In-vitro fertilization outcome in women with hydrosalpinx*

Human Reproduction vol.11 no.3 pp.526-530, 1996 In-vitro fertilization outcome in women with hydrosalpinx* Fady LSharara 1 * 3, Richard TScott Jr 2, Edward L-Marut 1 and John T.Queenan Jr 1 'Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynaecology, Michael Reese Hospital and Fertility Center, Chicago, IL, and 2 Division of Reproductive Endocrinology and Infertility, Saint Barnabas Medical Center, Livingston, NJ, USA ^o whom correspondence should be addressed at: Michael Reese Fertility Center, 60 East Delaware Place, 14th Floor, Chicago, IL 60611, USA Recent studies have suggested that the presence of hydrosalpinx has a negative effect on in-vitro fertilization (TVF) outcome, with markedly diminished implantation and pregnancy rates, and increased early pregnancy loss. We evaluated the impact of hydrosalpinx on IVF outcome in a large population with tubal factor infertility: 63 patients with hydrosalpinx and 60 without hydrosalpinx (no hydrosalpinx) underwent 103 and 89 IVF cycles respectively. Hydrosalpinx was diagnosed by hysterosalpingography and/or laparoscopy prior to IVF. Patients were further subdivided into those with or without elevated quantitative serum Chlamydia trachomatis IgG antibody (Ab) titres. All couples with elevated serum Ab titres (1: 16 or more) were treated with doxycycline (100 mg bid.) 10 days prior to the first IVF cycle. In all, 88 women (71.5%) had elevated C. trachomatis Ab: 47 women (74.6%) with hydrosalpinx had elevated titres, compared to 41 (683%) in the no hydrosalpinx group. There were no significant differences in mean age, number of mature oocytes obtained, and number of embryos transferred between the two groups. There was a trend for a higher implantation rate and ongoing pregnancy rate in the no hydrosalpinx group compared to the hydrosalpinx group (12.6 versus 9.8%, and 33.7 versus 24.8% respectively); however, this did not reach statistical significance. The incidence of early pregnancy loss was similar in the two groups. Two ectopic pregnancies were noted in the hydrosalpinx group compared to none in the no hydrosalpinx group. As expected, the prevalence of elevated titres of C. trachomatis IgG Ab in patients with tubal factor infertility presenting for assisted reproductive treatment was high. In contrast to recently published reports, our study did not confirm a negative effect of hydrosalpinx on IVF outcome when antibiotic treatment was given prior to assisted reproductive treatment. Prospective multicentre studies evaluating the Presented in part at the American Society for Reproductive Medicine 51st Annual Meeting, October 7-12, 1995, Seattle, WA, USA. 526 effect of hydrosalpinx and its treatment on IVF outcome are needed. Key words: Chlamydia trachomatis/hydiosaipiitx/imnmnofluorescent antibodies/implantation rate/tvf l outcome Introduction TubaJ factor infertility remains the most common indication for in-vitro fertilization (IVF). Tubal infertility encompasses multiple aetiologies, including unilateral or bilateral hydrosalpinx, distal or proximal obstruction and intraluminal or extraluminal adhesions. Recently, several investigations have examined the impact of hydrosalpinx on IVF outcome (Andersen et al, 1994; Kassabji et al., 1994; Strandell et al, 1994; Vandromme et al, 1995). Most of the published studies to date have suggested that the presence of hydrosalpinx is associated with decreased implantation rate, decreased pregnancy rate and increased early pregnancy loss. The mechanisms by which hydrosalpinx may affect IVF success include reflux of hydrosalpinx fluid into the uterine lumen (Mansour et al., 1991), endometrial damage simultaneous with acute-phase tubal damage (Strandell et al., 1994), or by altering endometrial integrin expression (Lessey et al., 1994). All those mechanisms can potentially affect endometrial receptivity, leading to implantation failure. Chlamydia trachomatis is the most common sexually transmitted disease in Western society, with an estimated 4 million cases in the USA annually, resulting in annual costs exceeding $2 billion (Pearlman and McNeely, 1992). Recognized sequelae of a chlamydial infection on the reproductive tract include tubal factor infertility, ectopic pregnancies, pelvic adhesions, chronic endometritis and salpingitis and a persistent carrier state (Walters et al, 1988; Reniers et al, 1989; Cates and Wasserheit, 1991; Ruijs et al, 1991; Pearlman and McNeely, 1992). Because up to 70% of pelvic infections with C. trachomatis can.be asymptomatic (Schachter et al, 1983), serum C. trachomatis antibody titres have been recently used as a non-invasive test to detect tubal disease; retrospective studies have demonstrated a consistent correlation between the presence of elevated serum antibody titres and tubal factor infertility (Meikle et al, 1994). Up to 75% of women with tubal factor infertility (diagnosed by laparoscopy and/or hysterosalpingography) have significantly elevated titres, as compared to 20-40% of fertile controls (Jones et al, 1982; Sellors et al, 1988). These antibody titres were found to be as predictive as hysterosalpingography in diagnosing tubal disease (Minassian and Wu, 1992; Meikle et al, 1994). C. trachomatis can cause severe tubal damage resulting European Society for Human Reproduction and Embryology

IVF outcome in women with hydrosalpinx in hydrosalpinx formation. Because of the recent studies associating hydrosalpinx with poor IVF outcome, we retrospectively examined: (i) the effect of unilateral or bilateral hydrosalpinx on IVF outcome in a large assisted reproduction treatment population, and (ii) the incidence of C. trachomatis IgG seropositivity and its treatment prior to IVF using prospectively collected data. Materials and methods Population Between January 1990 and December 1994, 123 women <40 years of age with tubal factor infertility undergoing a total of 192 IVF cycles were evaluated in our centre. All micromanipulation, donor cycles and cycles with no fertilization were excluded. Hydrosalpinx was diagnosed by hysterosalpingography and/or laparoscopy. Cervical cultures for gonorrhoea and C. trachomatis were performed upon initial presentation if clinically indicated. Blood was drawn within 1 month prior to the first IVF cycle for the detection of C. trachomatis IgG antibodies (Ab). Those samples with positive IgG Ab were then quantified serially. All patients and their sexual partners were pretreated with doxycycline, 100 mg bid., for 10 days prior to the first IVF cycle if their titres were elevated. Experimental design All patients underwent ovulation induction with exogenous gonadotrophins (Metrodin and Pergonal; Serono, Randolph, MA, USA) after down-regulation with leuprolide acetate (Lupron; TAP, Deerfield, IL, USA) using established protocols (Neveu et al, 1987; Scott and Rosenwaks, 1989). The patients continued their leuprolide acetate until the day of human chorionic gonadotrophin administration. All cycles were monitored with serial transvaginal ultrasound and senun oestradiol measurements. All patients received two doses of 100 mg of doxycycline on the day of retrieval. After transvaginal ultrasoundguided oocyte retrieval, the oocytes were graded using established criteria (Veeck, 1988). Fertilization was assessed the day after insemination, and embryo transfer was performed 48 h after retrieval. Ongoing pregnancy rate implies a pregnancy which has resulted in a delivery or progressed beyond 20 weeks. Early pregnancy loss implies a first trimester miscarriage. Laboratory assays Serum C. trachomatis IgG antibodies were detected using a commercially available immunofluorescence antibody (DFA) detection kit (Virgo IFA; SchiappareUi Biosystems, Columbia, MD, USA). The procedure was carried out in two steps. In step one, the serum to be tested was brought in contact with the and genie substrate. If Chlamydia IgG antibody were present in the test serum, an antigen antibody complex would form. The second step involved adding a fluoresceinlabelled anti-human antibody to the test wells. If the specific antigenantibody complex was formed in step one, the fluorescein-labelled antibody would attach to the antibody moiety of the complex in step two. If the serum tested contained C. trachomatis IgG antibodies, a bright apple-green fluorescence could be seen with the aid of a fluorescence microscope. Positive samples were then serially diluted and the titres quantified (<1:8 to > 1:512). According to the manufacturer, a positive result is a titre of 1:16 or more. The cross-reactivity with other Chlamydia strains is 25%, and high titres (& 1:256) are highly diagnostic of a past C. trachomatis infection. Statistical evaluation The data were analysed using Student's /-tests, x 2 anc * Fisher's exact test where appropriate, using SPSS statistical software (SPSS, Table I. The incidence of elevated Chlamydia trachomatis IgG antibodies (Ab) in women with and without hydrosalpinx (HSPX). Percentages are given in parentheses No. of patients Women with +Ab No. of IVF cycles Cycles with +Ab a versus b was not significant. HSPX* No HSPX* Total 63(51.2) 47 (74.6) 103 (53.6) 73 (52.9) 60 (48.8) 41 (68.3) 89 (46.4) 65 (47.1) 123 88 192 138 Table II. In-vitro fertilization outcome in 60 women with hydrosalpinx (HSPX) compared to 63 women with no HSPX. Two cycles resulting in ectopic pregnancies were not included in the analysis of the implantation rate, pregnancy rate and early pregnancy loss No. of cycles Mean age* (years) No. of mature oocytes/patient* No. of embryos transferred/patient 1 Implantation rate* (%) Pregnancy rate b (%) Multiple gestation rate c (%) Early pregnancy loss (%) Ectopic pregnancy rate (%) HSPX 103 33.7 ± 3.5 11.0 ± 5.1 4.5 ± 1.2 43/437 (9.8) 25/101 (24.8) 6/25 (24.0) 11/101 (10.9) 2/103 (1.9) Values are means ± SD. HSPX versus no HSPX: /> = 0.30, b P = 0.39, C P = 0.33. No HSPX 89 34.1 ± 3.3 12.3 ± 6.3 4.6 ± 1.5 50/3% (12.6) 30/89 (33.7) 12/30 (40.0) 8/89 (9.0) 0 Chicago, IL, USA). Data were expressed as means with their associated SD. P values <0.05 were considered significant. Results The distribution of the 123 women with tubal factor infertility is shown in Table I: 63 (51.29b) had unilateral or bilateral hydrosalpinx and 60 (48.8%) did not have hydrosalpinx. Of these patients, 71.5% (88/123) had elevated C. trachomatis Ab, of which 53.4% (47/88) had unilateral or bilateral hydrosalpinx compared to 46.6% (41/88) who did not have hydrosalpinx (difference not significant). Similarly, 74.6% (47/63) of women with hydrosalpinx had elevated Ab, compared to 68.3% (41/ 60) in the no hydrosalpinx group (difference not significant). In addition, 38.2% (47/123) of patients had both hydrosalpinx and elevated C. trachomatis Ab. In total, 71.9% (138/192) of the IVF cycles were in women with elevated titres. Table II summarizes the IVF outcome in the two groups. There was no difference in mean age, number of mature oocytes retrieved, or number of embryos transferred per patient There was a trend towards a higher implantation rate, higher pregnancy rate and higher multiple gestation rate in the group of patients with no hydrosalpinx, but this was not statistically significant A total of six multiple gestations (two sets of twins, three triplets and one quadruplet) were noted in the hydrosalpinx group (five of these were in the elevated Ab group), giving a multiple gestation rate of 24.0% (6/25). In contrast 12 multiple gestations were noted in the no hydrosalpinx group [eight sets of twins, two triplets, one quadruplet (reduced to twins) and one quintuplet (reduced to twins)], giving a multiple gestation rate of 40.0% (12/30; difference 527

F.l-Sharara et al Table IH. FVF outcome of 101 cycles with hydrosalpinx (HSPX) and 89 cycles with no HSPX subdivided according to seropositivity for Chlamydia trachomatis IgG antibodies (Ab). Two cycles resulting in ectopic pregnancies were not included in the pregnancy analysis HSPX + Ab HSPX - Ab No HSPX + Ab No HSPX - Ab Implantation rate (%) Ongoing pregnancy rate (%) Early pregnancy loss (%) No pregnancy (%) Total 32/304 (10.5) 18(25.4) 8(11.3) 45 (633) 71 11/133(8.3) 7(233) 3 (10.0) 20 (66.7) 30 41/304 (13-5) 21 (32.3) 6 (9.2) 38 (58.5) 65 9/92 (9.8) 9 (37.5) 2 (8.3) 13 (54.2) 24 Table IV. Review of the published literature on the effect of hydrosalpinx (HSPX) on implantation rate (IR), ongoing pregnancy rate (PR) and early pregnancy loss (EPL). PR includes both ongoing (Vandromme, Kassabji, Andersen and Sharara data) and clinical (Strandell and Lessey data) pregnancies. EPL includes spontaneous abortions without EP (Vandromme, Kassabji, Andersen and Sharara data), biochemical/spontaneous abortion/ep (Andersen), and spontaneous abortion with EP (Strandell) HSPX* No HSPX b n No. of cycles IR(%) PR(%) EPL (%) n No. of cycles IR(%) PR(%) EPL (%) Vandromme et al. (1995) Kassabji et al. (1994) Andersen et al. (1994) Strandell et al. (1994) Lessey et al. (1994) Sharara et al. (present study) 37 118 62 45 42 63 69 234 104 91 113 103 4.2* 7.6* 2.9* 6.6* x 9.8 10.1* 9.8* 6.6* 13.2* 10.0 24.8 0 46.5* 70.0* 50.0 10.9 41 157 493 173 127 60 67 223 813 285 327 95 10.4* 11.6* 10.3* 18.2*' 12.6 21.3* 25.0* 22.8* 26.0* 20.9 33.7 9.1 20.0* 36.0* 30.0 9.0 = not stated. *a versus b: P < 0.05. Delivery rate/% of embryos transferred. not significant); 11 of these were in the elevated Ab group. There was no difference in the incidence of early pregnancy loss between the two groups. Two ectopic pregnancies were noted in the hydrosalpinx group (1.9% of 103 cycles, 5.3% of pregnancies in the hydrosalpinx group, 2.7% of total pregnancies) compared to none in the no hydrosalpinx group. Table HI represents the IVF outcome in the hydrosalpinx and no hydrosalpinx groups subdivided according to seropositivity for C. trachomatis IgG Ab. There was a trend for higher pregnancy rate in the no hydrosalpinx compared to the hydrosalpinx group: 25.4 versus 32.3% in the elevated Ab subgroup, compared to 23.3 versus 37.5% in the negative Ab subgroup respectively (not significant for both groups). There was no difference in early pregnancy losses in the hydrosalpinx compared to the no hydrosalpinx group: 11.3 versus 9.2% in the elevated Ab subgroup, compared to 10.0 versus 8.3% in the negative Ab subgroup (difference not significant). Seven patients with hydrosalpinx undergoing 11 cycles had their hydrosalpinges aspirated transvaginally at oocyte retrieval, immediately after aspirating all follicles. Seven cycles (63.6%) were in women with elevated C. trachomatis Ab. Two ongoing pregnancies (18.2%) and three early pregnancy losses (27.3%) were noted in this group. No intra-operative antibiotics were used; however, all patients received doxycycline, 100 mg twice, on the day of oocyte retrieval. No infectious complications were noted after transvaginal aspiration of the hydrosalpinges. Discussion In this study, the impact of unilateral or bilateral hydrosalpinx on IVF outcome was assessed in an infertile population with 528 tubal disease. We also examined the incidence of seropositivity for C. trachomatis in our population with tubal infertility. As expected, the majority of women in both the group with hydrosalpinx and the no hydrosalpinx group had elevated Ab titres (74.6 and 68.3%, respectively), underscoring the devastating role of C. trachomatis in the genesis of tubal disease (Sellors et al., 1988; Reniers et al, 1989; Cates et al, 1991). In view of previous data suggesting the presence of asymptomatic salpingitis in some women with elevated Ab, as of 1989 we began treating all couples with elevated titres with doxycycline prior to IVF (Cumining et al, 1988; Sellors et al, 1988). We noted a trend for lower implantation and pregnancy rates in the hydrosalpinx group compared to the no hydrosalpinx group; however, this was not statistically significant We were also unable to detect a difference in the incidence of early pregnancy loss between the two groups. We further subdivided the hydrosalpinx and the no hydrosalpinx groups into those with elevated or negative Ab titres. A trend towards a lower pregnancy rate and a higher rate of early pregnancy loss was noted in women with hydrosalpinx and elevated Ab titres, compared to women with no hydrosalpinx but with elevated Ab titres. Our study has only 50% power to detect a difference in implantation rate and pregnancy rate, and only 15% to detect a difference in rates of early pregnancy loss, indicating that the sample size was too small to detect a difference. A power analysis revealed that 370 patients (185 in each group) would be needed to detect a 10% difference in pregnancy rate using an a of 0.05 and a P of 0.20. The impact of hydrosalpinx on IVF success has surprisingly been addressed only recently, despite the fact that IVF was originally devised for women with tubal infertility. Table

IVF outcome in women with hydrosalpinx IV summarizes all the published reports on the effect of hydrosalpinx on IVF outcome. In contrast to the previously published studies, we could not detect a statistically significant difference among the two groups. However, we cannot rule out a treatment effect in that all couples were given 100 mg of doxycycline for 10 days prior to the first IVF cycle. To our knowledge, in the only published report assessing the effect of the concurrent therapy on IVF outcome, Torode et al. (1987) attempted to give doxycycline (100 mg twice a day for 7 days starting on day 3 of the IVF cycle) to alternate couples. Tubal factor infertility was the aetiology in 55% of the couples presenting for assisted reproduction treatment, and of those 59% had elevated C. trachomatis IgG Ab. The incidence of hydrosalpinx in that group was not reported. Because of patient refusal or non-compliance, doxycycline was administered to only 81 (36%) of 224 assisted reproduction treatment cycles (159 IVF and 65 intra-fallopian transfer cycles). Of 34 women seropositive for C. trachomatis who received doxycycline (34/ 81, 42%), seven (21%) became pregnant compared to seven (12%) of 58 women who were seropositive and did not receive treatment That difference was not statistically significant; however, the number of patients evaluated was too small to have adequate statistical power. In our study, we did not randomize patients into treatment and no treatment groups, and thus a 'therapeutic' effect of doxycycline cannot be ruled out Seven women undergoing 11 IVF cycles had their hydrosalpinges aspirated at the time of oocyte retrieval without any noted morbidity, resulting in two ongoing pregnancies and three pregnancy losses. The use of transvaginal ultrasound to aspirate hydrosalpinges with the subsequent result of successful pregnancies has been reported previously (Aboulghar et al, 1990; Russell et al., 1991). More recently, Poe-Zeigler et al. (1995) studied the impact of laparoscopic salpingectomies on 12 patients with unilateral or bilateral hydrosalpinx who had failed multiple prior IVF attempts. Seven subsequently underwent FVF and embryo transfer, from which three ongoing pregnancies and one early pregnancy loss were noted. Five patients underwent six cryo-transfers, with three achieving ongoing pregnancies (Poe-Zeigler et al., 1995). Larger prospective studies are needed to evaluate both modes of therapy further. The proposed mechanisms by which hydrosalpinx may exert a negative impact on IVF success include (i) reflux of hydrosalpinx fluid into the uterine lumen (Mansour et al, 1991), (ii) irreversible endometrial damage simultaneously with the acute-phase tubal damage (Strandell et al, 1994), (iii) release of intrauterine cytokines, prostaglandins, leukotrienes and other inflammatory compounds directly to the endometrium, or via the circulatory or lymphatic system (Grifo et al., 1989; Ben-Rafael and Orvieto, 1992), (iv) alteration in integrin expression (Lessey et al., 1994), (v) delayed hypersensitivity response secondary to increased production of a 57-kDa heat-shock protein leading to pregnancy loss (Witkin et al, 1994), or (vi) chronic endometritis caused by C. trachomatis: 41% of endometrial biopsies obtained from asymptomatic women at risk for chlamydial infection were positive for C. trachomatis (Jones et al, 1986). All the above mechanisms may potentially act by altering endometrial receptivity. Of note, C. trachomatis DNA or antigens were recently detected in 19 of 24 women with post-infectious tubal factor infertility in tissue biopsies of fimbrial and peritubal adhesions by in-situ hybridization, immunoperoxidase staining or electron microscopy, suggesting a persistent infection in these women even after antibiotic treatment (Patton et al, 1994). More recently, Witkin et al (1995) detected C. trachomatis from the endocervix by polymerase chain reaction in 20/307 (6.5%) of women prior to IVF who had negative cervical cultures. Similar findings may be at play in the uterus causing chronic endometritis and leading to altered endometrial receptivity. The results of our study support the last mechanism in that marked improvement in implantation rate, pregnancy rate and early pregnancy losses were noted after antibiotic therapy prior to IVF. Hydrosalpinx increases the incidence of ectopic pregnancies after natural cycles and assisted reproduction treatment (Zouves et al, 1991; Dubuisson et al, 1991; Vasquez et al, 1995). In a prospective study using intra-operative microscopy to grade mucosal adhesions, Vasquez et al (1995) documented an intrauterine pregnancy rate of 58% and no tubal pregnancies when no mucosal adhesions were identified, compared to an intrauterine pregnancy rate of 22% and an ectopic pregnancy rate of 11% when mucosal adhesions were present Ectopic pregnancies were also encountered in 1.2% of 21 870 embryo transfers and 4.9% of all assisted reproduction treatment pregnancies in the US and Canada in 1992 (Society for Assisted Reproductive Technology, 1994). However, these cases were not broken down to identify those associated with hydrosalpinx. Of 640 embryo transfer cycles performed in patients with tubal factor infertility, Zouves et al. (1991) encountered 13 ectopic pregnancies (2.0% of cycles, 12% of total pregnancies achieved). Of these, 10 were in patients who had prior tubal reconstructive surgery, nine of whom had hydrosalpinx. Analysing the outcome of 556 pregnancies established in 3103 IVF/embryo transfer cycles, Dubuisson et al. (1991) noted 48 ectopic pregnancies (1.6% of cycles, 8.6% of established pregnancies). Of these, 43 (89.6% of all ectopic pregnancies, 11.1% of total pregnancies) were in women with tubal infertility. The incidence of ectopic pregnancies was markedly lower in cycles performed for other indications (2.1% in endometriosis, 3.4% in cases of unexplained infertility). When the ectopic pregnancies were broken down according to the type of tubal lesions, both hydrosalpinx and proximal obstruction were associated with an increased number of ectopic pregnancies (14.2% of total pregnancies for hydrosalpinx and 16.2% for proximal obstruction) compared to bilateral salpingectomy (4.0%). In our study, the two ectopic pregnancies occurred in the hydrosalpinx group (1.9% of 103 hydrosalpinx cycles, 5.3% of pregnancies in the hydrosalpinx group, 2.7% of total pregnancies), and both were in women who had very high C. trachomatis Ab titres (>1:512 in both). In view of our results and the fact that interstitial pregnancies can occur after IVF (Pavic et al, 1986), we do not agree with the recommendations of some investigators that the presence of hydrosalpinx necessitates surgical intervention (bilateral salpingectomy, surgical correction, tubal ligation, medial resec- 529

FJ.Sharara et al tion, or laparoscopic salpingostomy) prior to IVF (Andersen et al, 1994; Kassabji et al., 1994; Lessey et al., 1994; Strandell etal, 1994). Dubuisson etal (1991) estimated that performing bilateral salpingectomy in patients with tubal infertility to reduce the risk of ectopic pregnancy would result in an unnecessary procedure in 88.9% of cases. Performing bilateral salpingectomy prior to IVF to maximize pregnancy rates may also result in unnecessary surgery. In summary, this is the first study evaluating the role of prior C. trachomatis pelvic infection and hydrosalpinx on IVF outcome. In view of the improved results with antibiotic therapy in couples with elevated Ab titres, we recommend routine testing for serum C. trachomatis IgG Ab and that all couples with elevated serum titres of C. trachomatis IgG antibodies should be treated with doxycycline for at least 1 week prior to IVF to optimize pregnancy rates. 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