Ultrasound Obstet Gynecol 2009; 34: 715719 Published online 9 November 2009 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/uog.

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Ultrasound monitoring in patients undergoing in-vitro fertilization after methotrexate treatment for ectopic pregnancy
M. PROVANSAL*, A. AGOSTINI*, O. LACROIX, S. GERBEAU*, J.-M. GRILLO and M. GAMERRE*
*Department of Obstetrics and Gynaecology and Laboratory of Reproductive Biology, Conception Hospital, Marseille, France

K E Y W O R D S: ectopic pregnancy; endometrial quality; in-vitro fertilization; methotrexate; ultrasound monitoring

ABSTRACT
Objectives To compare sonographic characteristics of the endometrium and follicles during in-vitro fertilization (IVF) before and after methotrexate (MTX) treatment for ectopic pregnancy. Methods This retrospective study, conducted at Conception Hospital from January 2000 to July 2007, included all patients diagnosed with an ectopic pregnancy resulting from IVF treatment that was treated with MTX and who then underwent another IVF cycle. We compared the number and size of follicles and the endometrial thickness and quality on the day of human chorionic gonadotropin injection in the cycles before and after the MTX treatment to determine whether MTX had any effect. Results Eleven patients were included in the study. The median interval between the IVF cycle resulting in ectopic pregnancy and the rst IVF cycle after MTX therapy was 180 (range, 150900) days. There was no statistically signicant difference between the before and after MTX treatment groups with respect to number of follicles (14 (320) vs. 9 (416), P = 0.12), follicle size (16.5 (14.721.7) mm vs. 17.8 (14.919.8) mm, P = 0.37), endometrial thickness (10.0 (9.512.0) mm vs. 10.0 (7.514.0) mm, P = 0.31) or endometrial quality (P = 0.32). Four women became pregnant during the IVF cycle following MTX treatment. Conclusions Ultrasound monitoring showed no modication of the characteristics of the endometrium or follicles during IVF after MTX treatment for ectopic pregnancy. Copyright 2009 ISUOG. Published by John Wiley & Sons, Ltd.

INTRODUCTION
Methotrexate (MTX) is frequently used to treat ectopic pregnancy medically, thus avoiding surgery1,2 . Although the success rate is variable, it can be very high in carefully selected populations3,4 . This treatment appears to be highly appropriate for patients with ectopic pregnancy after in-vitro fertilization (IVF), as it avoids an additional surgical procedure for patients who often have already had several operations and repeated anesthesia5 . MTX is a folic acid antagonist that inhibits dihydrofolate reductase, the enzyme necessary for conversion of dihydrofolic acid to tetrahydrofolic acid. Because inhibition of this enzyme blocks the synthesis of purine bases, it prevents DNA synthesis and inhibits cell multiplication6,7 . MTX has harmful effects on ovarian function and can induce premature ovarian failure8 10 . Yet three recent studies evaluated its inuence on the ovarian response and endometrial thickness after ovarian stimulation for IVF and found no quantitative modication of either11 13 . These results must, however, be conrmed by other studies. Controlled ovarian hyperstimulation for IVF requires rst assessing the status of the ovarian follicles, then implementing a hormone strategy and nally monitoring follicle maturation. The ovarian examinations include ultrasound to evaluate the number of follicles and their size and hormone tests to measure folliclestimulating hormone (FSH) and estradiol (E2). Ultrasound is further used to monitor follicle maturation during ovarian stimulation14 as well as endometrial patterns. However, there are currently no data available on the sonographically detectable modication of endometrial thickness and quality and ovarian follicles during IVF stimulation after MTX treatment for ectopic pregnancy.

Correspondence to: Dr M. Provansal, Department of Obstetrics and Gynecology, Conception Hospital, 147 Boulevard Baille, 13005 Marseille, France (e-mail: magali.provansalcheylan@ap-hm.fr) Accepted: 27 April 2009

Copyright 2009 ISUOG. Published by John Wiley & Sons, Ltd.

ORIGINAL PAPER

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A recent publication reported clinical data conrming that MTX did not inuence endometrial thickness in a subsequent IVF cycle13 , but no previous study has evaluated its inuence on endometrial quality or on the number and size of follicles. The aim of this study was to evaluate the characteristics of the endometrium and ovarian follicles during IVF before and after MTX treatment for ectopic pregnancy.

METHODS
This retrospective study reviewed records of patients seen at Conception Hospital between January 2000 and July 2007, having been admitted to our department with a diagnosis of ectopic pregnancy following IVF treatment, who were successfully treated with MTX and who then underwent another IVF cycle. The criteria for ectopic pregnancy diagnosis and indications for MTX were similar to those used in our department for women with spontaneous ectopic pregnancy15 . Ectopic pregnancy was diagnosed from clinical data, human chorionic gonadotropin (hCG) course and ultrasound data15 . Patients were not eligible for MTX treatment if they met any of the following criteria: preference for surgical treatment, declining hCG levels, hemodynamic instability, ectopic pregnancy with cardiac activity, hepatic dysfunction, blood dyscrasia, kidney disease evidenced by aspartate aminotransferase levels greater than twice the upper limit of normal, white blood cell count less than 1500/ mL, platelet count less than 100 000/ mL, or serum creatinine level greater than 1.5 mg/dL. Administration of MTX was by injection intramusculary or into the site of the ectopic pregnancy under sonographic control16,17 . Patients received a single dose of MTX (1 mg per kg actual body weight) and were monitored according to a standardized clinical protocol18 . The study excluded all patients who required supplementary surgical treatment. All subsequent IVF cycles were performed at least 150 days after MTX treatment. We applied the protocol used by Lipscomb et al.19 for follow-up. The following characteristics of stimulation protocols before and after MTX treatment were recorded: duration of stimulation; FSH dosage; mean E2 and luteinizing hormone levels on day of hCG injection. We classied endometrial quality according to Gonen et al.20 , with three different types of pattern being distinguished20 : (I) a multilayered endometrium (Figure 1); (II) an intermediate-type endometrium (Figure 2); and (III) an entirely homogeneous, hyperechogenic endometrium (Figure 3). We also evaluated the ratios of E2 either to the number of follicles > 14 mm in diameter on the day of hCG administration (E2/follicle) or to the number of oocytes retrieved (E2/oocytes) during controlled ovarian hyperstimulation. All patients participating in the study provided written informed consent. The institutional review board was consulted and determined that, in accordance with French public health law, its approval was unnecessary

Figure 1 Longitudinal view of the uterine cavity demonstrating a day 14 endometrium, 11.1 mm thick (D1), with a triple-layer pattern.

Figure 2 Longitudinal view demonstrating an intermediate type endometrium on the day of human chorionic gonadotropin injection.

because the patient received standard management that involved no additional, unusual or innovative diagnostic or follow-up procedures. Results are presented as medians and ranges. Qualitative variables were compared by Wilcoxons test. Differences were considered to be signicant when P < 0.05.

RESULTS
Eleven patients met the inclusion criteria during the study period. Their median age at the IVF cycle leading to the ectopic pregnancy was 32 (range, 2741) years and at the cycle after MTX treatment it was 34 (range, 2742) years (P = 0.02). Indications for IVF were: tubal (n = 4), male (n = 2), tubal and male (n = 2), tubal and related to endometriosis (n = 1) or unexplained (n = 2). MTX was administered intramuscularly in all but one patient. During ectopic pregnancy follow-up, two patients required a second intramuscular injection of MTX. The

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Figure 3 Longitudinal views of the uterine cavity demonstrating entirely homogeneous, hyperechogenic endometrium, 29.4 mm thick (a) and 11.2 mm thick (b).

median interval between the IVF cycle resulting in ectopic pregnancy and the rst IVF cycle after MTX therapy was 180 (150990) days. All patients underwent the same controlled ovarian hyperstimulation protocol before and after MTX treatment (Table 1). There was no statistically signicant difference before and after MTX therapy with respect to the number and size of follicles. The E2/oocytes ratio was higher after MTX treatment but there was no signicant difference in fertilization rate. Nor was there any statistically signicant difference between groups for either endometrial thickness or endometrial quality. The number of oocytes per retrieval declined signicantly after MTX treatment. Four patients became pregnant during the IVF cycle following MTX treatment, giving a pregnancy rate of 36.4%. This fertilization rate was similar to the clinics general fertilization rate (30.1%). There was no recurrence of ectopic pregnancy among these pregnancies.

DISCUSSION
We found that women had similar IVF cycles, in terms of both endometrial thickness and quality and follicle

number and size, before and after MTX treatment of ectopic pregnancy. There are two plausible reasons why MTX treatment for ectopic pregnancy should not affect endometrial thickness and quality. The rst relates to the small dose of MTX used, too small to have had a longterm effect on the ovaries or endometrium6 . The second relates to the waiting time applied at our clinic: a new IVF cycle was not started until at least 150 days after MTX treatment. High-dose MTX was reported by Longhi et al.8 to have harmful effects on ovarian function; they found a premature ovarian failure rate of 4.2% in patients treated with MTX as adjuvant therapy for osteosarcoma. Lower et al.9 reported a high frequency of menstrual irregularities and premature ovarian failure in a group of older patients treated with MTX for breast cancer. Bower et al.10 also observed that menopause was advanced by 3 years in patients who maintained gonadal function after chemotherapy. However, at the doses of MTX used in the medical treatment of ectopic pregnancy and with the time intervals between treatment and the following IVF cycle, the patients reproductive future is not compromised. Stovall et al.21 rst reported that patients with ectopic pregnancy treated with MTX had a timely return of menses and better rates of both tubal patency on hysterosalpingography and pregnancy than did patients treated with conservative surgical management22,23 . Further studies specically examined reproductive outcome in infertile populations and showed no effect on ovarian reserve of a single dose of MTX12,13 . McLaren et al.13 reported that MTX had a reversible impact on endometrial thickness in the IVF cycle after treatment. In cycles occurring within 180 days after MTX exposure, they observed slightly decreased endometrial thickness and a statistically signicant decline in oocytes retrieved. Cycles more than 180 days after MTX exposure showed no signicant decrease in these variables. In our clinic, we defer fertility treatment for 150 days after MTX administration. Although the median number of oocytes per retrieval declined signicantly after MTX treatment, this reduction may have been related to patient age, the median age being signicantly higher in the group following MTX treatment24 26 . This age difference may also explain in part the reduction in number of oocytes retrieved and is a limitation of this study. With the exception of FSH level on day 3 and antral follicle count, biological markers of ovarian reserve (inhibin B, antiMullerian hormone) were not available for all patients. Absence of these data is another limitation of this study. Endometrial thickness is an easily measurable ultrasound parameter and indicates endometrial growth during the cycle. A linear correlation between endometrial thickness and the probability of conception has not been established denitively27 . In this study, we found that MTX did not inuence either endometrial thickness or quality. Welker et al. suggested that the endometrial pattern may be a predictor of implantation following IVF and embryo transfer28 . A triple-line pattern has been associated with cycles of conception more frequently than

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Table 1 Comparison of ovarian stimulation protocols and results of in-vitro fertilization cycles before and after ectopic pregnancy treated by methotrexate (MTX) Clinical characteristic Age (years) FSH level on day 3 (IU/L) Duration of stimulation (days) Dose of FSH administered (IU) Day of hCG injection E2 level (pg/mL) Endometrial thickness (mm) Endometrial quality Type I Type II Type III Follicle number E2/follicle (pg/mL) Follicle size (mm) Number of oocytes collected E2/oocyte (pg/mL) Number of embryos obtained Fertilization rate (%) Before MTX (n = 11) 32 (2741) 5.80 (2.0911.20) 12 1875 (11876000) 2230 (5063480) 10.0 (9.512.0) 7 3 1 14 (320) 193.33 (36.14313.75) 16.5 (14.721.7) 10 (321) 257.78 (42.50587.00) 8 (212) 88.9 (66.7100) After MTX (n = 11) 34 (2742) 7.90 (5.1013.70) 13 2325 (12374500) 1720 (3843330) 10.0 (7.514.0) 8 2 1 9 (416) 175.45 (81.80268.57) 17.8 (14.919.8) 5 (122) 333.00 (58.431060.00) 4 (119) 100 (25.0100)
P

0.02 0.71 0.73 0.57 0.59 0.31 0.32

0.12 0.93 0.37 0.01 0.03 0.07 0.67

Data are presented as n or median (range). E2, estradiol; FSH, follicle stimulating hormone; hCG, human chorionic gonadotropin.

has any other sonographic parameter and may thus be the one that best reects, albeit indirectly, endometrial receptivity29 . It is nonetheless at most a non-specic parameter for the prediction of conception following assisted reproduction treatment. The main limitation of our study was that the sample size may have been too small statistically. Further studies are needed to elucidate the inuence of MTX treatment on endometrial characteristics and ovarian follicles. In conclusion, the results of our study of fertility after medical treatment of ectopic pregnancy are similar to those in the literature. Our data conrm that MTX does not compromise the patients performance in her next IVF cycle and that no ultrasound modication is seen in the endometrial or follicular characteristics during IVF monitoring.

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Ultrasound Obstet Gynecol 2009; 34: 715719.