first step of the naxrsing process, which according to Roy is the nurse's first iJuiication of how the couple is managing to cope with their environmental changes (Andrews 8 Roy, 1986) . Second level assessment for the nurse in advanced Pracrtice, as seen in Figure 1, is the assessment of stimuli 24 from one's environment that may be influencing the observed behavior. In this study, the presence of bacterial vaginosis and/or Ureaplasma urealyticum are assessed for their potential influence on the couple's perceived infertility. The couple's reproductive physiologic coping mechanisms, given the presence of bacterial vaginosis and/or Ureaplasma urealyticum is measured by the couple's sperm cervical mucus penetration test outcome. Therefore, the nurse using the skills of scientific inquiry, observes measures and records behavioral responses under the condition proposed, i.e., the presence and absence of bacterial vaginosis and/or Ureaplasma urealyticum, and compares those responses observed to preestablished normal values. In the third step of the nursing process the nurse employs nursing judgments and formulates nursing diagnoses, as seen in Figure 1, about whether or not these behaviors observed under certain conditions or circumstances are adaptive or maladaptive. At the same time the nurse determines what stimuli are associated with what behaviors. Identification of those stimuli or circumstances that are most often associated with ineffective or maladaptive behaviors hold the key to the accomplishment of the nurses goal of promoting adaptation. Summary This chapter described Roy's conceptual framework and discusses those concepts inherent in the model. The 25 framework was then applied to the study's stated problem with a model offered showing the relationship of the study's major concepts used within the framework. The relationship of the model to Roy’s conceptual framework was also exemplified. CHAPTER III Literature Review Sheen/is! In this chapter the literature is reviewed and critiqued. Those studies supportive to the concept of bacterial vaginosis as a sexually transmittable infection are presented. In the same regard, those studies supportive to the concept of bacterial vaginosis (BV) as a pathogen are reviewed and summarized. The literature outlining and confirming the potential for bacterial vaginosis related organisms to ascend to higher structures in the female reproductive tract are also recapitulated. Finally those studies specifically related to the study of bacterial vaginosis and Ureaplasma urealyticum (UU), as they have been related to infertility are reviewed. In reviewing the literature specifically related to this study, it was found that no researcher to date has investigated the potential of a relationship between bacterial vaginosis and maladaptive sperm-cervical mucus interaction. A few researchers, however, have investigated the potential for bacteria to disrupt sperm survival in cervical mucus and the potential for bacteria to disturb sperm motility and penetration in-vitro. No study to date has specifically examined men or women with bacterial vaginosis for any possible adversity to sperm-cervical mucus interaction in couples experiencing infertility. 26 27 support for Bacterial vaginosis as a Pathogen The literature does support the concept of bacterial vaginosis as a shared infection between sexual consorts. Gardner and Dukes (1959) established many years ago the prevalence of organisms associated with bacterial vaginosis in male sexual partners of infected women. They found that 91 of 101 male consorts of infected women were culture positive for those organisms thought to be associated with bacterial vaginosis. Pheifer, Forsyth, Durfee, Pollock, and Holmes (1978) supported the idea of bacterial vaginosis as a sexually transmitted disease by documenting bacterial vaginosis related organisms by culture in 79% of the males studied who were sexual consorts of infected women. bacterial vaginosis has also been found to be associated with other sexually transmitted diseases (Josey 8 Lambe, 1976). Brown and associates (1984) conclude that the primary source of infection in the overwhelming majority of women is sexual intercourse. Secondly, there is the question of bacterial vaginosis's virulence or pathogenicity. Evidence to support the potential virulence or pathogenicity of bacterial vaginosis has only been recently investigated. Potential genital disorders suggested in the literature to be associated with bacterial vaginosis include: (1) premature rupture of the membranes (Gravett, Nelson et al., 1986; Minkoff et al., 1984); (2) premature labor (Gravett, Hummel et al., 1986; Martius et al., 1988; Morales et al., 1988); 28 (3) salpingitis (Eschenbach et al., 1988); (4) endometritis (Watts et al., 1989; Watts et al., 1990); (5) pelvic inflammatory disease (Paavonen et al., 1987); and (6) urinary tract infections (Josephson et al., 1988). These recent investigations stem from the theory that the increased concentration of various microorganisms found in the condition known as bacterial vaginosis may be more virulent than the number and type of organisms normally present in the vagina. For the female with bacterial vaginosis the increase in abnormal or pathogenic bacteria in the vagina is thought to reach a 100-1000 fold increase above the normal levels of flora. Eschenbach (1984) theorized that the increased numbers of bacteria present in bacterial vaginosis, and the polymicrobial nature of the condition is what causes the client with bacterial vaginosis to be susceptible to the spread or ascension of the infection beyond the vagina. The intravaginal concentration of Gardnerella vaginalis and Ureaplasma urealyticum are increased about loo-fold in women with bacterial vaginosis (Gravett, Nelson et al., 1986). There is also an increased prevalence of various vaginal anaerobes including, Bacteroides bivius, B. disiens, B. melaninogenicus, Peptostreptococcus, Peptococcus, Eubacterium, and a newly recognized species Mobiluncus (Blackwell et al., 1983; Spiegel et al., 1980; Spiegel, Eschenbach, Amsel, 8 Holmes, 1983; Spiegel, 1984; Taylor et al., 1982; Thomason et al., 1984). This increased 29 prevalence of anaerobes in women with bacterial vaginosis is thought to be a loco-fold increase from the prevalence of these organisms in the normal vagina (Gravett, Nelson et al., 1986). Therefore, there is not only an increase in the quantity of colonization of bacteria with bacterial vaginosis but a change in the variety of bacteria present in the vagina as well. This polymicrobial nature of bacterial vaginosis and the effectiveness of the patient's antibacterial immune mechanisms are thought to determine the client's vulnerability to the spread of this infection beyond the vaginal canal (Eschenbach, 1984; Wahbeh et al., 1984). Romero and colleagues (1987) outline the potential for the ascension of those bacteria associated with bacterial vaginosis into higher structures and describes the potential for the development of complications, when bacterial vaginosis is present in pregnancy. Their review and supportive evidence for the possibility of cervical invasion of these organisms associated with bacterial vaginosis certainly lends support to this investigation. Silver and . associates (1989) in their study provide evidence that link bacterial vaginosis to intraamniotic infection which also lends support to the concept of ascension of bacterial vaginosis related organisms into higher anatomic structures. Tan and associates (1987) were the first to introduce the concept or link between increased numbers of bacteria in 30 the cervix and sperm-cervical mucus hostility. They studied cervical mucus hostility and found that cervical mucus hostility was associated with increased total bacterial counts. They, however, only examined facultative or normal flora organisms for the vagina and cervix. Tredway and colleagues (1985) were also interested in the potential for bacteria in the cervix to interfere with sperm motility and survival. They examined common in vivo postcoital test outcomes within vitro sperm cervical mucus penetration test outcomes in an attempt to recheck the correlation between these two diagnostic evaluation tools. Concurrently Tredway determined that the presence of Ureaplasma urealyticum had no effect or association with adverse SCMPT and/or adverse postcoital test outcomes. In a similar study, Eggert-Kruse and associates (1987) examined the influence of microbial colonization of various. microorganisms on sperm-mucus interaction in vivo and in vitro. They found no marked differences in the different parameters evaluated by postcoital test and sperm meter penetration tests, except for a reduction in sperm density and a higher percentage of sluggish motility in semen samples colonized with potentially pathogenic anaerobes (i.e., Bacteroides sp., Gardnerella vaginalis, peptostreptococcus, and peptococcus). The findings of their investigation with respect to Ureaplasma urealyticum were also consistent with that of Tredway et al. (1985) in that no significant difference could be demonstrated for 31 Ureaplasma urealyticum and adverse postcoital test or sperm meter penetration test outcome. This may indicate that Ureaplasma urealyticum in and of itself does not influence sperm-mucus interaction. The importance of those anaerobes associated with bacterial vaginosis for adequate sperm penetration or sperm function is questioned by Eggert-Kruse and colleagues (1987) secondary to the significantly higher rate of cultivation of these organisms in the group with inadequate sperm penetration in vitro. Eggert-Kruse and associates (1987) suggest that more work be done to further substantiate their findings. The literature review to this point has outlined those studies that lend support to the concept of bacterial vaginosis as a shared polymicrobial condition for the couple afflicted. It, too, has been shown through the above literature review that those organisms associated with bacterial vaginosis are capable of ascending beyond the vagina to higher structures of the female reproductive tract, and have been significantly associated with adverse afflictions of these structures (i.e., endometritis, premature labor, PROM, cystitis, PID, and salpingitis). It is theorized that the syndrome of bacterial vaginosis may be capable of adverse influences in the environment of the vagina, endocervix, uterus, fallopian tubes, and pelvis. Therefore, the evidence in the literature on how the syndrome of bacterial vaginosis may cause this adverse 32 influence once established in these environments will now be reviewed. Mechanisms By Which Bacterial Vaginosis May Affect Sperm Mucus Interaction There is literature to support an association between bacterial vaginosis and prostaglandin synthesis. Bejar, Curbelo, Davis, and Gluck (1981) in their study documented high phospholipase A-2 activity for Corynebacterium vaginalis (Gardnerella vaginalis), peptostreptococcus, bacteroides fragilis, and other anaerobes typically associated with bacterial vaginosis, through culture studies and phospholipase A-2 assay testing. It is theorized, therefore, that these anaerobic microorganisms are capable of phospholipase A-2 activity which liberates free arachidonic acid, and thereby allows for prostaglandin synthesis (Bejar et al., 1981; Fingold 8 Sbarra, 1987). It is apparent from the current male reproductive literature that appropriate balancing of prostaglandins is important in maintaining human reproduction. Prostaglandins and their appropriate balancing in the reproductive tract are thought to play a vital role in human reproduction, including the regulation of sperm motility, sperm penetration into cervical mucus, and the sperms capacity to penetrate the oocyte (Aitken 8 Kelly, 1985; Gottlieb, Savanborg, Eneroth, 8 Bygdeman, 1988; Savanborg, Gottlieb, Bendvold, 8 Bygdeman. 1989). Ft 33 More recently, Sjoberg and Hakansson (1991) evaluated vaginal discharge of 28 females for the presence of endotoxins that are capable of activating various systems in the body (e.g. coagulation, fibrinolysis, complement activation, or prostaglandin synthesis). For the 19 women in the sample diagnosed with bacterial vaginosis the mean amount of endotoxin per milligram of absorbed vaginal fluid was significantly higher than for the women in the control group. Mean level in the group with bacterial vaginosis was nearly 40 times that found in the controls. These endotoxins are known to be produced by gram negative bacteria such as Gardnerella vaginalis and bacteroides species which are-associated with bacterial vaginosis. Endotoxins (i.e., those toxins within the body of bacteria namely large polysaccharides, proteins) are typically those substances of bacteria that initiate an immune response during genital infection. During genital infections, according to Soffer, Ron-El, Golan, Herman, Caspi, and Samra (1990), the complement system becomes activated which results in smooth muscle contraction and increased vascular permeability through the release of histamine from mast cells. Thereby, complement activation results in extravasation of spermatozoa into the interstitium, lymph vessels, or blood capillaries, and subsequent transfer of sperm properties to regional lymph nodes where sperm antibody formation may be initiated. Their study may possibly explain another avenue by which 34 bacteria may create a hostile environment for sperm in the reproductive track of the female. Ureaplasma Urealyticum As with bacterial vaginosis, the literature has limited information that supports the theory of a pathogenic role for Ureaplasma urealyticum (UU), with respect to infertility. Most studies review Ureaplasma urealyticum for its relationship to adverse pregnancy outcome. Birth weight lower than 2500 gram, birth before 36 weeks gestation, and chorioamnionitis have all been found to be associated with Ureaplasma urealyticum (Kundson, Driscoll 8 Monson, 1984; McCormack, Rosner, Lee, Munoz, Charles 8 Kass, 1987). This evidence of an association between Ureaplasma urealyticum and adverse pregnancy outcome however is not without dispute (DPhill, Alexander, Weinstein, Lewis, Nash, 8 Sim, 1983). An association between spontaneous pregnancy loss and Ureaplasma urealyticum has also been established by Quinn, Shewchuk, Shuber, Lie, Tyan, Shey, and Chipman, (1983); Quinn, Shewchyk, Shyber, Lie, Ryan, Chipman, and Nocilla, (1983). Quinn and his associates also raised questions regarding whether or not certain serotypes of Ureaplasma urealyticum are more pathogenic than others, and demonstrated that certain varieties (or serotypes) of Ureaplasma urealyticum were more often associated with pregnancy loss than others. Gravett and Eschenbach (1986) also suggest in their review of the literature related to bacterial vaginosis that the relationship between Ureaplasma 35 urealyticum and bacterial vaginosis can not be over locked by researchers. They, too, recommend that more research be done to explore whether or not the coexistence of bacterial vaginosis and Ureaplasma urealyticum represents a greater pathogenic state than the mere presence of either syndrome or organism alone. Furthermore, it was the opinion of these researchers that the combination of these organisms together may represent the greatest threat to maternal and infant mortality and morbidity. Given the findings of Quinn, Shewchuk, Shuber, Lie, Ryan, Chipman, and Nocilla, (1983) one might also wonder whether or not there is a certain serotype of Ureaplasma urealyticum that commonly coexists with bacterial vaginosis that is more virulent, since the literature does offer some evidence of a pathogenic role for bacterial vaginosis and Ureaplasma urealyticum. Currently, there is controversy in the literature regarding Ureaplasma urealyticum and infertility. Few authors support the role of Ureaplasma urealyticum as a pathogen to sperm (Swenson, Toth, 8 O'Leary, 1979; Toth 8 Lesser, 1982). These authors report that selective antibiotic therapy for males with Ureaplasma urealyticum was associated with improved, forward progression (Swenson et al., 1979), motility, quantity, quality and the percentage of oval and small forms of sperm (Toth 8 Lesser, 1982). Conversely, other researchers dispute these findings that support the concept of Ureaplasma urealyticum being a pathogen. In 1984, Gump, Gibson, and Askikasa followed and 36 reviewed case histories of 205 females from couples with infertility of at least one year's duration. Their findings did not support any positive correlations between the finding of Ureaplasma urealyticum and the following: 1) history or evidence of pelvic inflammatory disease; 2) cervical inflammation; 3) numbers and motility of spermatozoa on postcoital test; 4) pyosemia; 5) quality of cervical mucus; and 6) the occurrence and outcome of subsequent pregnancy. Thus, their study did not support a role for genital mycoplasmas in the cause of infertility according to the researchers. Similarly, Shalhoub, Abdel-Latif, Fredericks, Mathur, and Rust (1986) found no effect on sperm function, as assessed by seminal fluid analysis, when Ureaplasma urealyticum was isolated. They, too, found in vitro penetration of bovine cervical mucus and hamster sperm penetration assays to be free of any ill effect when Ureaplasma urealyticum was found. Furthermore, Hellstrom, Schachter, Sweet, and McClure (1987) studied males with positive urethral Ureaplasma urealyticum cultures and normal controls seeking vasectomy and found no correlation between the prevalence of genital Ureaplasma urealyticum and infertility. This premise was based on the fact that they found the same or greater prevalence of Ureaplasma urealyticum among the controls as the infertile group. Lumpkin, Smith, Coulam, and O'Brien (1987) also dispute the concept that Ureaplasma urealyticum, by virtue of a mere 37 positive culture in the male, plays any kind of independent role in male factor infertility. In this study by Lumpkin and associates, 24 males donating semen for artificial insemination were cultured for Ureaplasma urealyticum. The women inseminated with these Ureaplasma urealyticum positive and negative specimens were then followed for 10 pregnancy outcomes, with no differences found between the control and treatment group. Neither did these investigators find any significant difference between groups with respect to sperm count, grade, or motility. Eggert-Kruse and colleagues (1987) also found no relationship to exist between in vitro sperm penetration testing and the presence of Ureaplasma urealyticum in infertile couples. Eggert-Kruse, Hofmann, Gerhard, Bilke, Runnebaum and Petzoldt (1988) further explored a possible~ link between Ureaplasma urealyticum and sperm-mucus ‘ interaction and experimented with infertile couples who had no symptoms of lower genital tract infection to see if antimicrobial therapy would alter sperm-mucus interaction. Slight improvements were found in penetration distance, sperm density, quality and duration of motility for that group with very poor sperm penetration mucus test results prior to treatment. This difference, however, was not statistically significant. One other controversy that warrants review concerns the concept of Ureaplasma urealyticum as a facultative and commensal organism. Historically Ureaplasma urealyticum has ho DUI 38 been identified with Dienes and Edsole (McCormack et al., 1973) the first scientists to grow the pleuropneumonia-like organism from a bartholin abscess. Mycoplasma organisms were first recognized in veterinary medicine. Mycoplasma mycoides was the first mycoplasma to be isolated and is well recognized in veterinary medicine as the causative agent in a contagious bovine pleuropneumonia since 1898 (McCormack et al., 1973). Since 1898, mycoplasmas have been found to be common inhabitants of the oropharyngeal and genital mucous membranes of humans. Currently there are eight recognized species of human mycoplasmas. Mycoplasma hominis and Ureaplasma urealyticum (previously called T-mycoplasma) being the principal mycoplasmas isolated from the human genital tract. These two species in humans being the focus of much research in the past two decades. Epidemiologically, humans can be colonized with M. hominis and Ureaplasma urealyticum at birth. Theoretically, the organisms are acquired in the birth canal, with infants born by cesarean section found to be less colonized with these organisms than infants born vaginally. Infants, followed sequentially during their first year of life, were found to progressively decrease their colonization of M. hominis and Ureaplasma urealyticum as they matured (McCormack et al., 1973). _ Repopulation of the genital track in humans by M. hominis and Ureaplasma urealyticum is thought to occur after puberty with the onset of sexual contact (McCormack et al., 39 1973). McCormack et a1. (1973) also report that women without sexual maturity have only negligible rates of colonization with Ureaplasma urealyticum. Conversely, 37% of women with one sexual partner are colonized with these organisms. Seventy five percent of women with a history of three or more sexual partners are also found to be colonized with Ureaplasma urealyticum. M. hominis is generally less prevalent but tends to follow the same general trends as far as genital isolation is concerned (McCormack et al., 1973). A review of the literature from the late 1960's and the early 1970's by McCormack and colleagues (1973) points out that Ureaplasma urealyticum was reported more than half the time in studies where nonspecific vaginitis (BV) was diagnosed. Ureaplasma urealyticum, however, was as prevalent in women with nonspecific vaginitis (BV) as their matched normal controls in early studies, which has implied that Ureaplasma urealyticum is a commensal organism incapable of hurting the organism it lives with (i.e., the human body). The matched normal controls in these early studies, however, were most likely sexually active. Therefore, one can not assume from the literature available that Ureaplasma urealyticum is commensal. In the same regard, these early studies did not employ precise clinical diagnostic criteria to diagnose bacterial vaginosis for those women studied. Therefore, it becomes impossible to determine what true percentage of women studied in the early literature were normal and what 40 percentage had bacterial vaginosis. Furthermore, it must be understood that the rate of occurrence is only one factor to be considered. More important than the mere presence of Ureaplasma urealyticum is the issue of the degree of colonization of Ureaplasma urealyticum in the female tract, and perhaps the variety (or serotype) of Ureaplasma urealyticum present in the individual. The degree of colonization of Ureaplasma urealyticum and/or the serotype of Ureaplasma urealyticum, along with its possible coexistence with other organisms or syndromes like bacterial vaginosis, in the human genital tract appears from the literature to be the most important consideration. critique] Implications The literature as reviewed certainly lends support to the aim of this study in that the pathogenic nature of bacterial vaginosis or its associated anaerobes is questionable for some subgroups of patients. The inability of early researchers to appropriately identify bacterial vaginosis infected groups and their failure to identify coexisting infection with bacterial vaginosis and Ureaplasma urealyticum for those samples studied is also made clear. This lack of appropriate organism identification in the study samples makes accurate interpretation of the early literature difficult. More recently, with strict clinical criteria in place and improved methodology for anaerobic screening, researchers have begun to clarify more accurately the 41 association between bacterial vaginosis, anaerobes, such as, Ureaplasma urealyticum. Researchers just recently recognized the need for controlling for bacterial vaginosis independently and/or its connection with other associated organisms such as Ureaplasma urealyticum. For the future, researchers must continue to clarify what if any association exists between Ureaplasma urealyticum and bacterial vaginosis using strict diagnostic criteria for subject placement into treatment and control groups. Future work must examine whether or not bacterial vaginosis, with or without Ureaplasma urealyticum present, influences sperm-mucus interactions so that cause and effect studies can be justified. In this study, for the first time, bacterial vaginosis will be used as an independent variable and rigorously diagnosed. In a non-experimental study sperm-cervical mucus interaction responses will be examined in infected and noninfected infertile couples. The design of this study will allow a comparison of the effects of bacterial vaginosis with and without Ureaplasma urealyticum on sperm cervical mucus penetration test score outcome. This will allow the researcher to determine whether or not Ureaplasma urealyticum is significant only when it coexists with bacterial vaginosis when examining sperm-cervical mucus interaction responses in infertile couples. 42 Summary This literature review has examined the history of bacterial vaginosis and its association with recognized genital mycoplasmas. Gardner has been identified as the first to identify the condition as something other that nonspecific vaginitis. The classification of those organisms associated with bacterial vaginosis has been traced and examined, so that a greater understanding and appreciation for the syndrome now known as ”Bacterial Vaginosis” can be grasped by the reader. The relevant controversies that have surfaced regarding a probable symbiotic relationship between G. vaginalis, and other anaerobes has also been outlined. Relevant research has been reviewed that documents current evidence of the pathogenic nature of bacterial vaginosis and/or its related mycoplasma Ureaplasma urealyticum. In the review of the literature relevant to Ureaplasma urealyticum, it was established that these bacterial organisms are frequently present in the condition known as bacterial vaginosis and among sexually active adults. The question of the accuracy of early work that established associations between bacterial vaginosis and Ureaplasma urealyticum is also raised secondary to the limited clinical criteria used at that time to diagnose bacterial vaginosis. Four studies are offered (Tredway et al., 1985; Tan et al., 1987; Eggert-Kruse et al., 1987, 1988) as evidence that 43 no influence has been documented for Ureaplasma urealyticum alone having an independent influence on sperm-mucus interaction. However, the study by Eggert—Kruse (1987) does suggest that further study with respect to bacterial vaginosis and sperm-mucus interaction may be justified. CHAPTER IV Methods and Procedures marries This chapter includes an explanation of the methods and procedures applied in this study. Major variables are operationally defined, and the research hypotheses stated. The sample is described in detail, along with the instruments to be utilized. Furthermore, the data collection procedures, the setting for data collection, and the type of data analysis to be used are outlined. The provisions for agency consent, and human subjects review, along with a copy of the consent form to be used are described. Background An established theory exists regarding the condition known as Bacterial vaginosis (BV). This theory concludes that in the condition known as bacterial vaginosis there are increased concentrations of various micro-organisms that may be more virulent than the normal flora of the vagina (Eschenbach, 1984; Romero, et al., 1987; Tan, et al., 1987). It is also generally accepted that those organisms associated with bacterial vaginosis are capable of ascension into the cervix and higher structures of the female reproductive tract. Therefore, it is purported in this study that the presence of bacterial vaginosis and its component organisms in the cervix may create a hostile environment for the sperm. 44 45 The purpose of this study was to describe which differences, if any, could be produced by bacterial vaginosis and/or Ureaplasma urealyticum (UU) in sperm- cervical mucus interaction, as measured by the sperm- cervical mucus penetration test (SCMPT). This study also described the incidence of bacterial vaginosis and Ureaplasma urealyticum in the sample and explored the variation among the four groups as defined by two independent variables (presence of bacterial vaginosis and Ureaplasma urealyticum) on one dependent variable (SCMPT score). Methods The study utilized a nonexperimental design. That is to say, the independent variables, bacterial vaginosis and Ureaplasma urealyticum were present in the sample as they occurred naturally. Four groups of subjects were identified as follows: 1) subjects who had bacterial vaginosis only; 2) subjects who had Ureaplasma urealyticum only; 3) subjects who had both bacterial vaginosis and Ureaplasma urealyticum; and 4) subjects who had neither bacterial vaginosis or Ureaplasma urealyticum. Qpe;gtigngl_§efiigigign§ V ' os' . Bacterial vaginhsis was defined as the presence of three of the four criteria established by Amsel, Totten, Spiegel, Chen, Eschenbach, and Holmes (1983) to justify the diagnosis of bacterial vaginosis. Those criteria were: 1) homogeneous, white/gray, vaginal discharge; 2) vaginal fluid pH > 4.5; 3) a fishy odor on 46 addition of potassium hydroxide to a vaginal discharge wet mount; and 4) the presence of clue cells, vaginal epithelial cells coated with coccobacillary organisms. Confirmation of this diagnosis was by gram stain for any woman who presented with any three of the four criteria listed above. The absence of bacterial vaginosis was defined as any vaginal condition that revealed fewer than three of the four possible criteria.. The characteristic features found on the gram-stained smear of vaginal material from women with bacterial vaginosis was a predominance of small gram-variable rods, and only a few large gram-positive rods or white blood cells (Spiegel et al., 1983). The results of the gram stain that was considered consistent with the diagnosis of or the presence of bacterial vaginosis therefore, was one that showed a heavy mixed anaerobic type flora with, curved, variably staining gram negative rods. This confirmatory gram stain was reported from the laboratory as zero to two white blood cells and a mixed flora consistent with a diagnosis of bacterial vaginosis, i.e. curved variably staining gram negative bacilli seen (Mobiluncus morphotype). The presence of bacterial vaginosis in the male subjects for the purpose of this study was defined as the presence of any of the following anaerobic organisms found through prostatic fluid anaerobic culture: 1) Bacteroides bivis; 2) B. disens; 3) B. melaninogenicus; 4) .pt 47 Peptostreptococcus; 5) Peptococcus; or 6) Eubacterium (Spiegel, 1987; Spiegel et al., 1980). Ureapla§m§_gzgalytigum. Ureaplasma urealyticum was defined as the presence of the organism on culture for any subject. That is to say, those women and men studied were placed in the Ureaplasma urealyticum positive group if the 10B broth turned cherry red and the A7 agar grew Ureaplasma urealyticum colonies that were observable upon microscopic analysis as dark gold or deep brown colonies. Testing procedures were as recommended by The Manual of Clinical Microbiology (1986) and as followed and reported by Cascade Medical Laboratory. 5 e t t' s t . Sperm cervical mucus penetration test outcome (SCMPT) was defined according to three criteria: 1) linear penetration of the mucus by the sperm in centimeters; 2) density of sperm penetration or the number of sperm penetrating; and 3) the percentage of the total sperm penetrating or having progressive motility. A composite score was derived for each couple from the three criteria and scored according to Table 1. The range of scores was from 0-9, with 0 being the worst SCMPT score and 9 being the best score meaning good cervical mucus penetration. 48 Table 1 g.! . E . 5321 Score Criteria 0 1 g:g_____; Linear penetration (cm) 0 0-2 3-5 >5 Density of penetration 0 1-10 11-50 >50 (no. of sperm) Progressive motility 0 25% 26-50% >50% A Hete. Cumulative score interpretation: 7-9 excellent; 4-6 fair; 1-3 poor; 0 negative. From "Correlation of postcoital evaluation with invitro sperm cervical mucus determinations and ureaplasma cultures" by D- R- Tredway et 31-: 1985. W. 5.3.. p. 287. 49 Research Hypotheses The research questions of interest in this study were: I. Are couples with bacterial vaginosis more likely, than couples without bacterial vaginosis, to have lower SCMPT scores? II. Are sperm cervical mucus penetration test scores more likely to be lower in couples with bacterial vaginosis, when one or both partners are also positive for Ureaplasma urealyticum. Therefore, the following research hypotheses, stated in alternate form, were formulated: I. The composite sperm cervical mucus penetration test score among couples with bacterial vaginosis is lower than among couples without bacterial vaginosis. II. There is a difference in composite sperm cervical mucus penetration test score outcome between those couples with only Ureaplasma urealyticum and those without Ureaplasma urealyticum and without bacterial vaginosis. III. There is a lower composite sperm cervical mucus penetration test score among couples with bacterial vaginosis and Ureaplasma urealyticum combined, compared to couples with either bacterial vaginosis or Ureaplasma urealyticum alone. Sammie The sample consisted of 25 couples seen for their first infertility evaluation and care from September 1, 1990 to April 1, 1990 at a center for reproductive care in west 50 Michigan. The initial selection criteria for the subjects was a history of failure to achieve pregnancy, after one or more years of unprotected coitus. Data collection was restricted to seven months with the number of subjects accrued at that point utilized for data analysis. Subjects were self-selected after informed consent (Appendix A) which was reviewed with each couple seeking assistance from the reproductive center at their intake consultation. After informed consent, couples were instructed on scheduling sperm cervical mucus penetration test testing, and the use of a home urinary luteinizing hormone kit. The luteinizing hormone kit was used to help identify the periovulatory period so that sperm cervical mucus penetration test (SCMPT) testing could be done during this period. Couples were then assigned to one of four groups for data analysis depending on the results of their preliminary tests for the presence of mycoplasmas, gonorrhea, chlamydia, syphilis, bacterial vaginosis or any vaginitis. Classification or group assignment was therefore determined by those infections identified for each subject. Subjects with gonorrhea, chlamydia, and syphilis were excluded from SCMPT testing when encountered. Setting The site for data collection was a private ambulatory care office that services infertile couples, who by definition have been unable to achieve a pregnancy for a minimum of one year's duration. Laboratory tests for this 51 study were performed at a licensed medical laboratory, which is a specialty lab that performs those specialty laboratory studies deemed necessary by the providers of the reproductive center. These facilities were located in western Michigan and service couples from all of west Michigan and beyond. Full cooperation and consent for this study was obtained from the director of both facilities. We W- A detailed assessment of sperm-cervical mucus interaction was accomplished using an in-vitro sperm-cervical mucus penetration test. The capillary tube test was the instrument used in this study labeled as the sperm cervical mucus penetration test (SCMPT), as described by Tredway et al., (1985). This sperm-cervical mucus penetration test as modified by Tredway (1985) was similar to the sperm penetration meter test designed by Kremer (1980) and recommended by the World Health Organization. The test was simplified by Tredway (1985) so that Kremer's parameters which were classified as migration reduction and duration of the progressive movements were incorporated and simplified to represent progressive motility. The progressive motility for this study was measured at 30 minutes, according to WHO standards, and as utilized by Eggert-Kruse et al., (1987, 1988) and Tredway et al., (1985). The reliability of this instrument, or the degree of consistency with which it measured the attributes it was 52 supposed to measure, has not been documented in the literature but its extensive use and consistency at identifying suspected disorders of sperm-mucus interaction can be cited (Alexander, 1981; Blasco, 1984; Eggert-Kruse et al., 1987). The only type of reliability testing utilized in those studies found in the literature was the cross test (x-test), which is primarily repeated measurement. The X- test utilized donor sperm of good quality and donor mucus of good quality which are used simultaneously to check the results found in test subjects. Specifically the sperm penetration meter test is thought to reliably predict mucus quality and functional sperm quality. Broadly stated, this measurement tool should give the fertility prognosis of the infertile couple. When compared to the huhner's test, in which positive values reveal a cumulative conception rate of 84% and when negative values indicate a 16% conception rate, the SCMPT was found to be significantly correlated with the huhner's when both measures were dichotomized as good (or positive) and poor (or negative), according to Tredway and colleagues (1985). Furthermore, the huhner's and the SCMPT were found to agree on the findings in 87% of the cases studied for sperm-mucus interaction. The validity, or the degree to which the instrument measures what it is supposed to be measuring, of the capillary tube test has been documented by Alexander, (1981), Blasco (1984), and Eggert-Kruse et al., (1987), and 53 found to be superior to sperm analysis alone with regard to fertility prognosis. Comparisons have also been made between the in vivo postcoital test (the huhner's test) and the sperm-cervical mucus penetration test (or sperm penetration meter test) for concurrent validity checks with good results (Eggert-Kruse et al., 1987, 1988; Tredway et al., 1985). These studies provide give concurrent validity checks to this instrument by simultaneously running a cross- test (x-test) of the capillary tube test using donor sperm and donor mucus for quality control. Q;am_§tgin. A gram-stained vaginal smear was used to confirm the diagnosis of bacterial vaginosis when at least three out of the four clinical criteria for bacterial vaginosis were manifested by the female subject. The gram- stained vaginal smear can be used to diagnose bacterial vaginosis by identifying the extracellular bacterial flora associated with this condition. Those smears considered normal were those with a semi-quantification scheme, and a 3-4+ Lactobacillus morphotype (large gram-positive rods) predominance (Eschenbach, 1984). The characteristics of the gram-stained vaginal smear that are diagnostic for bacterial vaginosis include indications of predominantly small gram- variable rods, with only a few large gram-positive rods or white blood cells (Spiegel et al., 1983). The reliability of this instrument has been tested by Spiegel et al., (1983) and found to have 100% specificity for diagnosing bacterial vaginosis. Eschenbach (1984) also 54 suggests the gram stain as a reliable instrument for the diagnosis of bacterial vaginosis because of its low intraobserver variability and low cost. The validity of the gram stain has been partially documented through studies that have found it to be highly sensitive (100%) at detecting bacterial vaginosis (Spiegel et al., 1983). Another group of investigators verified validity through a correlational study, and they found the gram stain to detect bacterial vaginosis in 90% of the samples that were positive by DNA probe (Roberts, Hillier, Schoenknecht 8 Holmes, 1985). Eschenbach and his colleagues (1988) also found the gram stain, through correlational research on 640 randomly selected females, to correlate better with the four clinical criteria of bacterial vaginosis, than the results of semi-quantitative cultures for Gardnerella vaginalis. Ureaplama_flrealytigum. Ureaplasma urealyticum testing was measured according to the medical lab protocol and procedure manual, which follows those recommendations of the Manual of Clinical Microbiology (1986). That is to say, moist cervical, and urethral cultures were taken with a sterile plastic polystyrene stick culturett from the cervical as or urethral as on the male. Semen samples were also used for culture, with 3 drops of semen added to the 10B broth from a sterile 1 ml. pipette, within 1-2 hours of its collection into a sterile semen cup through masturbation. A 10B broth (10B Lyophilized media) was used to presumptively identify 55 Ureaplasma urealyticum, with broths checked twice daily for 7 days while stored at 37 degrees centigrade in an incubator. Negative cultures were clear amber color and positive cultures cherry red in color. The reliability and validity of Ureaplasma urealyticum testing has been investigated by Kundsin, Parreno and Poulin (1978). These investigators state that the ultimate criterion for Ureaplasma urealyticum is the characteristic growth of the organism on agar, and that the most common problems with isolating Ureaplasma urealyticum involves four testing factors: 1) the type of specimen used; 2) the type of media used; 3) the conditions of incubation; and 4) the experience of the technician in identifying isolates. Therefore, according to Kundsin and colleagues (1978), given the type of specimens, the type of media, the conditions planned for. incubation, and the experience of the technicians to be used, one might expect a 9% failure to isolate Ureaplasma urealyticum in women and a 13% failure at isolating Ureaplasma urealyticum in males. These projected errors for the present study on bacterial vaginosis were primarily based on plans to utilize urethral, semen, and cervical swabbings rather than urine specimens, otherwise those procedures followed were as those recommended by Kundsin (1978). The error rate for the technician reading the colony growth, however, can not be estimated from the literature and was not determined through the course of the present study. This potential for error or the 56 misclassification of Ureaplasma urealyticum in men and women meant that differences between Ureaplasma urealyticum couples and non-Ureaplasma urealyticum couples was made smaller. WWW After obtaining informed consent, the couples received instructions for the SCMPT procedure. No examination or testing was done at the intake interview. The first task for the test subjects was to utilize a first response ovulation predictor kit. Testing for luteinizing hormone (LH) was self monitored by each couple at home according to kit instructions. More specifically, subjects were instructed to begin LH testing two days prior to the females shortest ovulation, according to reported menstrual cycle lengths. Ovulation was suspected by a subject reported LH surge but confirmed by documentation of a mature ovarian follicle (>16 mm diameter) by transvaginal ultrasound, which was done the day of SCMPT testing. The procedures for testing were as follows: I. Home urinary luteinizing hormone kit beginning two days prior to the earliest possible ovulation day of the menstrual cycle. II. Call office for appointment the day of the positive LH test and bring semen sample to office at scheduled appointment time. Semen specimen to be collected by masturbation no more than one hour prior to the appointment. 57 III. At appointment: A. female - 1. Transvaginal ultrasound - if follicular cyst > 16 mm preform speculum exam, if no follicular cyst observed cancel SCMPT. 2. Speculum exam - wet mount, gram stain, and pH taken from lateral vaginal wall. Endocervix Cultured for Ureaplasma urealyticum, and gonorrhea. Endocervical cells were collected for microtrack analysis with an endocervical brush for rule out of chlamydia. 3. Collection of mucus - portio vaginalis of the cervix was cleansed with a cotton swab remove the external pool of vaginal contaminants. Two specimens of endocervical mucus were collected by aspiration with a tuberculin syringe. 4. Blood sample - all females were tested for syphilis by RPR analysis. male - 1. Semen analysis - semen parameter were assessed by a licensed medical technologist using a Hamilton-Thorn M2000 and reported in concentration of total cells, motile cells, and percent of rapid motile cells, i.e. the percent of sperm with a pat velocity > 25 (see Appendix B). 2. Urethral swabs - cultures for Ureaplasma urealyticum and gonorrhea. Cell collection for Micro Track analysis for chlamydia. IV. VI. 58 3. Prostate exam - prostate massage performed on each male subject by the physician for the sterile collection and culture of anaerobic bacteria. 4. Blood test - RPR drawn for rule out of syphilis. The first specimen collected from the female was used for evaluation of cervical mucus quality and scored. Mucus scores could be as high as 15 with mucus scores dependent on volume, consistency, ferning, spinnbarkeit, and cellularity (see Appendix C). Subjects with mucus scores < 10 were excluded. Sperm cervical mucus penetration test - the second endocervical mucus specimen collected was loaded into a Tru-Trax for SCMPT testing. The mucus chamber of the Tru-Trax was filled with 0.2 cc of mucus. The semen reservoir was than filled with a drop of the semen specimen, taking care to avoid an air bubble at the mucus - semen interface. The Tru-Trax was incubated at 37 degrees centigrade in a covered petri dish, with damp sponges on the sides, to maintain humidity. After incubation for 30 min. the Tru-Trax was examined microscopically and evaluated according to the criteria noted in Table 1. After test results were available subjects were excluded if chlamydia, gonorrhea, or syphilis were 59 found. Remaining subjects were grouped according to test results: 1) those with bacterial vaginosis only; 2) those with Ureaplasma urealyticum only; 3) those with bacterial vaginosis and Ureaplasma urealyticum combined; 4) those with neither bacterial vaginosis or Ureaplasma urealyticum. Scaring One rater who had no knowledge of other test results was used for all SCMPT scoring. A second rater was used to check the reliability of the SCMPT scoring, by repeating the score every 6th SCMPT. W Descriptive statistics were completed to describe the sample according to the age of subjects and the number of years of infertility for each couple. Semen parameters, standardized according to WHO, on the male subjects were also determined and recorded. In the same regard female test subjects were assessed for ovulation parameters by ovarian follicle size (i.e., a mature follicle indicated by > 16mm diameter) and the presence of an LH surge. Test subjects were also scrutinized for the presence of anaerobes, chlamydia, syphilis, and gonorrhea so that the prevalence of infection in the sample could be determined. Data analysis of the research hypotheses was carried out through analysis of variance procedures (ANOVA), which allowed the researcher to test the significance of the differences between mean SCMPT scores for each of the four 60 groups. The variation between groups was then contrasted with the variation within groups to yield an F-ratio. The F-values were then used to establish the probability of the SCMPT score differences being merely the result of group selection processes. ANOVA therefore was utilized primarily to determine the main effect bacterial vaginosis and Ureaplasma urealyticum had on SCMPT score outcomes and the interaction effect that may exist for a combined infection of bacterial vaginosis and Ureaplasma urealyticum in test subjects evaluated by SCMPT. Furthermore, it was necessary to run specific contrast tests (one-way ANOVA) in order to test hypothesis II and III. Human Subjects The study was approved by the University Committee On Research Involving Human Subjects. A copy of the consent form and letter of approval from the human subjects review board are provided in appendix A. Summary In this chapter the methods and the procedures of the study were reviewed. Operational definitions of the major variables are given and research hypotheses are stated. The sample, too, was described along with the instruments utilized. Finally, the data collection procedures and planned data analysis were outlined. CHAPTER V Results IDLIQQEQEiQn This chapter includes a description of the results of the data analysis. First the descriptive statistics are presented, followed by the inferential statistics for testing the hypotheses. Tables are provided throughout to summarize the background data and to address the major hypotheses. Descriptive Data m1: During the seven month data collection period, only twenty eight couples were available to be approached concerning participation in the research study. Of these, three couples did not consent. The remaining 25 couples for the study were fewer than originally projected. This was probably related to the unforeseen absence of the physician at the center responsible for new client intake consultations. Of the 25 couples in the sample, women were between 26 and 41 years of age (§= 31.28 years), while men were between 28 and 48 years of age (i= 33.28 years). The couples in this sample had a 2 to 8 year history of infertility, and all of the women were nulliparous. Eligibility for participation in SCMPT testing for the women was determined by markers of ovulation i.e., follicular cyst sizes greater than 16mm and positive LH tests. There were 19 (76%) women 61 62 whose follicular cysts met the criteria and 20 (80%) women met the criteria LH tests the day SCMPT testing was attempted. Therefore, 19 women were eligible to proceed with the research protocol. Of these 19 women, however, only 14 had mucus scores greater than 10 allowing them to proceed with SCMPT testing. To control for semen parameters as an extraneous variable a complete semen analysis was done for each male on the day of the SCMPT testing. Semen parameters met WHO standards (see Appendix B) in 68% of the sample (17/25 males). Of these 17 couples that met WHO semen parameters, 16 of the wives met ovulation criteria and one had no demonstrable follicular cyst by ultrasound. Out of the total sample of 25 couples 11 did not complete SCMPT testing (see Table 2). In addition to the six couples who did not meet mucus overall criteria, five (20%) couples could not complete the research protocol for various reasons, such as, cost, difficulty getting to the office the day of LH surge, and having an LH surge on the weekend when the lab was unavailable. Therefore, the final sample for testing the hypotheses was 14 or 56 percent of the original sample. For those couples completing SCMPT testing (n=14) 13 males (93%) met WHO standards for semen analysis and all the females met the ovulation criteria. The couple that did not meet WHO semen parameters that completed SCMPT testing were included in the group without bacterial vaginosis or 63 Ureaplasma urealyticum. The only bacteria found, following completion of the research protocol, for this couple was peptostreptococcus. . In assessing those six couples with inadequate mucus scores, who were excluded from further study, it was noted that 4/6 (or 66%) had bacterial vaginosis by gram stain, with anovulation explaining the inadequate mucus score for the remaining two couples. This finding of a higher . prevalence for bacterial vaginosis in those couples found to have inadequate mucus scores prompted further analysis which revealed only a non-significant relationship (phi = 0.34, df = 17) between the presence of bacterial vaginosis and inadequate mucus score outcome. However, given the sample size, the power of this test is rather weak. The prevalence of bacteria in the initial sample and final sample is summarized in Table 3. The presence of bacterial vaginosis without coexisting bacteria was found in 12% of the total sample and in 7% of those couples completing SCMPT testing. Ureaplasma urealyticum had a prevalence of 16% for the total sample and was present in 28% of those couples completing SCMPT testing. Bacterial vaginosis and Ureaplasma urealyticum coexisted in only 16% of the total sample and coexisted in 14% of those couples who completed SCMPT testing. Chlamydia was present in 12% of the total sample, but was found in none of those 14 couples that completed SCMPT testing. Gonorrhea and syphilis were also tested for but not present in this 64 Table 2 W sunbeam Initial sample (couples) 25 100 Loss Inadequate mucus 6 24 Unable to complete protocol 5 20 Final sample 14 56 Table 3 f - . ‘lCT o, =a _- Ta i. _;e .7t'-. .1- S.u-.e Bacteria Initial Sample Final Sample (n= 25) (n= 14) n % n % Bacterial Vaginosis alone 3 12 1 7 Bacteria Vaginosis total 7 28 3 21 Ureaplasma Urealyticum alone 4 16 4 28 Bacterial Vaginosis 8 Ureaplasma Urealyticum 4 16 2 14 Chlamydia 3 12 0 0 Anaerobes 12 48 6 42 None 2 8 3 21 Unable to complete 5 20 4 28 No e. Chlamydia and anaerobes can coexist with BV and UU thereby the total percentage is greater than 100. 65 sample. The total sample was also scrutinized for the prevalence of Ureaplasma urealyticum in those known to be infected with bacterial vaginosis. For those couples diagnosed with bacterial vaginosis by gram stain (n=7), Ureaplasma urealyticum was found to coexist in four cases (57.1%). The presence or absence of those anaerobes typically associated with bacterial vaginosis (i.e., Peptostreptococcus, Bacteroides species, Peptococcus, 8 Eubacterium) were also examined to see if they were related to SCMPT score outcome. This was necessary, since 48% of the sample was found to have between one and six of these anaerobes present according to male anaerobic cultures (Table 4). There was a significant pearson correlation (r = -.3274, p < .001 df = 13) between SCMPT score outcomes and the number of anaerobes present indicating that as the number of anaerobes increased the SCMPT score decreased. Eight couples were found to have no anaerobes present after prostatic cultures were completed. There SCMPT scores ranged from five to nine with scores from 0 to 3 considered poor, 4 to 6 considered fair, and 7 to 9 considered excellent. Three couples had one anaerobe present and their SCMPT scores varied from 6 to 9. Two couples had two anaerobes present and their SCMPT scores were 3 and 7. One couple had six anaerobes present and had a SCMPT score of 4{ Therefore, even though the number of couples with anaerobes present were few in number there was a demonstrable tendency 66 or lower SCMPT scores among those with anaerobes present (Table 4). Hypothesis Testing The hypothesis derived for the study were tested on a sample of 14 couples completing SCMPT testing. Two-way analysis of variance was used to compare differences between group means on the dependent variable, SCMPT score outcome. 'The independent variables in the study, bacterial vaginosis status and Ureaplasma urealyticum status, were categorized into four groups: 1) those couples with bacterial vaginosis and Ureaplasma urealyticum; 2) those couples with bacterial vaginosis but without Ureaplasma urealyticum; 3) those couples with Ureaplasma urealyticum but without bacterial vaginosis; and 4) those couples with neither bacterial vaginosis or Ureaplasma urealyticum. Three hypotheses were tested in the study by means of analysis of variance: I. The composite sperm cervical mucus penetration test score among couples with bacterial vaginosis is lower than among couples without bacterial vaginosis. II. There is no significant difference in composite sperm cervical mucus penetration test score outcomes between those couples with only Ureaplasma urealyticum and those without Ureaplasma urealyticum and without bacterial vaginosis. III. There is a lower composite sperm cervical mucus penetration test score among couples with bacterial 67 vaginosis and Ureaplasma urealyticum combined, compared to couples with either bacterial vaginosis or Ureaplasma urealyticum alone. The mean SCMPT scores for each of the four groups: 1) those couples with bacterial vaginosis and Ureaplasma urealyticum; 2) those couples with bacterial vaginosis but without Ureaplasma urealyticum; 3) those couples with Ureaplasma urealyticum but without bacterial vaginosis; 4) those couples with neither bacterial vaginosis or Ureaplasma urealyticum, can be seen in Table 5. There was no significant difference between the mean SCMPT score for the group with bacterial vaginosis and the group without bacterial vaginosis (F = 2.75, df 1, 2), therefore Hypothesis I was not supported. There was also no significant mean SCMPT score difference between the group with Ureaplasma urealyticum and those without Ureaplasma urealyticum (F = 1.25, df 1, 5), nor in the interaction of the four groups (F = .811, df, 3, 13). Therefore, presence .or absence of infection is not, in this sample, associated with difference in outcome when analyzed as a whole. To accurately test hypothesis II it was necessary to run a specific contrast test (one-way ANOVA) that allowed for a comparison of mean SCMPT score outcome between the group with Ureaplasma urealyticum alone (if= 6.0) and the group without Ureaplasma urealyticum or bacterial vaginosis (§'= 6.57). This analysis revealed no significant difference (F = .5881, df 1) on mean SCMPT score between pl 68 Table 4 Co w t o t S o 0 e W r293; Fair Excellent 0 (n=8) 6 2 1 (n=3) 2 1 2 (n=2) 1 1 6 (n=1) 1 Total 9 4 Table 5 4‘ Hell 0 P ‘S‘ e 4-08‘ 0 $.10 U a s t'c lath milieu: 3With 3.5 6.0 4.33 m a (2) (1) <3) gWithout 6.00 6.57 6.36 H 5 (4) (7) (11) d) U U m m 5.17 6.5 5.93 (6) (8) (14) '69 these two groups and hypothesis II was thereby supported. Couples with Ureaplasma urealyticum alone and with bacterial vaginosis alone have the same mean SCMPT score. Hypothesis III was tested as a planned contrast. This contrast, tested the difference between the mean SCMPT score of a created combined group of couples with bacterial' vaginosis or Ureaplasma urealyticum alone (X'= 6.00, n = 5), compared to the group with combined bacterial vaginosis and Ureaplasma urealyticum (i = 3.5, n = 2). One-way analysis ’of variance revealed a significant difference between these two groups (F = 9.9 , p = .03, df 1). Therefore hypothesis III was supported. Couples with bacterial vaginosis and Ureaplasma urealyticum combined infections have significantly lower SCMPT scores than do couples with single bacterial vaginosis or Ureaplasma urealyticum infections. Summary Twenty eight couples were approached concerning participation in the research study. Of these, three couples did not consent. The study sample, therefore, was made up of 25 couples. The demographics of this sample and the particular characteristics (i.e., semen parameters and ovulation parameters) that were important to the study were reviewed. Five couples were unable to complete SCMPT testing, for various reasons and excluded from the sample as were six couples that could not complete SCMPT testing secondary to 7O inadequate mucus volume. A significant relationship was not found between bacterial vaginosis and mucus score outcome. The prevalence for bacterial vaginosis alone in the sample (n = 25) was 12% while 16% of the sample had Ureaplasma urealyticum alone. The percentage of the sample with a shared infection of bacterial vaginosis and Ureaplasma urealyticum was 16%. Anaerobes were also present in 48% of the sample they were significantly related to SCMPT score outcome. Three hypotheses were tested on the final sample of 14 couples, out of 25, that completed SCMPT testing. There were no significant differences on mean SCMPT score outcome for couples with bacterial vaginosis or Ureaplasma urealyticum and no interaction effect. Couples with Ureaplasma urealyticum alone were also not significantly different on SCMPT score outcome than couples without bacterial vaginosis or Ureaplasma urealyticum. Further contrast tests, however, did reveal statistical significance and difference between mean SCMPT score outcomes for couples with bacterial vaginosis and Ureaplasma urealyticum combined infection when compared to couples with single bacterial vaginosis and Ureaplasma urealyticum infections. CHAPTER VI Summary and conclusions In this chapter the study is summarized, the findings are interpreted and recommendations are made based on the findings. The implications of the findings for advanced nursing practice and research are also discussed. Conclusions are stated and discussed. Summary of The Study A retrospective nonexperimental descriptive study was conducted to examine and analyze the potential virulence or physiological maladaptation that may be associated with bacterial vaginosis alone or in combination with the organism Ureaplasma urealyticum, with respect to sperm- cervical mucus interaction. The variables in this study were bacterial vaginosis, as the primary independent variable, and Ureaplasma urealyticum and the outcome of the sperm cervical mucus penetration test (SCMPT), as the _ dependent variable. This investigation was based on the biological theory that with bacterial vaginosis there are increased concentrations of various micro-organisms which may be more virulent than the normal lactobacillus dominated flora of the normal vagina and cervix, thereby representing .a more hostile environment to the sperm (Eschenbach, 1984; Tan et al., 1987). The independent variable, Ureaplasma urealyticum, was primarily analyzed for any confounding effect on sperm mucus interaction secondary to an established link between 71 72 Ureaplasma urealyticum and bacterial vaginosis in the current literature (Bump 8 Buesching, 1988; Gravett, Nelson et al., 1986). For this study the problem of interest was: Are infertile couples infected with bacterial vaginosis and/or Ureaplasma urealyticum more likely to have adverse sperm- cervical mucus penetration test outcome than those infertile couples not infected? This problem was studied according to Roy's Adaptation Model. This framework allowing the couple to be viewed as an adaptive system, taking in input and processing it to produce responses that are adaptive or maladaptive. A maladaptive response as defined in this study is the state of infertility, or a lack of reproductive physiologic integrity. Defined by Roy (1984) ineffective or maladaptive responses are those that do nothing to promote the couples' or individual's purposeful functioning. Infertility, therefore, is conceptually defined in this study as a state of compromised fertility for the couple, which is physiologically represented in this study by a poor or fair SCMPT score outcome. A comprehensive review of the literature demonstrated that the study of bacterial vaginosis as biologically antagonistic to sperm-mucus interaction leading therefore to infertility, has been neglected in the empirical literature. There were only two studies documenting a possible association between bacterial vaginosis related anaerobes 73 and poor sperm meter penetration test outcome (Eggert-Kruse et al., 1987, 1988). The method of the study was non-experimental and correlational. Four groups were scrutinized for sperm -cervical mucus interaction, utilizing an in-vitro sperm cervical mucus penetration test. In other words, those . subjects studied were grouped according to infections as they occurred naturally. The four groups were, those with bacterial vaginosis and Ureaplasma urealyticum, those with bacterial vaginosis without Ureaplasma urealyticum, those with Ureaplasma urealyticum without bacterial vaginosis, and those without either bacterial vaginosis or Ureaplasma urealyticum. The study sample was comprised of 25 couples. The age range for the sample was between 26 and 48 years, with women ranging from 21 to 41 years and men from 28 to 48 years. The women in the sample were all nulliparous, and the couples had a 2 to 8 year history of infertility. The 14 couples completing SCMPT testing met WHO semen parameter standards and ovulation indicators as tested by LH surge and follicular cyst size. The attrition for the sample was 44%. The prevalence of bacterial vaginosis in the sample was 12% (n=3) and the prevalence of Ureaplasma urealyticum was 16% (n=4). Bacterial vaginosis and Ureaplasma urealyticum were found to coexist in 16% (n=4) of the total sample (n=25). For those couples testing positive for bacterial vaginosis (n=7), four were culture positive for Ureaplasma 74 urealyticum. The prevalence of anaerobes typically associated with bacterial vaginosis was 48% (n=12) in this sample. No significant relationship between the presence of these anaerobes and SCMPT score was found. The research questions on bacterial vaginosis and Ureaplasma urealyticum for SCMPT outcome were tested by analysis of variance with SCMPT score as the dependent variable and with the presence and absence of bacterial vaginosis and the presence and absence of Ureaplasma urealyticum as the grouping variables. There were no significant differences found on SCMPT score outcome between the groups by two-way ANOVA with respect to the presence or absence of either of these bacteria either alone or in combination. There was, however, a significant difference found between those couples with bacterial vaginosis and Ureaplasma urealyticum combined (n=2), when compared to those with bacterial vaginosis and Ureaplasma urealyticum alone (n=5). Discussion Given the effects of bacterial vaginosis reported in the literature, such as the association of bacterial vaginosis with prostaglandin synthesis and vaginal endotoxin activity, it was hoped that the presence of bacterial vaginosis alone or in combination with Ureaplasma urealyticum would be associated with a significant difference between group means on SCMPT score outcomes. But in this study, only the presence of bacterial vaginosis and 75 Ureaplasma urealyticum combined, when compared to bacterial vaginosis and Ureaplasma urealyticum alone, was found to be significantly associated with adverse SCMPT score outcome. The possible reasons for the two-way ANOVA finding of no significant differences between mean SCMPT score outcomes for those with and without bacterial vaginosis are probably the small sample size and potentially the sensitivity of the SCMPT. The sample size for those couples found to have bacterial vaginosis alone, was only three, which may be grossly inadequate to show a significant difference in mean SCMPT score outcome for that group with bacterial vaginosis present. The mean for a small sample size is less likely to be representative of the true population mean than the mean ofia large sample with any research data. The sperm cervical mucus penetration test (SCMPT) may also not be sensitive enough to show an effect for bacterial vaginosis on sperm migration. Possible reasons for sperm cervical mucus penetration tests to lack sensitivity are their short lengths of testing time (i.e., 30 min. to 2 hours) and their in-vitro nature. That is to say, sperm cervical mucus penetration tests attempt to replicate the sperm mucus activity found in the female endocervix and therefore, may lack some important characteristic peculiar to the endocervix which could make a difference in the effects observed. It was not expected from the review of the literature that Ureaplasma urealyticum would play a significant role in 76 sperm cervical mucus interaction and this premise was supported, in that when Ureaplasma urealyticum was looked at alone there was no significant findings. That is to say, for the group with Ureaplasma urealyticum alone the SCMPT score mean did not differ significantly from that group without Ureaplasma urealyticum. As stated previously with respect to bacterial vaginosis, it was hoped that a combined infection of bacterial vaginosis and Ureaplasma urealyticum would show a joint effect on SCMPT score outcome. Again this hope of an effect on the dependent variable by bacterial vaginosis and Ureaplasma urealyticum combined was based on the literature regarding these infections and their potential for pathogenicity because of their association with endotoxin activity and prostaglandin synthesis. When bacterial vaginosis and Ureaplasma urealyticum were looked at together, no statistically significant interaction effect could be found. However, when one considers the mean SCMPT score for the group with bacterial vaginosis and Ureaplasma urealyticum combined (i = 3.5) there is difference which has some practical significance when compared to the groups with bacterial vaginosis (i = 6.0) and Ureaplasma urealyticum (x = 6.0) alone. Practically speaking the mean SCMPT score for those couples with bacterial vaginosis and Ureaplasma urealyticum combined infections (i = 3.5) would be classified as a poor outcome, suggesting of course inadequate sperm mucus 77 interaction and possibly infertility. Conversely, those couples with bacterial vaginosis and Ureaplasma urealyticum alone were found to have a mean SCMPT score outcome of 6.0 which would be classified as fair and not necessarily suggestive of infertility. There is, therefore, a practical difference between the reformulated groups i.e., those with bacterial vaginosis and Ureaplasma urealyticum alone combined; those with bacterial vaginosis and Ureaplasma urealyticum combined infections; and those without infection eliminated, that can not be ignored. This practical difference between those with bacterial vaginosis and Ureaplasma urealyticum alone and bacterial vaginosis and Ureaplasma urealyticum combined infections is further supported by a significant contrast test. 0 d' e With respect to the current literature, this study’s findings of a significant correlation between SCMPT outcome and the number of bacterial vaginosis associated anaerobes present supports the findings of Eggert-Kruse and colleagues (1987) who reported statistical evidence that those anaerobic organisms thought to be associated with bacterial vaginosis where associated with inadequate sperm penetration in vitro. However, it should be noted that in this study the numbers of anaerobes present in the couples tested and the number of couples tested were very small, and only three couples probably came close to representing the 100-1000 .fold increase of colonization of anaerobes typically found A' AA“ 78 in couples with bacterial vaginosis (Gravett, Nelson et al., 1986). The study by Tredway and colleagues (1985) was supported by the findings of this study in that no main effect for Ureaplasma urealyticum alone was found by two-way ANOVA. This study, therefore, lends further evidence that Ureaplasma urealyticum as a single entity may not have a pathologic effect on sperm cervical mucus interaction responses for those couples carrying certain varieties of this organism. No other study in the current literature can appropriately be compared to the findings of this study but the consistency of the findings of this study can be discussed in view of what is known about the syndrome known as bacterial vaginosis. That is to say, the practical and statistically significant implications of this study are that those couples with bacterial vaginosis and Ureaplasma urealyticum combined (i=3.50) may experience lower SCMPT scores than those couples without bacterial vaginosis and Ureaplasma urealyticum (i=6.57) or those couples with bacterial vaginosis alone (336.0) or Ureaplasma urealyticum alone (i=6.0) in their reproductive tracts. The potential impact of a combination infection of bacterial vaginosis and Ureaplasma urealyticum on maladaptive reproductive physiological responses is supported by several studies in the current literature (Bejar et al., 1981; Feingold, 8 Sbarra, 1987; Gravett, Hummel et al., 1986; Sjoberg 8 79 Hakansson, 1991). These studies support the implication of bacterial vaginosis and Ureaplasma urealyticum having a potential for being related to pathological sperm cervical mucus responses in infertile couples because of their established association with prostaglandin synthesis and increased levels of vaginal endotoxins. E J !i ll E I] E' 3' ! I] H l 1 With respect to the conceptual model presented in Figure 1 and the findings of this study, one cannot state emphatically that the model was supported. From a practical stand point one might also say that there is reason to believe that bacterial vaginosis at least when combined with Ureaplasma urealyticum may be considered focal stimuli of a maladaptive reproductive physiological process such as poor sperm cervical mucus responses in some infertile couples. This hypothesis again is based on the lower mean SCMPT score outcome reported for that group with bacterial vaginosis and Ureaplasma urealyticum combined when compared to those without bacterial vaginosis and Ureaplasma urealyticum, or bacterial vaginosis and Ureaplasma urealyticum alone. The small sample size and possibly a lack of sensitivity in the SCMPT may explain the partial failure of this study to show statistical evidence that would lend the appropriate support to this conceptual model. A larger sample size may, in fact, assist the researcher to more accurately report a closer estimate of the population mean SCMPT score outcome for those couples with bacterial 80 vaginosis and Ureaplasma urealyticum infections combined. An emphatic statement in support of the conceptual model from this study, therefore, cannot be made at this time. Implications for Nursing This study has specific implications for nursing theory and education. Nurses in advanced practice need to utilize current nursing theory within the context of systematic and scientific research endeavors so as to examine the practical applicability of these conceptual frameworks in the study of actual or potential clinical problems. For this study the application of Sister Callista Roy's conceptual framework served as a useful model by which the nurse in advanced practice could systematically examine reproductive physiological mode functioning in couples with known infertility. Roy's Adaptation Model, however, is the most sophisticated nursing model that exists currently that allows and encourages the nurse to consider scientifically investigating those physiological questions that exist in advanced practice. Roy's model of Adaptation from the physiological perspective of this study, therefore, represents an essential component of the current theoretical basis of nursing practice and one that deserves more consideration from the profession of nursing. Nursing education has always demanded expert physiologic knowledge from it's professionals but has done little to encourage those prepared professionals to utilize this physiological 81 knowledge in scientific inquiry. This becomes very apparent when one reviews any leading nursing journal. I This study continues the effort by few to break that trend in nursing and serves to encourage nurses with expert skill in reproductive physiology to explore alone or through collaborative research those practice dilemmas that have more traditionally been explored by professionals in the medical field. Nursing has much to offer in this regard since the CNS practicing in primary care and reproductive health brings with her/him a whole repertoire of role characteristics that facilitate new perspectives and thoughts on physiologic concerns that other professionals may not have considered. This study has specific implications for the Clinical Nurse Specialist (CNS) and current practice standards of all primary care providers who care for women. As a researcher and primary care provider, the CNS must pursue systematically and scientifically through investigation those clinical problems, as formulated, that would advance current clinical nursing knowledge. The implications for clinical practice are that, the mean SCMPT score outcome reported for the interaction effect of bacterial vaginosis and Ureaplasma urealyticum combined was found to be poor (i=3.50) in comparison to that mean SCMPT score reported for those with Ureaplasma urealyticum alone (i=6.00), bacterial vaginosis alone (i=6.00), and those without either infection (i=6.50). This tendency for lower SCMPT score outcome among ‘ /_flh 82 infertile couples with bacterial vaginosis and Ureaplasma urealyticum combined may be contributing to their overall infertile state, but to what extent at the present time unknown. The CNS, therefore, in infertility practice could utilize these findings to try to promote reproductive health for infertile couples by being aware that the presence of these organisms in infertile couples may not promote their adaptive reproductive physiological response. The CNS may also logically assume, as evidenced by the current. literature, that the way in which the presence of these organisms may affect reproductive physiological responses in infertile couples may be through their tendency to promote the development of prostaglandins and endotoxins in the reproductive tracts of their victims. Therefore, the CNS has the right and the responsibility to assess, diagnose, and treat those infertile couples infected with bacterial vaginosis and Ureaplasma urealyticum, so as to potentially facilitate their reproductive health. The CNS to has a continued responsibility to investigate through scientific inquiry how bacterial vaginosis and Ureaplasma urealyticum may be directly or indirectly related to maladaptive reproductive physiological responses in infertile couples. More broadly stated, the implications of this study have been to continue the work of establishing a broader knowledge base for the possible risks for women with 83 bacterial vaginosis and Ureaplasma urealyticum. This knowledge deemed necessary to facilitate a change in the clinicians' perceptions of bacterial vaginosis and its potential virulence, so that appropriate attention can be given to the many women afflicted. That is to say, more evidence is needed from research to convince providers that they should have a greater respect for, and routinely screen for, bacterial vaginosis.. Establishment of cause and effect research evidence of bacterial vaginosis and Ureaplasma urealyticum combined infections causing a pathological response in SCMPT outcome would especially affect the current practice standards for ruling out possible causes for adverse postcoital test outcomes in the evaluation of infertile couples. This study has in a small way begun to establish a body of knowledge that will influence the reproductive health clinicians assessment and evaluation of couples seeking infertility health care, so that bacterial vaginosis is appropriately considered, ruled out, evaluated, and treated when present. More specifically this study has begun the process of change in the existing health care system and its' providers so that their perception of bacterial vaginosis, and it's potential virulence, are appropriately modified to foster a greater overall concern for the potential presence of the condition in infertile clients. As for future research, the findings of this study have failed to answer satisfactorily the question of whether or 84 not there is a significant association between the presence of bacterial vaginosis alone and maladaptive sperm cervical mucus interaction. The findings of this study have, however, shown some statistical evidence that bacterial vaginosis when combined with serotypes of Ureaplasma urealyticum that tend to coexist with bacterial vaginosis, is associated with maladaptive sperm cervical mucus interactions. Therefore continued work in this area needs to be done. Replication of the study with a larger sample, over a longer period of time, and utilizing varying sites is, therefore, encouraged. Further work in this area is encouraged because of the suggestion from this study that significant findings would impact current practice standards for ruling out possible causes for adverse postcoital test outcome in the evaluation of infertile couples. If significant associations can be established between bacterial vaginosis alone or in combination with Ureaplasma urealyticum and sperm or cervical mucus hostility other important questions regarding this syndrome and the process by which it induces hostility will have to be answered. Most importantly future research will need to assess for any adverse affect by bacterial vaginosis, and its related organisms, on sperm or perhaps early gestations in the uterus, since these organisms are known to invade these environments. Bacterial vaginosis once established in the environment of the uterus possible causing an adverse affect 85 on sperm and/or early gestations through its known link to prostaglandin/endotoxin synthesis. Conclusions Out of a total sample of 25 infertile couples 14, completed SCMPT testing. The effect of both bacterial vaginosis and Ureaplasma urealyticum were tested for a main effect and interaction effect on SCMPT score outcome, without significance. SCMPT scores were not, therefore significantly influenced by bacterial vaginosis, Ureaplasma urealyticum or bacterial vaginosis and Ureaplasma urealyticum combined when tested by two-way ANOVA in this sample. The sample did however reveal a significant influence by the presence of bacterial vaginosis and Ureaplasma urealyticum combined on adverse SCMPT score outcome when compared to bacterial vaginosis alone and Ureaplasma urealyticum alone, as analyzed by one-way ANOVA. Failure of this sample to show a significant difference in group means for those with bacterial vaginosis and Ureaplasma urealyticum combined by two-way ANOVA probably was do to the small sample size, or potentially the sensitivity of the SCMPT as an instrument of measure for maladaptive sperm cervical mucus responses. The literature as reported by Eggert-Kruse (1987) was supported in that a tendency for a relationship to exist between those anaerobes typically associated with bacterial vaginosis were found to have a significant relationship to SCMPT score outcome. Similarly, the study done by Tredway 86 and colleagues, (1985) which found no significant relationship between Ureaplasma urealyticum and SCMPT outcome was supported in that no main effect could be found in this study for the presence of Ureaplasma urealyticum alone, with respect to SCMPT score outcome. The conceptual model presented could not be emphatically supported by this study because no significant main or interaction effect was found for bacterial vaginosis, Ureaplasma urealyticum or bacterial vaginosis and Ureaplasma urealyticum together on SCMPT score outcome. Practically speaking, however, there was some support for the model in that the reported mean for the interaction effect of bacterial vaginosis and Ureaplasma urealyticum did reveal a poor or maladaptive SCMPT score outcome response for the presence of the organisms together. This reported mean for the interaction effect of bacterial vaginosis and Ureaplasma urealyticum together much lower than that mean reported for those with bacterial vaginosis alone, Ureaplasma urealyticum alone, and the mean reported for those with neither bacterial vaginosis or Ureaplasma urealyticum. There was, however, statistical significance for.SCMPT score outcome between a combined presence of bacterial vaginosis and Ureaplasma urealyticum (n=2) when compared to a reformulated group composed of those with bacterial vaginosis and Ureaplasma urealyticum alone (n=7). The specific practical implications of this study for clinical practice are that mean SCMPT score outcomes may be 87 lower in those infertile couples with combined infections of bacterial vaginosis and Ureaplasma urealyticum. The CNS in clinical practice could utilize these findings to try to promote reproductive health for infertile couples by assessing, diagnosing, and treating bacterial vaginosis and Ureaplasma urealyticum combined infections, so as to potentially promote their reproductive physiologic health. The broader implications of this study for nursing practice are to establish a broader knowledge base with respect to bacterial vaginosis and the possible risks that may be associated with the infection. Specifically establishing this broader knowledge base will influence all clinicians in their assessment and evaluation of all clients seeking health care. Further research on this subject should focus on a larger sample that could provide more empiric evidence that an association exists between the presence of bacterial vaginosis and Ureaplasma urealyticum combined infections and maladaptive SCMPT outcome. Secondly, with significant associations, established researchers must examine whether or not increased prostaglandin synthesis is responsible for this adverse reaction of sperm mucus interaction or an immune response. This study builds on the existing knowledge base that exists on the topic of bacterial vaginosis, and encourages further research in this area. Furthermore this study will assist in changing the current health care system, it's providers, and their perception of bacterial vaginosis and 88 the syndromes potential virulence, as it relates to the reproductive physiologic health of those infertile clients they serve. LI ST 01" REFERENCES List of References Aitken, R., 8 Kelly, R. (1985). 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WW Diseases. 118(5). 817-822. 98 Svangorg, K., Gottlieb, C., Bendvold, E., 8 Bygdeman, M. (1989). Variation in, and inter-relationship between, prostaglandin levels and other semen parameters in normal men. Intcrnaticncl_lcnzncl_cf_hndnclccx. 12. 411-419. Swenson, C. E., Toth, A., 8 O'Leary, W. M. (1979). Ureaplasma urealyticum and human infertility: The effects of antibiotic therapy on semen quality. W191. 3.1. 660. Tan, S. L., Scammell, H. E., 8 Houang E. (1987). The midcycle cervical microbial flora as studied by the weighted swab method, and its possible correlation with results of sperm cervical mucus penetration tests. Ecrtilitx_cnd_§cczllicx. 11(6). 941-946- Taylor, E., Blackwell, A. L., Barlow, D., 8 Phillips, I. (1982). Gardnerella vaginalis, anaerobes, and vaginal discharge. Lancer, i, 1376. Taylor-Robinson, D., 8 McCormack, W. M. (1980). The genital mycoplasmas. Ihe_Ney_Englgng_ggurngl_gr Medicine. 322(18). 1003-1010. Thomason, J. L., Gelbart, S. M., 8 Broekkuizen, F. R. (1989). Advances in the understanding of bacterial vaginosis. 1curnal_cf_Bccrccnccixc_ncdicinc. 31(8). 581-587. 99 Thomason, J. L., Schreckenberger, P. C., Spellacy, W. N., Riff, L. J., 8 LeBeau, L. J. (1984). Clinical and microbiological characterization of patients with nonspecific vaginosis associated with motile, curved anaerobic rods. lnrecrlgug_nl§eg§e, lgg, 801. Toth, A., 8 Lesser, M. L. (1982). Ureaplasma urealyticum and infertility: The effect of different antibiotic regimens on the semen quality. Ihe_JQgrnal_gr_nrglggy, lag, 705-707. Tredway, D. R., Wortham, J. W., Condon-Mahony, M., Baker, D., 8 Shane, J. M. (1985). Correlation of postcoital evaluation with in-vitro sperm cervical mucus determinations and ureaplasma cultures. Eerrlllry_gg§_ firerlllry, 11(2), 286-289. Wahbeh, D. J., Hill, G. B., Eden, R. D., 8 Gull, S. A. (1984). Intra amniotic bacterial colonization in premature labor. Ancriccn_Icurncl_cf_cbscctricc_and EYnccclcQY. 115. 739-743- Watts, D. H., Eschenbach, D. A., and Kenny, G. E. (1989). Early postpartum endometritis: The role of bacteria, genital mycoplasmas, and chlamydia trachomatis. Ohsterrics and Gynecolggy,,1;(1), 52-59. Watts, D. H., Krohn, M. A., Hillier, S. L., 8 Eschenbach, D. A. (1990). Bacterial vaginosis as a risk factor for post-cesarean endometritis. cbscccricc_and_cxnccclccx. 15(1), 52-58. 100 World Health Organization. (1987). W§Q_lghgrgrgry_m§nual mugu§_lnreragrlgn. Cambridge University Press: Cambridge. APPENDICES h; MICHIGAN STATE UNIVERSITY UNIVERSITY COMMITTEE ON RESEARCH INVOLVING EAST LANSING - Mia-SCAN - «IN-tit: HUMAN SUBJECTS «mm 106 BERKS? MALI. ($17) 353-913! July 24, l990 IRB# 90-289 Penny L Waltman 5250 Windmill. N.E. ' Rockford, Ml 4934i Dear Ms. Waltman: Re; I 'THE ASSOCIATION OF BACTERIAL VAGINOSIS WITH SPERM CERVICAL MUCUS PENETRATION TEST OUTCOME IRB# 90-289' UCRIHS’ review of the above referenced project has now been completed. I am pleased to advise that since reviewer comments have been satisfactorily addressed. the conditional approval given by the Commttee at its July 9. l990 meeting has been now changed to full approval. You are reminded that UCRlI-ls approval is valid for one calendar year. If you plan to continue this project beyond one year, please make provisions for obtaining appropriate UCRIHS approval one month prior to July 9,!991.‘ Any changes in procedures involving human subjects must be reviewed by UCRIHS prior to initiation of the change. UCRIHS must also be notified promptly of any problems t(tr'lnexpected side effects, complaints, etc.) involving human subjects during the course of 8 work. 1hank you for bringing this project to our attention. If we can be of any future help, please do not hesitate to let us know. . Sincerely. Acting Co-Chair, UCRIH KOM/sar cc: R. Schiffman MSU is as A/lm Adios/Equal Way Inuit-scion 101 Appendix A Investigator: Penny Waltman RNC MSU Family Clinical Nurse Specialist Graduate Student Phone: 616-942-5180 The study in which you are being asked to participate is designed to measure sperm-mucus interaction in the presence and absence of two common infections found in infertile couples. This study will provide more information about factors involved in infertility for couples like yourself. Participation in the study will require: 1. The first office visit be for the collection of a mid- cycle sample of cervical mucus. This visit will take the place of your required post-coital test (Huhner's). 2. All the tests for infection normally done for infertility evaluation be done on both the male and female partner the day of the mucus test. 3. All participating couples to purchase a luteinizing hormone ovulation predictor kit, which will be used, according to office recommendations, to appropriately time the mucus test with ovulation. 4. ' The female partner to have an ultrasound, at no charge, for confirmation of ovulation the day of the LH surge. S. The male partner to submit a semen specimen no more than one hour prior to the appointment time for the mucus test, after 48 hours of abstinence. This specimen is to be collected in a sterile container, ,which will be supplied by West Michigan Reproductive Institute. This research study investigating the interaction of sperm and female cervical mucus in the presence and absence of specific infections has been explained to me. We understand that if we participate we can expect that: 1. All information is confidential i.e., test results will be made available to you, Dr. Eward and myself, and no attempt will be made to identify you in any manner through the reporting of the study findings. 2. Our participation is voluntary. 102 ' Our decision to participate or not participate will not affect in any way our care at WMRI. We are free to withdraw our consent and to discontinue participation in the study at any time without adverse affect on our care at WMRI. We understand that if we are injured as a result of our participation in this research project, Michigan State University will provide emergency medical care if necessary. We further understand that if the injury is not caused by the negligence of MSU we are personally responsible for the expense of this emergency care and any other medical expenses incurred as a result of this injury. The benefits offered us by participating include having a more specialized test for sperm-cervical mucus interaction and having a vaginal ultrasound done to verify ovulation at no charge. We have fully read the consent form, understand what it says, and agree to participate in this research study. Signature of participant Signature of participant Signature of witness Date 103 Appendix B ssmen_2arameter_§tandard§ zzu., 01- 1° 1 1 BIC - : 0 v 2 r ' ° 9 0! Standards: M New 6 Total Cells 20-250 X 10 [ml 20 M (count) Motile Cells >40 % 50 % (motility) Velocity >20 Rapid V>25 25 % Medium 1060 % normal Volume 1.5 - 5.5 ml Liquefaction 10 - 30 minutes Note. Values represent the normal range of reference for semen analysis values uSing an objective microcomputerized multiple exposure photography system (Hamilton-Thorn M2000). 104 Appendix C §§I21931_M2£n§_§2221n9 Cervical mucus properties will be scored according to the system suggested by the World Health Organization (1987). The maximum score for cervical mucus is 15. A score greater than 10 will be considered good or as favoring sperm penetration. A score less than 10 may represent cervical mucus that is unfavorable to sperm. Scoring I categories are as follows: 1. Volume will be scored as follows: 0= 0 ml 1= 0.1 m1 2= 0.2 ml 3= 0.3 ml or more 2. angigtgngy will be scored as follows: O= thick, highly viscous mucus 1= intermediate type (viscous) 2= mildly viscous 3= normal midcycle mucus (preovulatory) 3. ferning will be scored as follows: 0= no crystallization 1= atypical fern formation M II primary and secondary stem fern 3= tertiary and quaternary stem ferning 4. Spinnbazkgit will be scored as follows: = < 1 cm 1 1 to 4 cm 105 2= 5 to 8 cm 3= > 9 cm 5. lelglgnity will be scored as follows: O= > 11 cells/HPF 1= 6-10 cells/HPF 2= 1-5 cells/HPF 3= 0 cells/HPF * Adapted from World Health Organization (1987) Laboratory Manual for the examination of human semen and semen-cervical mucus interaction (1987) pp. 20-22. 106 "Illlllllllllllllllllllllll“