Abstract. Introduction. RBMOnline - Vol 19. No Reproductive BioMedicine Online; on web 12 October 2009

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1 RBMOnline - Vol 19. No Reproductive BioMedicine Online; on web 12 October 2009 Article Significance of positive Chlamydia serology in women with normal-looking Fallopian tubes Valentine Akande is a Consultant Gynaecologist at Bristol centre of reproductive medicine at Southmead Hospital in Bristol. His academic training in reproductive medicine includes having been a Research Fellow under the late Professor Michael Hull, which eventually led to the attainment of a PhD at the University of Bristol in He was later a Clinical Lecturer in Reproductive Medicine also with the University of Bristol. He has published widely and continues to undertake research. His research interests are in the assessment, causes and treatment of pelvic tubal infective damage and endometriosis. Dr Valentine Akande Elsamawal A El Hakim 1, Mathias Epee 2, Timothy Draycott 2, Uma D Gordon 1, Valentine A Akande 1,2,3 1 Bristol Centre of Reproductive Medicine, Southmead Hospital, Bristol BS10 5NB, UK; 2 Division of Women s Health, Southmead Hospital, Bristol BS10 5NB, UK 3 Correspondence: vakande@gmail.com Abstract Chlamydia trachomatis poses a potential threat to the fertility of women by causing tubal damage. Many women with serological evidence of past Chlamydia infection have normal tubal appearances on laparoscopic assessment. The aim of this study was to assess if serological evidence of past chlamydial infection affects the likelihood of conception in women with normal tubes. Infertile couples in which the female partner was under the age of 40 years, with normal ovulatory function and a male partner with normal sperm function were studied. All women had normal tubes as assessed by laparoscopy. Serum Chlamydia antibody titres were assayed using the immunofluorescence test. Pregnancy rates were related to grouped Chlamydia antibody titres (<64, and P512). A total of 174 women were studied. The cumulative pregnancy rates (SE) according to these titres were 45.1% (6.2), 42.6% (9.3), 59.1% (11.8) and the risk ratios (95% confidence interval) were 1, 1.59 ( ) and 1.04 ( ) respectively. The differences were not statistically significant. Therefore, in women with normal-looking tubes, serological evidence of past chlamydial infection does not appear to have an adverse effect on pregnancy rates. These findings suggest that laparoscopic findings and not Chlamydia serological titres are the key to prognosis. Keywords: chlamydia, infection, normal, serology, tubes Introduction Most couples with subfertility have one or more obvious reasons for their difficulty in conceiving. Evidence of past chlamydial infection as indicated by positive serology is highly prevalent in the infertile population. However, there is wide human variation in the response to genital chlamydial infections, such that many women exposed to the infection actually have no evidence of tubal or pelvic damage when investigated by laparoscopy. In addition, the amount of tubal damage if present can vary greatly in extent, anatomical location and nature and hence may influence the couple s management (Akande, 2007). However, whilst Chlamydia antibody titres (CAT) may correlate with the amount of pelvic adhesions, it may not necessarily with tubal adhesions and tubal damage and, hence, may not reflect tubal function (Sonmez et al., 2008). Various screening strategies and approaches are available to estimate the risk of tubal damage and subfertility, laparoscopy has been advocated as a standard reference in pelvic assessment. This is particularly important in patients with significantly high Chlamydia antibody titre (den Hartog et al., 2008). This study was carried out to investigate whether women who have normal tubes on laparoscopy, including those with unexplained subfertility, may have silent, latent or unidentified tubal mucosal damage, which may affect the functional ability of the tubal mucosa to transport gametes, thus impairing the potential for pregnancy. In addition, women with evidence of previous chlamydial infection may still harbour the organisms even after treatment. The objective of this study was to assess if evidence of past chlamydial infection 847 Ó 2009 Published by Reproductive Healthcare Ltd, Duck End Farm, Dry Drayton, Cambridge CB23 8DB, UK

2 848 affects the likelihood of pregnancy in women with normal tubes. Materials and methods Local research ethics committee approval was obtained for the study. This was a retrospective study of infertile women who attended the Reproductive Medicine Clinic at St Michael s Hospital in Bristol between 1985 and This period was chosen because this was the only reproductive medicine unit in Bristol at that time, and also during this time the same protocol was used by all the clinicians to investigate pelvic pathology. The clinic is run as a subspecialty service dealing with infertile couples referred by general practitioners in the Bristol area and tertiary care to couples referred by specialists in Bristol, the south west of England and parts of south Wales. All couples studied had a diagnostic laparoscopy for assessment of tubal patency, fibrosis, distortion or the presence of endometriosis or pelvic adhesions. If tubal patency was not confirmed at the time of laparoscopy, this was attempted by hysterosalpingography. Couples who had a distinct cause of infertility, such as ovulatory dysfunction with no index of pelvic disease, would not have had a routine laparoscopy and some others conceived before laparoscopy was necessary or arranged. Although different clinicians carried out the laparoscopies over this period of time, each clinician employed the same technique because they were trained and supervised initially by one of two consultants (Professor MGR Hull and Mr PG Wardle) prior to being allowed to assess the pelvis independently. All clinicians undertaking laparoscopy were accredited specialists or experienced senior trainees. Findings were recorded in a standardized way and no surgical therapy was undertaken to treat any endometriosis found at this time. Biopsies were not taken, therefore the diagnosis of endometriosis required one or more lesions to be visually identified at laparoscopy as defined by the revised American Fertility Society classification for endometriosis (American Fertility Society, 1985). In women found to have endometriosis, only those with minor lesions referred to as minimal/mild endometriosis by the revised classification were studied. To avoid any adverse influence of anatomical factors, women with adhesive disease or cysts were also excluded. For the purposes of this study, women were included if menstruation in four of five spontaneous menstrual cycles had a duration of days irrespective of cycle variability. Ovulation was investigated by estimation of serum progesterone concentration in the mid-luteal phase, which was retrospectively confirmed, by the patient telephoning the clinic, to be within 5 10 days of the onset of menstruation. A progesterone concentration of 30 nmol/l (9.4 ng/ml) or greater was taken to indicate a normal ovulatory cycle, as previously established by the observation of untreated conception cycles (Hull et al., 1982). Semen analysis was considered normal if the sperm concentration was / ml with normal motility 50% and normal morphology 30% (World Health Organization, 1992). The post-coital test was used as a screening test for coital and ejaculatory competence, mucus receptivity and sperm function. This test was carried out by instructing couples to have intercourse at mid cycle, preferably when they recognized the typical pre-ovulatory mucus secretion, 6 18 h before the test, as described by (Hull et al., 1982). A post-coital test was considered favourable if one or more progressively motile sperm were seen per high-power field. The women with unexplained infertility satisfied the criteria of having a normal pelvis, normal sperm function and normal ovulatory cycles as described above. The women with endometriosis included in this study fulfilled the criteria as those with unexplained infertility, the only difference being the presence of minor endometriosis at laparoscopy. The factors explored in this study were: (i) history of previous pregnancy by the couple (primary or secondary infertility); (ii) duration of infertility and age of the woman at the time of laparoscopy; (iii) frequency of intercourse; and (iv) smoking. All couples had a minimum of 1 year duration of infertility. The effect of duration of infertility was analysed in two ways: initially women were divided into four groups of <3, 3 3.9, and 5 years of infertility and then later dichotomised into groups of <3 years and 3 years of infertility, both at the time of laparoscopy. The frequency of intercourse was divided into women who had intercourse once or less a week and those who had intercourse twice or more a week. A woman was said to be a smoker if she admitted to any smoking at the time of initial consultation. The effect of age was analysed in two ways: dividing women into three groups (<30, and years) and as a continuous variable (range years). The duration of follow-up from laparoscopy was censored at 3 years and women who were 40 years or older were excluded from the analysis. However, women who were 40 years were included if their laparoscopy was undertaken when they were under the age of 40 years. All women were managed expectantly as treatment of these conditions at the time was considered to be of doubtful benefit (Hull, 1992). Following laparoscopy, a 3-year duration of follow-up was sought to assess fertility outcome. If the follow-up details of the woman were not available in her infertility records or if the duration of follow-up was shorter than three years, women were sent a questionnaire to obtain details on whether and when they conceived and if they had any form of further treatment. For the patients who conceived, the time interval of interest was from the date of diagnostic laparoscopy to the last menstrual period before conception. For the patients who did not conceive, the time interval of interest was until the time of the last follow-up or correspondence with the patient. For analytical purposes, the patients who did not become pregnant were censored at this time. Pregnancy was defined by a positive pregnancy test and confirmed by the presence of an intrauterine gestational sac by ultrasonography. A live birth was defined as a pregnancy known to go beyond the 24th week of pregnancy. Statistical methods and analysis A number of factors may affect time to outcome. Analyses of these factors provide concomitant information that may improve the description and interpretation of the data.

3 Although the (univariate) log-rank test used in this study was useful for comparing time-to-pregnancy or live birth curves between two or more groups and taking into account differences in follow-up times and drop outs. To allow adjustment for other factors (multivariate analysis), a series of Cox s proportional hazards regression models were used. Results A total of 209 women with normal Fallopian tubes on laparoscopy were identified. After excluding those who were 40 years or over (n = 17) and those who did not have a serum Chlamydia antibody assay done (n = 19) (one woman was excluded for both reasons), 174 women were studied. Subsequent analysis is based on these 174 women. These were women with either unexplained infertility (n = 106) or who had minimal mild endometriosis (n = 68) in the absence of pelvic or other adhesions. The number of women in relation to antibody titres <1:64, 1:64, 1:128, 1:256, 1:512, 1:1024 and 1:2048 were 107 (61.5%), 16 (9.2%), 13 (7.5%), 12 (6.9%), 6 (3.4%), 16 (9.2%) and 4 (2.3%) respectively. For comparative reasons, women were also grouped in relation to negative (<1:64), low (1:64 1:256) and high (1:512) titres, with 107 (61.5%), 41 (23.6%) and 26 (14.9%) women in these groups respectively (Table 1). Figure 1. Cumulative pregnancy rates in women with normal tubes (unexplained and superficial endometriosis) in relation to grouped Chlamydia antibody titres (CAT) (n = 174). There was only one ectopic pregnancy in a woman with unexplained infertility and a titre of <64. Three women had miscarriages, two of these women had negative titres of <64, while the third had a high titre of 512. The log-rank test was used to compare the Kaplan Meier estimated cumulative pregnancy rates at 3 years following laparoscopy. The pregnancy rate did not differ significantly amongst the groups of women with different antibody titres as shown in Figure 1. Multivariate analysis was carried out using Cox s proportional hazards regression method to study the relative risk of pregnancy in relation to CAT levels after controlling for age, duration of subfertility, smoking, and primary infertility. As shown in Figure 2, this was not statistically significant. Discussion The main finding of the present study was that in women with normal-looking tubes, serological evidence of past chlamydial infection and CAT levels does not appear to have an adverse effect on pregnancy rates (Table 1, Figures Figure 2. Relative risk of pregnancy in relation to Chlamydia antibody titre controlling for age, duration of subfertility, smoking and primary infertility. 1 and 2). These findings suggest that laparoscopic findings on pelvic assessment and not Chlamydia serological titres are the key to assessing the likelihood of pregnancy. There is a recognized quantitative relationship between CAT levels and severity of tubal damage in infertile women. However, none of the previously published studies have explored the relationship between pregnancy rates in women with normal-looking tubes and CAT. The present Table 1. Pregnancy outcomes for groups of women with different Chlamydia antibody titres. Chlamydia antibody titre < Number of women Cumulative pregnancy rate (SE) 45.1% (6.2) 42.6% (9.3) 59.1% (11.8) Risk of pregnancy (95% CI) ( ) 1.04 ( ) 849

4 850 study explores the relationship of CAT levels in laparoscopically normal-looking tubes and the likelihood of pregnancy. The number of live births in women with normal tubes was small; hence, the independent effect of titres on this outcome was not studied. However, it is likely to have been similar to that of pregnancy rates because there was only one ectopic pregnancy and three miscarriages in this group of women. Furthermore, it would be of interest to ascertain if women who have normal pelvis on laparoscopy such as those with unexplained infertility may have silent, latent or unidentified tubal mucosal damage affecting the functional ability of the tubal mucosa to transport gametes and thus impair the potential for pregnancy. In addition, women with evidence of previous chlamydial infection may still harbour the organisms even after treatment. There is, therefore, the hypothetical possibility that the latent endometrial infection may be reactivated in pregnancy leading to an increase in miscarriage rates. It is likely that variation in pelvic pathology and tubal damage reflects the known variation in immune response to Chlamydia trachomatis infection; this may have a genetic element related to the genes determining the receptor s function (den Hartog et al., 2006). This would explain why many women with serological evidence of past infection have no laparoscopic evidence of tubal damage. It is helpful for fertility clinicians to determine the severity of the tubal damage in women with evidence of past chlamydial infection before deciding on what treatment to offer a woman or couple (Akande, 2002, 2007; Elstein, 2008). Evaluation of the pelvis by laparoscopy in patients with positive chlamydial serology is important because the incidence of peritubal adhesions and tubal occlusion is high in this group; other tubal patency tests including hysterosalpingogram and hysterocontrastsonography are not suitable in this group (Shibahara et al., 2001) because of their limitations in not being able to assess adhesions. Considering that laparoscopy is an expensive and invasive test, screening for Chlamydia beforehand, as part of the initial fertility investigations, is useful in order to ascertain those at highest risk of tubal damage (Thomas et al., 2000). Studies have evaluated the significance of persistent chlamydial infection in tubal pathology in comparison to previous infection, as well as other biochemical or serological factors (den Hartog i, 2005). These may help in differentiating those with active or persistent infection, the most commonly used is C reactive protein. This was not examined in this study population. Also, this study used multivariate analysis with Cox s proportional hazards regression method to control for the influence of common confounding variables such as age, duration of infertility, smoking, and primary or secondary infertility (type). The differences were not statistically significant for the different CAT groups (Figure 2). Perquin et al. (2007) studied of 178 subfertile women, and assessed the value of C. trachomatis-specific immunoglobulin G antibody testing for prediction of tubal pathology and occurrence of pregnancy. The poor performance of this test agrees with the data from this study. Furthermore, screening for tubal patency or Chlamydia may be of limited clinical value in low-risk couples with a known cause of subfertility such as anovulation or significant sperm dysfunction (van Tetering et al., 2007). In clinical practice, a number of tests are often needed to establish the clinical diagnosis and plan future management. Very often second-line investigations are needed to provide diagnostic discrimination between alternative states of a condition, such as extent of damage and the prognosis. The findings of this study suggest that the chlamydial serological titres have no influence on fertility outcome if the Fallopian tubes look normal at laparoscopy where there is no structural damage or adhesions. Further studies in immune responses to Chlamydia infection and its effect on tubal damage are warranted. References Akande VA 2007 Tubal disease: towards a classification. Reproductive BioMedicine Online 15, Akande VA 2002 Tubal pelvic damage: prediction and prognosis. Human Fertility 5(suppl. 1), S15 S20. American Fertility Society Revised American fertility society classification for endometriosis. Fertility Sterility vol. 43, pp den Hartog JE, Lardionjie CM, Severens JL et al Screening strategies for tubal factor subfertility. Human Reproduction 23, den Hartog JE, Morre SA, Land JA et al Chlamydia trachomatis associated tubal factor subfertility: immunogenetic aspects and serological screening. Human Reproduction Update 12, den Hartog JE, Land JA, Stassen FR et al Serological markers of persistent C trachomatis infections in women with tubal factor subfertility. Human Reproduction 20, Elstein M 2008 Tubal disease and fertility outcome. Reproductive BioMedicine Online 16, Hull MGR 1992 Infertility treatment: relative effectiveness of conventional and assisted conception methods. Human Reproduction 7, Hull MG, Savage PE, Bromham DR et al The value of a single serum progesterone measurement in the midluteal phase as a criterion of a potentially fertile cycle ( ovulation ) derived form treated and untreated conception cycles. Fertility and Sterility 37, Perquin DA, Beersma MF, de Craen AJ et al The value of Chlamydia trachomatis-specific IgG antibody testing and hysterosalpingography for predicting tubal pathology and occurrence of pregnancy. Fertility and Sterility 88, Shibahara H, Fujiwara H, Hirano Y et al Usefulness of transvaginal hydrolaparoscopy in investigating infertile women with Chlamydia trachomatis infection. Human Reproduction 16, Sonmez S, Sonmez E, Yasar L et al Can screening Chlamydia trachomatis by serological tests predict tubal damage in infertile patients? New Microbiology 31, Thomas K, Coughlin L, Mannion PT et al The value of Chlamydia trachomatis antibody testing as part of routine infertility investigations. Human Reproduction 15, van Tetering EA, Bourdrez P, Koks CA et al Routine Chlamydia antibody testing is of limited use in subfertile women

5 with anovulation. Reproductive BioMedicine Online 14, World Health Organization World Health Organization Laboratory Manual for the Examination of Human Semen and Sperm Cervical Mucus Interaction 3rd ed. Cambridge University Press, Cambridge. Declaration: The authors report no financial or commercial conflicts of interest. Received 5 January 2009; refereed 27 January 2009; accepted 17 August