Co-treatment of gonadotropin and letrozole in infertile women with endometriosis: A double-blind randomized clinical trial

Abstract Background The common causes of infertility in women with endometriosis are folliculogenesis alternation, steroidogenesis and fertilization impairment, oocyte and embryo quality reduction, and implantation defect. Objective To compare in vitro fertilization (IVF) cycle success rates of women with endometriosis who were treated with letrozole + gonadotropin (LA) vs. placebo + gonadotropin (PA). Materials and Methods This double-blind, randomized clinical trial study was conducted with 94 infertile women with endometriosis (47 in the LA group and 47 in the PA group) who were candidates for IVF, from April-June 2021. For all participants, the long agonist protocol was applied. In both groups, gonadotropin-releasing hormone agonist was prescribed in the mid-luteal stage and from the third day of the cycle, and gonadotropin was started and its doses were regulated based on the patient's age, serum anti-Mullerian hormone and follicle-stimulating hormone. From the third day of the menstrual cycle, 5 mg of letrozole daily for 5 days was prescribed for the LA group, while the placebo was prescribed for the PA group on the identical days and duration. After embryo transfer, biochemical and clinical pregnancy were measured in the 2 groups. Results The gonadotropin dosage (p < 0.01) and estradiol level (p = 0.02) on the human chorionic gonadotropin administration day were significantly lower in the LA group compared with in the PA group. Fetus transfer was done for 32 women. No significant differences were detected between the study groups regarding biochemical or clinical pregnancy (p = 0.72 for both). Conclusion Letrozole as a co-treatment drug in the IVF cycle of women with endometriosis can significantly reduce the gonadotropin dosage and estradiol level with the same pregnancy rates.


Introduction
The prevalence of endometriosis, tissue and glands of endometrium placed outside the uterus, in infertile women is 25-50%, and it can cause the failure of in vitro fertilization (IVF) treatment (1,2). The usual causes of infertility in women with endometriosis are folliculogenesis alternation, steroidogenesis and fertilization impairment, oocyte and embryo quality reduction, implantation defect, and pelvic adhesions (3). Endometriosis is an estrogen-dependent disease associated with increased aromatase enzyme expression and concentration and some pathologic mediator secretion, such as of estradiol and prostaglandin E 2 (1). These pathologic mediators have an important role in promoting the growth and invasion of endometriotic tissue, pain, inflammation, and infertility (4).
Nowadays, assisted-reproductive technology can have a considerable role in resolving infertility problems in most couples. However, previous studies have shown significantly lower successful fertilization rates in endometriosis rather than in other causes of infertility in IVF cycles (5)(6)(7).
Letrozole is a selective aromatase inhibitor, that causes a decrease in the estrogen concentration.
The result of a decline in estrogen level is an increase in follicle-stimulating hormone secretion, ovarian follicle-stimulating hormone receptor affinity, antral follicle growth, follicle phase enhancement, and follicle development. The other effect of letrozole is reducing estradiol and prostaglandin E 2 production, which affects oocyte quality (8,9). Letrozole has been recommended in some research for improving fertility results in poor responder women, treatment of endometriosisrelated pelvic pain, treatment of hormone receptor-positive breast cancer, and fertility preservation in women with breast cancer (10)(11)(12).
However, studies about the application of letrozole in the IVF cycles of women with endometriosis are rare and more studies are needed on this topic. This study was designed to compare IVF cycle success rates of women with endometriosis treated with letrozole + gonadotropin (LA) vs. placebo + gonadotropin (PA).

Statistical analysis
All of the statistical analyses were done using the Statistical Package for the Social Sciences (SPSS) version 24.0. P-values < 0.05 were considered statistically significant. The independent t test and non-parametric Mann-Whitney U test were used to evaluate the differences in means. A Chi-square test and Fisher's exact test were applied to assess the differences in proportions.

Results
100 infertile women were assessed for eligibility; of them, 6 women were excluded due to: severe azoospermia in their partners (n = 2), leiomyoma (n = 1), and declined to participate (n = 3). In total, 94 women were randomized to the LA and PA groups. During the study, 12 participants (6 from each group) did not refer for follow-up and were considered lost to follow-up.
Finally, data from 82 women with endometriosis were analyzed (Figure 1).
The mean age and infertility duration were 31.49 ± 4.64 yr and 5.01 ± 3.37 yr, respectively. The basic characteristics of participants did not differ significantly between the 2 study groups (Table   I).
Gonadotropin dosage (p < 0.01) and estradiol level (p = 0.02) on the hCG administration day were significantly lower in the LA group in comparison with the PA group. The other cycle characteristics did not differ significantly between the 2 study groups (Table II).
Embryo transfer was done for 32 women.

Discussion
Our study showed that gonadotropin dosage was significantly lower in the LA group compared to the PA group. Furthermore, the estradiol level on the hCG administration day was significantly lower in the LA group in comparison with the PA group. However, no significant differences were detected between the study groups regarding biochemical or clinical pregnancy.
Infertile women with endometriosis have a poorer response to infertility treatment than women with other causes of infertility; the main reasons may be a reduction in embryo quality, endometrial reception and implantation rates, and enhancement in inflammation and aromatase synthesis (14)(15)(16).
Previous research has attempted to find solutions to counter the adverse impact of infertility in women with endometriosis, such as by evaluating the effect of a post-operative systematic GnRH agonist prescription (17,18) and pre-treatment with GnRH agonist (19); however, this problem remains unresolved, and further studies are needed.
This study assessed the effect of a combination of letrozole and gonadotropin compared with gonadotropin alone on the outcomes of IVF treatment in infertile women with endometriosis. The results showed that this combination was associated with significantly lower estradiol levels and dose of Cinnal-f. However, it had no significant influence on oocyte number or quality, embryo quality, biochemical pregnancy, or clinical pregnancy rates.
Our results showing that letrozole application was associated with a significantly lower dose of gonadotropin and estradiol level are in line with studies conducted on normal responders (20), women with breast cancer (21), and poor responders (22,23). However, other studies found that the gonadotropin dose was similar in the letrozole vs. control groups that they examined (12,24); this could be due to the lack of randomization in these studies and because they enrolled women with laparoscopic-approved endometriosis.
Some studies (2,12,(20)(21)(22), similarly to this study, indicated that adding letrozole to the IVF regimen had no significant effect on stimulation duration, oocyte number, or embryo number or quality. In contrast, in other studies, letrozole was able to significantly affect the number and quality of oocytes (23,24) and the length of stimulation (23,25).
Although some previous studies (2,(20)(21)(22)(23)(24), in line with our study, indicated no significant variation in pregnancy proportions with letrozole application, Piedimonte et al. (25) indicated that pregnancy and live-birth rates increased significantly following administration of a letrozole and GnRH agonist combination. Given the varied findings of previous studies, future studies seem needed to assess the effect of varying doses of letrozole in infertile women with endometriosis, as well as to compare the effect of a combination of letrozole with agonist vs. antagonist IVF protocol.

Limitation
The small sample size was a limitation of our study.

Conclusion
According to the study findings, using letrozole as a co-treatment drug in the IVF cycle of women with endometriosis can significantly reduce gonadotropin dosage and estradiol level with the same pregnancy rates.