Effect of Single and Multiple Methotrexate Doses Treatment on Ovarian Reserve in Ectopic Pregnancy Women: A Prospective Cohort Study

Background One of the main concerns for women diagnosed with ectopic pregnancy (EP). This study has been designed to evaluate the potential effects of methotrexate (MTX) single and multi-dose exposures on the ovarian reserve.

This was a prospective observational cohort study that was held in the Imam Khomeini Hospital, Ahvaz, Iran, from June 2021 to October 2022. Demographic information such as (age, body mass index (BMI), gravid, and parity) were recorded. Follicle stimulation hormone (FSH), estradiol plasma levels (on day 3 of the cycle), and Anti-Mullerian Hormone (AMH) were measured two times before MTX administration (pre-treatment) and then eight weeks after treatment (post-treatment). Also, antral follicle count (AFC) was evaluated by trans-vaginal ultrasound pre- and post-treatment.

One hundred and seventy-two patients were recruited into the study. There was no difference in levels of FSH, AMH and AFC between single-dose and multiple-dose groups. There was only a statistically significant difference in estradiol levels between the two groups (P: 0.02), and estradiol concentrations were higher in the multiple-dose MTX group.

Administration of MTX (regardless of number of doses) has no negative effect on ovarian reserve and is safe to be used as a treatment for EP.

Fertility, Ovarian reserve, Pregnancy complications, Ectopic pregnancy, Aminopterin, Methotrexate

One of the main concerns for women diagnosed with ectopic pregnancy (EP) is the potential adverse effect of the treatment techniques on ovarian reserve and fertility preservation [1]. EP complicates about 1–2% of all pregnancies, which could lead to potentially life-threatening hemorrhage that seriously affects maternal mortality and morbidity [2, 3]. Recently, maternal mortality rates have subsided due to advances in early diagnosis and treatment [4]. While laparoscopic approaches (salpingectomy or salpingostomy) are the gold standard and mainstay of managing EP, some patients are candidates for medical treatment [5]. The primary agent used for medical treatment is methotrexate (MTX) [6, 7]. Single- or multiple-dose MTX administration currently is used for the treatment of patients with EP [8]. MTX is an antimetabolite agent, acting as an antifactual acid antagonist against the growth of active cells [9], which might devastate proliferating cells within the ovaries and may lead to diminished ovarian reserve [10]. The ovarian reserve determines the size, the quality of the remaining ovarian follicular pool, and the functional potential of the ovary, which predicts the fecundity of a woman [11]. They use both biochemical and ultrasonic methods for their tests. Follicle stimulation hormone (FSH) concentration was one of the earliest tests used to evaluate ovarian reserve; its result was interpreted along with estradiol concentration [12]. Anti-Mullerian hormone (AMH) is an endocrine marker produced by granulosa cells of preantral and small antral follicles. The AMH marker is considered to be one of the best ovarian reserve markers, and its level has slight variation during different stages of the menstrual cycle [13, 14]. Antral follicle count (AFC) correlates with oocyte yield and is accepted as an excellent marker for assessing ovarian reserve [15]. Due to the low diagnostic performance of FSH and estradiol measurements, they are widely replaced by AMH, and AFC [16]. The impact of multiple-dose MTX on ovarian reserve has not been assessed in a few studies, their results are controversial [17–19]. Thus, this study has been designed to evaluate the potential effects of MTX single and multi-dose exposures on the ovarian reserve.

Setting This was a prospective observational cohort study that was held in the Imam Khomeini Hospital, Ahvaz, Iran, from June 2021 to October 2022 based on the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) Statement [20]. Moreover, this study was approved by the Ahvaz Jundishapur University of Medical Sciences (AJUMS) Ethics Committee (IR.AJUMS.HGOLESTAN.REC.1400.068(. To help clinicians choose the best treatment option with the lowest potential adverse effect on the ovarian reserve, this study aimed to determine whether MTX has a dose-dependent effect on ovarian reserve. Population Patients who had beta-hCG levels 3000 IU/ml, adnexal mass measuring more than 2 cm in diameter received multi-dose MTX. Inclusion Criteria All 18–44 years patients who were referred to the hospital with hemodynamically stable, absent fetal heart activity on ultrasound, beta-hCG levels < 5000 IU/ml, had no contraindication for MTX use ectopic mass, and measuring less than 4 cm in diameter, were selected for medical treatment enrolled in this study. The aim of the experiment was thoroughly explained to the patients, and written consent to take part in the study was obtained from them. All patients were informed about anonymity and confidentiality during data processing and that they had the right to quit the study at any time. Exclusion Criteria The history of tuba-ovarian surgery, chemotherapy, and smoking, or active peptic ulcer, active pulmonary disease, clinically significant hepatic, pre-existing blood dyscrasias, breastfeeding, renal dysfunction, and emergency laparotomy for ruptured EP [21]. Patients with a history of any kind of infertility, endometriosis, tubo-ovarian surgery, and systemic chemoradiotherapy that could have potentially altered ovarian physiology and ovarian reserve were excluded from the study. Patients treated with surgical techniques such as salpingostomy, salpingectomy, and milking techniques were excluded [22]. Sample Size The sample size was calculated by G power software which was determined by a pilot study done on twenty subjects. Given the means of AFC post-treatment in single- and multi-dose MTX groups (7.26 and 6.85, respectively), 0.88 standard deviations with an estimated drop of 15%, and it was calculated as 170 in total [19]. Variables Demographic information such as (age, body mass index (BMI), gravid, and parity) were recorded. FSH, estradiol plasma levels (on day 3 of the cycle), and AMH were measured two times before MTX administration (pre-treatment) and then eight weeks after treatment (post-treatment) [21]. Also, AFC was evaluated by trans-vaginal ultrasound pre- and post-treatment. Data Measurement Blood samples (6 mL) were collected before administration of MTX and eight weeks after. Samples were kept at −20 °C until assayed. Serum samples were assayed for AMH and FSH plasma levels using sandwich ELISA (Pishtaz Teb kits, Tehran, Iran), and estradiol was assayed using Vidas Estradiol II kit (bioMerieux SA, France). All samples were assayed with the same kit. Furthermore, a GE-E6 7 mHz vaginal transducer was used for trans-vaginal ultrasounds (TVS). As a whole number of AFCs in each patient, follicular units measuring 2 to 10 mm on both ovaries were recorded and reported. Statistical Analysis Statistical analysis was done by SPSS statistical software. The Kolmogorov–Smirnov test was evaluated for the normality of quantitative data. The paired t test was used to evaluate any differences in AMH, LH, FSH, estradiol, and AFC values between pre- and post-treatment data (normal distribution) in each group. Student t test was used to evaluate any differences between the two groups (single-dose and multi-dose MTX groups) which a P value of < 0.05 was considered to indicate statistical significance.

Participants One hundred and seventy-two patients were recruited into the study. Group 1 (single-dose MTX group) included 85 patients; eight patients underwent emergency surgery due to ruptured EP and were excluded from the study. Group 2 (multi-dose MTX group) was comprised of 85 patients, of which, eleven patients were removed from the study due to the need for emergency surgery. Persistent EP or heterotopic pregnancy were not reported in any of the patients. Descriptive Data Patient age, BMI, gravid, parity, pre- and post-treatment biochemical markers (AMH, FSH, and estradiol) and AFC were shown (Tables 1 and 2). Table 1. | Group 1 (n: 79) | Group 2 (n: 74) | | |---|---|---| | Age (years) | 30.12 (4.02) | 29.37 (4.16) | | BMI (kg/m2) | 24.53 (2.56) | 24.78 (2.50) | | Gravid | 2.03 (0.08) | 2.02 (0.96) | | Parity | 1.03 (0.08) | 1.01 (0.95) | Data are expressed as mean (SD) Table 2. | Variable | Group | Pretreatment | Post-treatment | P value | |---|---|---|---|---| | FSH | Single-dose MTX | 7.32 (1.11) | 7.37 (1.06) | 0.607 | | Multiple-dose MTX | 7.55 (1.19) | 8.40 (1.44) | < 0.001 | | | Comparing two groups | P: 0.91 | P: 0.20 | || | Estradiol | Single-dose MTX | 22.18 (4.39) | 22.45 (4.35) | 0.376 | | Multiple-dose MTX | 22.35 (4.23) | 23.01 (5.89) | 0.325 | | | Comparing two groups | P: 0.96 | P: 0.02 | || | AMH | Single-dose MTX | 2.78 (0.75) | 2.73 (0.69) | 0.079 | | Multiple-dose MTX | 2.40 (0.73) | 2.37 (0.56) | 0.607 | | | Comparing two groups | P: 0.27 | P: 0.06 | || | AFC | Single-dose MTX | 7.46 (1.27) | 7.31 (1.26) | 0.164 | | Multiple-dose MTX | 7.06 (1.13) | 7.10 (1.02) | 0.789 | | | Comparing two groups | P: 0.06 | P: 0.32 | Data are expressed as mean (SD) There were no statistically significant differences in demographic features, including age, and BMI, between the two groups (P: 0.24, 0.3, respectively) (Table 1). Also, there was no significant difference between the two groups’ biochemical parameters and AFC baseline (pre-treatment) measurements (Table 2). Data are expressed as means (SD). Outcome Data There was no loss to follow-up, and all patients were followed precisely after eight weeks of MTX administration. In the follow-up patients, ovarian reserves were assessed one more time to compare the pre- and post-treatment data and evaluate the potential effect of MTX administration on ovarian reserve. Neither the single-dose nor the multiple-dose MTX regimens showed changes in ovarian reserve post-treatment, except for the FSH level in the multiple-dose MTX group, which was significantly higher after treatment (P < 0.001). The single-dose MTX group had baseline FSH levels of 7.32 (1.11), and 7.37 (1.06) before and after treatment, respectively (P: 0.607); estradiol values of 22.18 (4.39) and 22.45 (4.35) before and after treatment, respectively (P: 0.376); AFC values of 7.46 (1.27) before and 7.31 (1.26) after treatment (P: 0.164); and AMH levels of 2.78 (0.75) and 2.73 (0.69) before and after treatment, respectively (P: 0.07). The multiple-dose MTX group had basal levels of 7.55(1.19) and 8.40 (1.44) before and after treatment, respectively (P < 0.001); estradiol values of 22.35 (4.23) and 23.01 (5.89) before and after treatment, respectively (P: 0.325); AFC values of 7.06 (1.13) before and 7.10 (1.02) after treatment (P: 0.789); and AMH levels of 2.40 (0.73) and 2.37(0.56) before and after treatment (P: 0.60). In the present study, two groups of post-treatment data were compared to evaluate if there was a dose-dependent effect of MTX on ovarian reserve. Mann–Whitney showed no differences in levels of FSH, AMH and AFC between single-dose and multiple-dose groups. The statistically significant difference was observed only in estradiol levels between the two groups (P: 0.02); estradiol concentrations were higher in the multiple-dose MTX group. However, the serum estradiol levels alone have little value as an ovarian reserve test. Table 2 compares biochemical parameters and AFC between two groups, pre- and post-treatment. Moreover, the variables correlation matrix was represented as a hierarchically clustered heatmap (Fig. 1).

One of the main concerns of patients undergoing MTX medical treatment for EP is the potential for some adverse effects such as ovarian reserve and the possibility of fertility. The main finding of this study was that there is no effect on the ovarian reserve by MTX dose-dependent. The effects of single-dose MTX on ovarian reserve have been evaluated in various literature studies [1, 21, 23, 24], several studies have been conducted to assess the effects of multiple doses of MTX on ovarian reserve [17–19], and two of them are performed on rats [18, 19]. However, the data from human studies are limited and the results of these studies are controversial. MTX is commonly used as a chemotherapeutic agent and has been reported to be associated with early ovarian failure in childhood cancer survivors in a dose-dependent manner [1]. Therefore, there is a demand for more investigations and experiments to see whether administrating a multiple-dose MTX regimen to treat EP affects ovarian reserve. American Society for Reproductive Medicine (ASRM) asserted that MTX is a safe drug and its success rate is similar to conservative surgery, when used to treat EP [19]. The most preferred and appropriate therapy for hemodynamically stable patients with unruptured EP is the single-dose MTX administration [19, 25]. Uyar et al. reported that single-dose MTX treatment has no statistically significant effect on ovarian reserve [24]. Boots et al. and Shirazi et al. reported similar results [21, 23], concluding that single-dose MTX administration does not affect ovarian reserve and subsequent IVF success. Sahin et al., the conclusion was consistent with Boots et al., and Shirazi et al., [16]. By measuring the AMH level, their study showed that a single dose of MTX does not have any side effects on ovarian reserve. Although the single-dose MTX is the most preferred and the most straightforward medical protocol for EP treatment, about one-quarter of patients need an extra dose to ensure the resolution [26]. Multiple-dose MTX administration is an alternative treatment protocol for EP [25, 26]. Hill et al., stated that the number of MTX doses does not correspond with changes in FSH and AFC values. [17]. In addition, the authors of Benian et al. also found no significant differences in AMH after administration of a single dose of MTX [18]. However, AMH samples were taken from the rats only twenty-four hours after MTX injection. By contrast, Ulug and his colleagues have demonstrated that ovarian reserve was diminished after administration of single-dose or multiple-dose MTX [19]. Regarding this study, Çetin et al., showed that multiple-dose MTX administration may not be indicated significant differences in AMH levels between patients [27]. The strength of the current study was evaluating both highly accepted AMH level and AFC tests [19, 28] for assessing ovarian reserve and comparing two groups to determine whether MTX has a dose-related effect on ovarian reserve. The AMH levels were not evaluated in the Hill et al., study [17]. Also, Benian et al., only measured AMH levels pre- and post-treatment, while administrating multiple-dose MTX regimen without a control group [18]. Consistent with previous studies, these results showed that single-dose and multiple-dose MTX treatment has no significant effects on ovarian reserve and antral follicle count.

Finally, MTX administration has no adverse effect on ovarian reserve and can be considered a safe treatment for EP. Performing clinical trials can be helpful to further evaluate the potential adverse effects of MTX in a dose-dependent manner on ovarian reserve. Acknowledgment The authors wish to acknowledge the support of the deputy of research affairs of the Ahvaz Jundishapur University of Medical Sciences as part of Shiva ShahAli’s thesis under the research code FIRC-0012. Author Contributions M.Z conceived the original idea, designed the scenarios, and collected the data. SH.SH, F.M carried out the analysis of data, drafted the manuscript. N.SH and SH.SH revised it, drafted the manuscript, and approved the final version that was submitted. Funding Ahvaz Jundishapur University of Medical Sciences Number: FIRC-0012. Declarations Conflict of interest The authors declare that they have no conflict of interest. Ethical Approval IR.AJUMS.HGOLESTAN.REC.1400.068. Footnotes Mahvash Zargar and Farideh Moramezi are Associate Professor; Shiva ShahAli is an MD; and Nahid Shahbazian is a Professor Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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