Abstract. Introduction. RBMOnline - Vol 10. No Reproductive BioMedicine Online; on web 18 February 2005

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1 RBMOnline - Vol 10. No Reproductive BioMedicine Online; on web 18 February 2005 Article Effect of inner myometrium fibroid on reproductive outcome after IVF Luca Gianaroli is the scientific director at the S.I.S.Me.R. Reproductive Medicine Unit in Bologna. He graduated at the Medical School of the University of Bologna in After graduation as a specialist in Gynaecology and Obstetrics in the University of Genova in 1982, he was appointed as Senior Research Fellow in the Department of Obstetrics and Gynaecology, Monash University, Melbourne. His experience there lasted two years under the supervision of Carl Wood and Alan Trounson and included in-vitro fertilization and embryo transfer techniques in humans and animals. Returning to Bologna, he began his clinical activity in reproductive medicine expanding his research to the most advanced techniques of the field, including preimplantation genetic diagnosis and stem cell research. Dr Luca Gianaroli Luca Gianaroli 1,4, Stephan Gordts 2, Arianna D Angelo 1, M Cristina Magli 1, Ivo Brosens 2, Carlo Cetera 3, Rudi Campo 2, Anna Pia Ferraretti 1 1 SISMeR Reproductive Medicine Unit, Via Mazzini, Bologna, Italy; 2 LIFE, Leuven Institute for Fertility and Embryology, Leuven, Belgium; 3 Ospedale Civile, Unit of Obstetrics/Gynaecology, Pieve di Cadore, Belluno, Italy 4 Correspondence: Fax: ; sismer@sismer.it Abstract To evaluate the influence of inner myometrium fibroids (myomas) on the outcome of IVF cycles, a retrospective agematched controlled study was performed at SISMeR Reproductive Medicine Unit. The study group included 129 IVF/intracytoplasmic sperm injection cycles in 75 patients with one or more intramural and/or submucosal fibroids, while the control group consisted of 129 cycles in 127 patients without fibroids. The two groups were similar for mean oestradiol concentration at human chorionic gonadotrophin administration ( ± 874 versus 1395 ± 821 pg/ml), mean number of transferred embryos (2.02 ± 0.4 versus 2.14 ± 0.6) and clinical pregnancy rate (34.9 versus 41.1%). Conversely, the implantation rate was significantly lower in the study group (18.0%) than in the control group (26.5%; χ 2 = 4.81, P< 0.05), whereas the rate of spontaneous abortion demonstrated an opposite trend (40 versus 18.9%; χ 2 = 4.34, P < 0.05). Further research should be aimed at classifying fibroids on the basis of their location, especially when they are positioned in the junctional zone of the myometrium. Whether this classification will be superior in predicting the impact of fibroids on the reproductive outcome should be elaborated in a large multicentric study. Keywords: abortion, assisted reproduction, fibroid, inner myometrium, reproductive outcome Introduction The incidence of fibroids (myomas) in women of reproductive age is reported to be between 20 and 40% (Verkauf, 1992; American Society for Reproductive Medicine, 2001). Their presence could cause failure to conceive, but no scientific evidence supports improvement after the surgical removal of the fibroid. This was recently highlighted in a review (Donnez and Jadoul, 2002) reporting a pregnancy rate after myomectomy in infertile women varying between 10 and 80%. A number of mechanisms by which fibroids could cause reduced fertility have been suggested and these include mechanical factors associated with the deformation of the uterine contour (for reviews see Verkauf, 1993; Wallach and Vlahos, 2004). This could interfere with sperm access to the cervix and uterine transport, distort or occlude the tubal ostia, or alter uterine contractility with consequent effect on normal sperm transport. An increased risk of spontaneous abortion has been attributed to augmented uterine contractions and growth or degeneration of fibroids. However, none of these potential mechanisms has been clearly demonstrated to adversely affect the outcome of the feto maternal relationship, or to cause pregnancy wastage. Factors influencing transport and fertilization are eliminated in assisted reproductive technologies and the evaluation of implantation and pregnancy rate and outcome have been investigated in retrospective and prospective studies. Seven case control trials published during the last decade have provided conflicting information regarding the impact of fibroids on these outcome measures. Two studies found that pregnancy rate is decreased when uterine leiofibromyomas cause deformation of the uterine cavity (Farhi et al., 1995; 473

2 474 Ramzy et al., 1998). Two other studies (Eldar-Geva et al., 1998; Stovall et al., 1998) reported significantly reduced pregnancy and delivery rates after IVF in the presence of fibroids in the inner myometrium, despite the absence of uterine deformation. Accordingly, Hart and co-workers (2001) found that an intramural fibroid of 5 cm or less in size halves the chances of an ongoing pregnancy after assisted reproduction. On the other hand, Surrey and coworkers (2001) reported a significant decrease in the implantation rate in women younger than 40 years with intramural fibroids, but no significant difference in the live birth rate, while Check and colleagues (2002) found similar rates of implantation and delivery. The purpose of this study was (i) to report the general outcome in assisted reproduction cycles performed by patients with fibroids in the inner myometrium and (ii) to discuss the current problems in evaluating the impact of uterine fibroids on IVF/intracytoplasmic sperm injection (ICSI) results. Materials and methods This retrospective case control study was based on the analysis of the results generated by patients undergoing IVF/ICSI cycles at the SISMeR Reproductive Medicine Unit from 1996 to Before being referred to the assisted reproduction unit, patients underwent physical examination and vaginal ultrasound to evaluate the presence of uterine fibroids and signs of uterine cavity distortion. In suspected cases, hysteroscopy was performed. The study included 129 treatment cycles performed in 75 patients with fibroids (study group) and 129 cycles in 127 patients without fibroids (control group) matched for age and cause of infertility. The study group also included 14 patients with recurrent fibroids after a previous myomectomy. Results were analysed in terms of clinical pregnancies (defined as presence of a gestational sac with fetal heart beat), implantation rates and spontaneous abortions. These figures were related to size and number of fibroids by dividing the patients into subgroup A, with three or fewer fibroids, and subgroup B, having more than three fibroids. In five patients with a negative outcome in the first assisted reproduction cycle, myomectomy was performed prior to further attempts. The reproductive outcome was analysed before and after myomectomy. All patients received the same hormonal ovarian stimulation protocol after suppression with long acting gonadotrophinreleasing hormone (GnRH) agonists (Ferraretti et al., 1996). Oocyte retrieval was performed h after human chorionic gonadotrophin (HCG) administration and embryo transfer was programmed 2 or 3 days later. The extracorporeal phases have been previously described (Gianaroli et al., 1996). Supernumerary embryos were cryopreserved and thawed in following cycles (Ferraretti et al., 1999); these results were included in the present study giving cumulative data per cycle performed. Statistical analysis Comparisons were made by Student s t-test and chi-squared analysis 2 2 contingency tables when appropriate. P < 0.05 was considered as statistically significant. Results There was no statistical difference between study and control groups regarding mean maternal age (35.8 ± 4.9 versus 35.7 ± 4.8 years), mean oestradiol concentration at HCG administration (1205 ± 874 versus 1395 ± 821 pg/ml), mean number of embryos transferred (2.02 ± 0.4 versus 2.14 ± 0.6) and cause of infertility (Table 1). The mean number of fibroids diagnosed in the inner myometrium was 2.46 ± 2.8, with a mean diameter of 1.84 ± 1.4 cm (Table 2). The clinical pregnancy rate per transferred cycle between the two groups was similar (34.9 versus 41.1%). However, the implantation rate was significantly lower in the study group compared with the control group (18.0 versus 26.5%; χ 2 = 4.81, P < 0.05), while the rate of spontaneous abortions demonstrated an opposite trend (40.0% in the study group and 18.9% in the control group χ 2 = 4.34, P< 0.05) (Table 3). Accordingly, the take-home baby rate per transferred cycle was higher in the control group (33.3%) compared with the study group (21.0%, χ 2 = 4.41, P < 0.05). The incidence of multiple pregnancies was 6.7% in the study group and 18.9% in the controls. Excluding patients with multiple pregnancies, the mean birth weight of the infants born was 2804 ± g in the study group versus 3070 ± g in the control group with a preterm birth rate in the study group of 33.3% (9/27) versus 18.6% (8/43) in the control group (not significant). In the study group, 112 transfers were performed in patients with three or fewer fibroids (subgroup A) and 17 transfers in patients with more than three fibroids (subgroup B). The mean number of fibroids was 1.2 ± 0.5 in subgroup A and 5.7 ± 1 in subgroup B. The two subgroups were homogeneous in terms of mean maternal age (36.4 ± 5.1 versus 37 ± 2.7 years) and mean number of transferred embryos (2.0 ± 0.9 versus 2.6 ± 0.9). The rate of clinical pregnancies was 35.7% in subgroup A and 29.4% in subgroup B versus 41.1% in the control group, while the abortion rate was 35.0, 80.0 and 18.9% respectively (subgroup B versus controls: χ 2 = 6.28, P < 0.01) (Table 4). The results were evaluated according to the size of the fibroids (<1, 1 <3, 3 cm). In case of single fibroids, no statistical differences were noted in the rates of pregnancy (40, 37, 28%) or abortion (13, 13, 8%), whereas when multiple fibroids were present with a diameter 3 cm, the abortion rate was significantly higher compared with controls (80.0 versus 18.9%, P < 0.01). In five patients with a mean maternal age of 39.5 ± 5.1 years, data pre- and post-myomectomy were analysed. The cause of infertility was tubal in two patients, ovulatory in two patients and male factor in one patient. Before myomectomy, five embryo transfers resulted in one spontaneous abortion, while after myomectomy, seven transfers resulted in four term pregnancies and one spontaneous abortion.

3 Table 1. Cause of infertility. Study Control group (%) group (%) Unexplained Endometriosis Male factor Tubal factor Male + tubal factor Ovulatory factor Genetic factor + recurrent abortions Table 2. Patient characteristics in the study and control groups. Study group Control (n = 75) group (n = 127) No. transferred cycles Age (mean ± SD, years) 35.8 ± ± 4.8 Oestradiol (mean ± SD, pg/ml) 1205 ± ± 821 No. retrieved oocytes (mean ± SD) 942 (8.7 ± 4.5) 1191 (9.3 ± 4.1) No. fertilized oocytes (%) 491 (67) 671 (56) No. of fibroids (mean ± SD) 2.46 ± 2.8 Size of fibroids (mean ± SD, cm) 1.84 ± 1.4 Table 3. Reproductive outcome. Study group Control group No. transferred embryos (mean ± SD) 2.02 ± ± 0.6 No. clinical pregnancies/transfer (%) 45/129 (34.9) 53/129 (41.1) No. multiple pregnancies (%) 3/45 (6.7) 10/53 (18.9) Implantation rate (%) 48/267 (18.0) a 63/238 (26.5) a No. spontaneous abortions (%) 18/45 (40.0) b 10/53 (18.9) b Take-home baby rate/transfer (%) 27/129 (21.0) c 43/129 (33.3) c Take-home baby rate/patient (%) 27/75 (36.0) 43/127 (33.9) a,b,c Values with the same superscript are significantly different: χ 2 = 4.81 (P < 0.05), χ 2 = 4.34 (P < 0.05), and χ 2 = 4.41 (P < 0.05) respectively. Table 4. Pregnancy and abortion rates in relation to the number of fibroids. 3 fibroids >3 fibroids Control No. transferred cycles Age (mean ± SD, years) 36.4 ± ± ± 4.8 No. of fibroids (mean ± SD) 1.2 ± ± 1 Size of fibroids (cm) (mean ± SD) 2.1 ± ± 1.2 No. of transferred embryos (mean ± SD) 2.0 ± ± ± 0.6 No. of clinical pregnancies (%) 40 (35.7) 5 (29.4) 53 (41.1) No. of abortions (%) 14 (35.0) 4 (80.0) a 10 (18.9) a Implantation rate (%) 43/221 (19.5) 5/46 (10.8) b 63/238 (26.5) b a,b Values with the same superscript are significantly different: χ 2 = 6.28 (P < 0.01), χ 2 = 4.33 (P < 0.05) respectively. 475

4 476 Discussion The data from this study confirm that small fibroids of the inner myometrium with no manifest impingement on the uterine cavity are associated with a reduced implantation rate and an increased incidence of spontaneous abortions in assisted reproduction. Although the presence of fibroids did not seem significantly to affect the pregnancy rate when compared with the performance observed in the control group, the take-home baby rate per transfer was significantly higher in the controls (P < 0.05). Interestingly, this difference disappeared when the take-home baby rate was calculated per patient, suggesting that women with uterine pathology need to undergo more cycles to reach a term pregnancy compared with the controls. According to the present results, the increase in spontaneous abortions became even more pronounced when more than three fibroids were present reaching an abortion rate of 80.0%. These data are in agreement with previous studies claiming a higher rate of pregnancy loss in the presence of uterine fibroids (Eldar-Geva et al., 1998; Li et al., 1999; Hart et al., 2001). For correct evaluation of the current results, it is important to consider that, as in other studies, some major limitations were confronted. The first is represented by the retrospective design of this case controlled study, including patients who were treated during the same period and were matched for age and cause of infertility. However, control of the uterine cavity was not systematically performed by hysterosalpingography, hydrosonography or hysteroscopy in all patients entering the study. On the other hand, controversy still persists regarding the most appropriate technique to exclude uterine impingement (Nawroth and Foth, 2002). Secondly, the sample size was not large enough for a robust statistical evaluation of the differences detected in the two groups, suggesting that multicentric studies are required. Comparison with the results in previous studies was hampered by differences in pathology definition and treatment modalities. It has been reported that the duration and depth of ovarian suppression by GnRH agonist treatment may affect the rate of implantation, implying that similar stimulation protocols should be followed to allow comparability. There is considerable variation in the mean number of transferred embryos, with a range from two to three or more. It is remarkable that studies with a mean number of three embryos transferred did not observe any effect on the clinical outcome due to the presence of fibroids in the inner myometrium (Surrey et al., 2001; Check et al., 2002), while studies in which a mean number of two embryos were transferred (Hart et al., 2001) reported a negative effect related to fibroids. Finally, in most studies the outcome measures included implantation and pregnancy rates, but not the full spectrum of pregnancy outcome ranging from spontaneous abortion to the birth of a healthy infant. Surrey and co-workers (2001) reported no differences in live birth rate, but did not indicate the rate of multiple pregnancies and premature births. As shown by recent studies (Koudstaal et al., 2000a,b; Schieve et al., 2002), multiple pregnancies may mask a deleterious effect due to fibroids, such as the reduced development in relation to the gestational week, which is apparent in singletons. Interestingly, the only study recording birth weights (Eldar- Geva et al., 1998) reported that the mean birthweight of singletons was significantly lower in the fibroid group than in the control group. A major problem in the current classification of uterine fibroids is the distinction between intramural and submucosal fibroids. The distinction may be important for the feasibility of hysteroscopic surgery, but its pathophysiological significance is unsupported by experimental data, and is very questionable. While subserosal and submucosal leiomyomas are likely to originate respectively from the outer myometrium and junctional zone myometrium, intramural fibroids may originate from the outer myometrium as well as from the junctional zone of the myometrium. The heterogeneous composition of the intramural group may explain the uncertain effect of these fibroids in current studies. On the other hand, there are major clinicopathological differences between outer myometrium and junctional zone fibroids (Brosens et al., 1998b), including frequency and types of cytogenetic abnormalities, vascularization, expression of sex steroid hormone receptors and response to GnRH agonist treatment. Recent reports on the ultrastructural examination of normal myometrium, host myometrium of fibromatous uteri and fibromyomata have demonstrated that myocytes of fibromyomata are different from those in the normal myometrium and that the host myometrium shows intermediate changes (Richards et al., 1998). Through the menstrual cycle and under the influence of cyclic hormonal changes, distinctive alterations occur in the myometrium of the junctional zone, but not in the outer myometrium (Brosens et al., 1998a). These changes suggest that fibroids of the junctional zone have an impact on the reproductive outcome that is different from that associated to fibroids of the outer myometrium. Accordingly, several observations have indicated that the junctional zone myometrium is relevant to both fertility and placenta formation. Lesny and colleagues demonstrated a correlation between zona thickness and subsequent implantation in assisted reproduction cycles (Lesny et al., 1999). Focal disruption of the junctional zone myometrium in a conception cycle has been reported by others (Turnbull et al., 1995), with endometrial decidualization extending to the junctional zone spiral arteries, which are involved in the subsequent process of placentation (Brosens, 1977; Pijnenborg et al., 1980). As a consequence, disruption of the junctional zone myometrium can have significant effects on uterine functionality. In light of these considerations, there is no agreement on the degree at which the anatomical impingement of the uterine cavity by the fibroid could negatively affect the reproductive outcome. Alternatively, as proposed by Brosens and co-workers (2003), the classification of uterine fibroids in clinicopathological studies may be based on their relationship with the myometrium of the junctional zone rather than on the impact on the uterine cavity. It has been suggested that T2- weighted magnetic resonance imaging scans and highresolution ultrasound would be useful to delineate the junctional zone and to determine whether the junctional zone myometrium is involved in the disease process. MR imaging has been proved to be superior to ultrasound and hysterosalpingography for sensitivity and accuracy in the

5 localization and measurement of uterine fibroids (Dudiak et al., 1988). The results of the current case control study are in favour of a myomectomy in case of submucous fibroid prior to an assisted reproduction cycle, whereas for subserosal fibroid an operative procedure seems not to be necessary. A myomectomy would be especially indicated for intramural fibroid with a diameter of >3 cm or for multiple intramural fibroids <3 cm in diameter. For intramural fibroids (<1 cm), there is no evidence of any benefit derived from a myomectomy, whereas in cases of repeated assisted reproduction failures or early pregnancy loss, an operative correction can be considered. As previously suggested, further research should be dedicated to classify fibroids based on their involvement of the junctional zone myometrium. Only large multicentric studies having as outcome measures the full range of reproductive events from implantation to birth will clarify whether this classification is superior in predicting the impact of fibroids on reproductive outcome. It is remarkable that after 25 years of assisted reproduction, the basic question of whether fibroids affect the pregnancy outcome is still unanswered. References American Society for Reproductive Medicine 2001 Myomas and Reproductive Function. A practice committee report. Released November Practice Committee Guidelines are accessible to ASRM members only. Brosens IA 1977 Morphological changes in the utero placental bed in pregnancy hypertension. Clinical Obstetrics and Gynaecology 4, Brosens J, Campo R, Gordts S et al Submucous and outer myometrium leyomyomas are two distinct clinical entities. 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