The effect of letrozole as an adjunct in GnRH-antagonist protocol on IVF/ICSI outcome in women with endometriosis: a randomized clinical trial

Background To evaluate the effect of adding letrozole to the antagonist ovarian stimulation protocol (COS) on in- vitro fertilization/intracytoplasmic sperm injection (IVF/ICSI) outcome in endometriosis patients.

This randomized clinical trial was carried out in the department of infertility treatment at Arash Women’s Hospital from May 2019 to May 2021. The eligible women with normal ovarian reserve tests who had endometriosis diagnosis and underwent IVF/ICSI cycles were evaluated. A flexible regimen of GnRH-antagonist protocol was used for COS. In the experimental (n = 34), the patients received 5 mg letrozole daily for the first 5 days in combination with 150 IU of recombinant follicle-stimulating hormone (rFSH). In the control group (n = 30), the patients received only the same dose of rFSH. The treatment cycle was compared between groups.

Analysis of demographic characteristics, severity of endometriosis, and baseline hormonal tests of patients showed that the two groups were similar and comparable. The means of total used gonadotropins ampoules and serum E2 level on oocyte trigger day in the letrozole group were significantly lower than those of in the con- trol group (P = 0.03 and P = 0.004, respectively). No statistically significant difference in terms of the total number of retrieved and MII oocytes as well as the total numbers of obtained and top-quality embryos, and cryopreserved embryos was found.

The co-treatment of letrozole with gonadotropins during the antagonist protocol was associated with a reduction in the total dose of gonadotropins, although it had no effect on the oocyte or embryo yield in patients with endometriosis. Trial registration The study was registered in the Iranian Registry of Clinical Trials on 2018 -07-13 (IRCT20150310021420N4 at www. irct. ir, registered while recruiting).

In vitro fertilization, Letrozole, An aromatase inhibitor, Live birth, Oocyte maturity Open Access © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Middle East Fertility Society Journal *Correspondence: Ashraf Moini [email protected]; [email protected] Full list of author information is available at the end of the article Page 2 of 8Mojtahedi et al. Middle East Fertility Society Journal (2023) 28:26

In recent years, one of the most important goals of assisted reproductive cycles (ART) in women is to focus on optimizing the method of ovarian stimulation with the least destructive effect on the endometrium. Individ - ualization of ovarian stimulation protocol based on the patient’s age, ovarian reserve tests, the cause of infertility, and the previous treatment history can allow for a safer and more effective ART practice [1]. One of the causes of infertility that has been reported to have negative effects on both the ovaries and the endometrium is the diagno - sis of endometriosis. It can be associated with ovulation disorders and decreased oocyte quality due to adverse changes in the process of folliculogenesis and steroido - genesis of granulosa cells as well as low-quality embryos, decreased implantation rate, sperm phagocytosis, and toxic environment for fetus due to pelvic adhesions in advanced stages [2–5]. It is important to select an appro - priate ovarian stimulation protocol for these patients, which in turn improves endometrial receptivity. A type of hormonal manipulation involves the use of aromatase inhibitors (AIs) along with COH standard protocol [6]. The aromatase p450 is a key enzyme in the biosynthesis of estradiol (E2) by ovarian granulosa cells in premenopausal time, whose expression in both eutopic and ectopic endometrial tissue in patients with endome - triosis is significantly higher than in non-endometriosis women [7]. Abnormal expression of aromatase leads to the production of estrogen locally at the site of implan - tation of ectopic endometrial cells. Since endometriosis is an estrogen-dependent disease, AIs appear to be good candidates for the treatment of endometriosis [7–10]. The third generation of Als, mainly letrozole (LZ) is a selective and non-steroidal AI which is superior to clo - miphene citrate for ovulation induction in patients with polycystic ovary syndrome [11]. Lu et  al. in  vivo study showed that LZ significantly reduces E2 production and aromatase p450 gene expression in luteinized granulosa cells belonging to women with advanced stages of endo - metriosis [12]. Recently, the beneficial effects of adjuvant therapy with LZ in GnRH antagonist-controlled ovarian stimu - lation (COS) protocol have been reported in patients with poor ovarian response [13, 14]. Some studies have shown favorable effects of AIs in the treatment and pre - vention of recurrence of pain and other complications of endometriosis [15]. Furthermore, Miller et  al. proposed this hypothesis that lack of endometrial ανβ3 integrin expression is associated with a poor prognosis for IVF in endometriosis patients that might be improved with LZ co-treatment [16]. Only one retrospective study has eval - uated the use of LZ in combination with gonadotropin on IVF outcomes in endometriosis patients and concluded that the combination therapy with LZ and gonadotropin produces similar oocyte and embryo yield to the conven - tional IVF protocol in women with endometriosis [16]. Since the use of AIs for improvement of infertility treatment in women with endometriosis is an interesting subject and clinical trial studies are still necessary for this area, The researchers designed a randomized clinical trial to evaluate the effect of adding LZ to the antagonist ovar- ian stimulation protocol on IVF outcome in endometrio - sis patients.

This randomized clinical trial (RCT) was carried out in the department of infertility treatment in Arash Wom - en’s Hospital from May 2018 to May 2021. The women in the age range of 18 to 42 years old with endometriosis diagnosis who underwent in  vitro fertilization/intracy - toplasmic sperm injection (IVF/ICSI) cycles were evalu - ated. Endometriosis diagnosis was defined according to sonographic evaluation by two experienced sonographers or the pathologic result of the previous laparoscopy. Endometriosis staging is done according to Enzian clas - sification by sonographic features (TVS and endo-anal ultrasound) done by 2 expert radiologists) [17, 18]. The patients with body mass index > 25  kg/m2, dimin- ished ovarian reserve (i.e., antral follicle count (AFC) < 5 follicles or anti-Mȕllerian hormone (AMH) < 1.1 ng/ml), donor/recipient or surrogacy treatments, metabolic, or endocrine disorders including (diabetes, hypo/hyperthy - roidism, hyperprolactinemia, hypothalamic amenorrhea, etc.), immunologic diseases (lupus, rheumatoid arthri - tis, antiphospholipid syndrome, cardiovascular, liver and kidney disease), congenital uterine anomalies and endo - metrial cavity disorders (Asherman syndrome, myoma, polyps, etc.), recurrent IVF failures (more than three consecutive failures and azoospermic male partner were not included in the study. The eligible patients on 2nd or 3rd day of the menstrual cycle were allocated into two groups randomly by strati - fied (based on the polycystic ovary syndrome (PCOS) diagnosis) block randomization method. The random allocation list for patients was solely available to the epi - demiologist and the number of blocks was considered 6. The type of study group was written on 72 cards, respec - tively, and then placed inside sealed envelopes. When the physician announced the eligibility of a patient, the methodologist provided the doctor with the envelope. The random allocation process and type of intervention were concealed from the assessor of the final outcome and also the data analyzer. The same controlled ovarian stimulation protocol (a flexible regimen of GnRH-antagonist) was used for all study populations. The ovarian quiescence was confirmed Page 3 of 8 Mojtahedi et al. Middle East Fertility Society Journal (2023) 28:26 by documenting the absence of ovarian cyst or lead folli - cle > 10 mm and the serum E2 concentrations < 50 pg/mL through baseline ultrasounds and hormonal assessment which were performed on the 2nd or 3rd day of the men - strual cycle. In the experimental (LZ group), the patients received 5  mg LZ (Letrofem ®; Iran hormone, Tehran, Iran) for the first 5  days of ovarian stimulation with 150 IU of recombinant human FSH (Cinnal-f, Cinagen), In the control group, the patients received only 150  IU of rFSH. The follicular maturation monitoring was done by serial vaginal ultrasound (sonographic device: Phil - lips, affinity 70) assessments. According to the ovarian response in each patient, the dosage of gonadotropins was adjusted. The administration of GnRH antagonist (Cetrotide ®, Serono International, Geneva, Switzer - land) (0.25  mg/day subcutaneously) was initiated when follicle(s) ≥ 13  mm in average diameter were observed, and it was continued until the day of final oocyte trig - gering. When at least two follicles measuring ≥ 18 mm in diameter were observed, the final stage of oocyte matu - ration was induced by two doses of recombinant hCG (250  μg) (Ovitrelle; Merck Serono). Transvaginal ultra - sound-guided oocyte retrieval was performed 34–36  h after final oocyte triggering. The serum levels of estradiol and progesterone were measured on the day of oocyte trigger and if the amount of progesterone was more than 1 ng/ml, the plan for freezing all the embryos was made. In vitro fertilization and/or intracytoplasmic sperm injection (IVF/ICSI) was performed with ejaculated sperm to metaphase II (MII) oocytes through standard procedure. The obtained Embryos were cultured in a commercially available culture medium until the day of transfer. Embryo quality was determined according to the number and regularity of blastomeres and the degree of embryonic fragmentation. Two or 3 days after oocyte retrieval, a maximum of two good-quality embryos at the cleavage stage were transferred under ultrasound scan guidance by a catheter (Guardia ™, Access ET Catheter, Cook Medical). The luteal phase was supported by 400 mg vaginal progesterone suppository twice daily (Cyc - logest, Actavis, Barnstaple, UK) starting on the evening of the oocyte retrieval and it was continued for 10 weeks in cases with a positive pregnancy test. A serum ß-hCG analysis was done 14  days after the ET, and the clinical pregnancy (presence of gestational sac with heartbeat) was documented by ultrasound scan four weeks later. Ongoing pregnancy was considered when the pregnancy was continued over 20 weeks of gestation. Statistical analysis The primary outcomes in the present study were the number of oocytes retrieved, the number of MII oocytes, total number and quality of obtained embryos. The secondary outcomes were clinical pregnancy and live birth rates. The Statistical Package for the Social Sci - ences, version 22, SPSS Inc, Chicago, IL, USA (SPSS) was used for the statistical analysis. The Kolmogorov– Smirnov test was applied to detect the normality of quantitative variables and it was determined that all of these variables had normal distribution. The independ - ent Student t-test and chi-square test were used for the comparison of the quantitative and qualitative variables between groups respectively. The descriptive data were presented as mean ± standard deviation (SD) or number (percent). The statistical significance level was considered as p value < 0.05. The sample size was estimated based on Kim et al. (a retrospective study) using NCSS-PASS soft - ware (version 2007; NCSS Inc., Kaysville, UT, USA) and it was determined that 70 subjects were needed in each study group considering α = 0.05, and 80% power. How - ever, in the sampling process, we found that the number of patients with a diagnosis of endometriosis who have a normal ovarian reserve and consented to participate in the study was very limited. Due to the long duration of the project, it was decided to end the study; since this study is one of the first RCTs in patients diagnosed with endometriosis, it can be reported as a pilot with a mini - mum sample size according to Julious’s study [19].

Among 124 women who were screened during the study period, 70 were eligible and enrolled in the study after obtaining their informed consent (35 patients in each group), after follow-up finally the result of treatment cycles were compared between groups (Fig.  1). The base- line characteristics and hormonal profiles of the patients are illustrated in Table  1. The analysis showed that there was no significant difference in terms of women’s age and BMI, duration and type of infertility, PCOS diagnosis, and AFC as well as basal serum levels of LH and FSH, serum AMH and, TSH between groups. There was no significant difference in the type and severity of endome - triosis between groups (P = 0.528 and P = 0.405). The outcomes of the ovarian stimulation cycle are com- pared between groups in Table 2. The means of total used gonadotropins ampoules and serum E2 level on oocyte trigger day in the LZ group were significantly lower than that of the control group (P = 0.03 and P = 0.004, respec- tively); however, the duration of ovarian stimulation was similar in two groups (P = 0.58). The analysis indicated that the two groups had no statistically significant differ - ence in terms of the number of follicles with 14–17 mm in diameter at trigger, total number of retrieved and MII oocytes as well as total number of obtained embryos, number of top-quality and total number of cryopreserved embryos. Page 4 of 8Mojtahedi et al. Middle East Fertility Society Journal (2023) 28:26 In the following, the number of cases with fresh and frozen embryo transfers was comparable between groups. It is worth noting that 10 (29.4%) cases of the patients in the intervention group and 9 (30%) patients in the control group had not been referred for the frozen embryo transfer until the manuscript preparation time.

The use of LZ as an adjunct in GnRH-antagonist ovarian stimulation protocol for infertile women with endome - triosis was associated with a significantly lower dosage of administrated gonadotropins compared with the stand - ard GnRH-antagonist protocol. The trend toward improvement in the total number of retrieved and meta - phase II oocytes as well as the total number of obtained embryos was observed in the LZ group; however, it was not statistically significant. Interestingly, in the follow- up after ET, the clinical pregnancy rate was significantly higher than the control group. Until now, some studies have been designed and con - ducted to investigate the effect of LZ as an adjunc - tive treatment in the COS protocol in patients with poor ovarian [11, 13, 20–24], normal [25, 26], and high responses [27] as well as in patients with breast cancer for fertility preservation [28]. The reported results in var- ious studies have been conflicting. Bülow et al. in a meta- analysis study concluded that co-administration of LZ in IVF cycles in patients with a poor ovarian response may be associated with improved outcomes; however, studies regarding normal patients or high responders are lim - ited, and further randomized clinical trials are required in this field [29]. The LZ increases ovarian response to stimulation protocol through mediation in reducing serum estrogen levels and temporary rising in intraovar - ian androgen concentrations that cause prolongation of the follicular phase, enhance the affinity of FSH recep - tors, preantral and antral follicle growth [30, 31]. Besides, the reduced serum E2 concentration attributed to LZ may justify the negative impact of excessive E2 levels on Fig. 1 Flowchart of the study sampling Page 5 of 8 Mojtahedi et al. Middle East Fertility Society Journal (2023) 28:26 oocyte quality and endometrial receptivity in ART cycles [21]. In line with the results of the present study, Eft - ekhar et al., in a clinical trial reported that co-treatment of LZ with gonadotropins reduced the total amount of consumed gonadotropin in patients with normal ovar - ian reserve; however, it did not improve the pregnancy Table 1 Demographic and clinical characteristics of study participants in two groups TSH thyroid-stimulating hormone, AMH anti-Müllerian hormone, OMA ovarian endometriosis, DIE deep infiltrating endometriosis Descriptive data were compared using independent Student’s t-test and presented as mean ± SD. P value ≤ 0.05 was considered statistically significant. No. number, FSH follicle-stimulating hormone, LH luteinizing hormone Variables Letrozole group (N = 34) Control group (N = 30) P value Female age (years) 31.38 ± 5.06 33.41 ± 4.51 0.10 Body mass index (kg/m2) 21.9 ± 2.2 21.5 ± 2.4 0.56 Duration of infertility (years) 2.2 ± 2.34 3.7 ± 3.5 0.06 No. of couples with primary infertility, n (%) 28 (82.4) 22 (73.3) 0.65 No. of women with dysmenorrhea, n (%) 17 (50) 16 (53.3) 0.78 PCOS diagnosis, n (%) 5 (14.7) 4 (13.3) 0.91 Endometriosis type, n (%) OMA 24 (52.9) 23 (76.7) 0.528 DIE 10 (47.1) 7 (23.3) Endometriosis stage, n (%) Stage III 29 (85.3) 28 (93.3) 0.405 Stage IV 5 (14.7) 2 (6.7) Basal serum level of FSH (IU/L) 4.0 ± 2.2 4.9 ± 2.4 0.13 Basal serum level of LH (IU/L) 4.5 ± 2.3 5.4 ± 2.2 0.11 Serum level of AMH (ng/ml) 2.4 ± 1.1 2.1 ± 1.5 0.31 Serum level of TSH (IU/mL) 1.3 ± 1.3 1.5 ± 1.1 0.52 Antral follicle count 7.3 ± 2.1 6.9 ± 3.1 0.43 No. of previous failed IVF cycles 0.14 ± 0.34 0.41 ± 0.73 0.10 Table 2 Comparison of stimulation and cycle outcomes in the two study groups Descriptive data were compared using an independent Student’s t-test and presented as mean ± SD. P value ≤ 0.05 was considered statistically significant. No.: number a 10 (29.4%) cases of the patients in the intervention group and 9 (30%) patients in the control group had not been transferred the frozen embryos until the manuscript preparation time Variables Letrozole group (N = 34) Control group (N = 30) P value Total gonadotropins ampoules (75 IU) 28.4 ± 8.5 34.3 ± 12.6 0.03 Duration of stimulation (day) 10.8 ± 1.9 11.2 ± 2.5 0.58 Estradiol level on trigger day (pg/ml) 1837.5 ± 577.6 2283.7 ± 606.4 0.004 No. of follicles with 14–17 mm in diameter at trigger 2.4 ± 2.1 2.8 ± 2.3 0.48 No. of follicles > 17 mm in diameter at trigger 2.7 ± 1.2 2.2 ± 0.74 0.11 No. of retrieved oocytes 11.5 ± 8.9 8.2 ± 7.7 0.12 No. of metaphase II oocytes 8.4 ± 7.1 6.5 ± 6.3 0.29 No. of obtained embryos 4.4 ± 2.6 4.0 ± 3.4 0.63 No. of top-quality embryo 3.1 ± 2.3 2.8 ± 2.8 0.62 No. of cryopreserved embryos 4.0 ± 3.0 3.6 ± 3.7 0.61 No. of all freeze cases, n (%) 30 (88.2) 28 (93.3) 0.84 No. of cases with fresh embryos transferred, n (%) 4 (11.8) 2 (6.7) 0.80 Chemical pregnancy rate/total ET (%) 12/ 24 (50) 3/21 (14.3) 0.01 Clinical pregnancy/total ETa (%) 10/24 (41.6) 3/21 (14.3) 0.04 Miscarriage rate/total ET (%) 2/24 (8.3) 0/0 0.39 Live birth rate/total ET 8/24 (33.8) 3/21 (14.3) 0.12 Page 6 of 8Mojtahedi et al. Middle East Fertility Society Journal (2023) 28:26 outcomes [25]. In a similar way, Haas et al. demonstrated that co-treatment with LZ improves the IVF outcome in normal responders in terms of the increased number of obtained blastocysts without changing the pregnancy rate or the risk of OHSS [26]. Elsewhere, et al., reported that total dose of administrated rFSH and risk of OHSS were significantly decreased in patients with male factor infertility who received LZ as an adjunct to gonadotro - pins. Furthermore, Yang and colleagues in a pilot RCT, concluded that LZ supplementation could not reduce the incidence of the premature rising of progesterone during the late follicular phase in patients with expected high ovarian response to standard stimulated IVF cycles, which was associated with producing a harmful effect on the pregnancy outcome. Finally, Bülow et al., in a multi - center double-blinded RCT evaluated 129 women with expected normal ovarian reserve to this question of whether LZ supplementation during COS with gonado - tropins for IVF reduces the proportion of women with premature progesterone levels above 1.5  ng/ml at the time of final oocyte maturation triggering?. The results of their study showed that although the use of LZ has no impact on the proportion of women with a premature rise in progesterone on the day of oocyte triggering, the increased progesterone in the mid-luteal phase due to LZ may contribute to optimizing the luteal phase endocri - nology [32]. The effect of LZ on increasing androgens and reducing gonadotropin consumption might be applied in poor responders. It was concluded that the impact of LZ on implantation and pregnancy outcomes should be assessed in a meta-analysis or larger RCT [32]. Based on our knowledge, the studies that are specific to patients diagnosed with endometriosis are limited. Recently, Kim et al., in a retrospective study, compared IVF outcomes of 38 patients who received standard COS protocol along with co-treatment of LZ versus 26 patients with standard COS protocol alone. It was concluded that the combination therapy with LZ and gonadotropin was associated with a significantly lower peak estradiol level and similar oocyte and embryo yield to the conventional IVF protocol in endometriosis patients [16]. In agreement with their study, we also did not find a positive effect of LZ on the number and qual - ity of oocytes and embryos. Although in the follow-up of pregnancies in the LZ group, the clinical pregnancy rate per total embryos transferred cycles was higher than the control group, due to the fact that the major - ity of embryo transfers were of the frozen type, we could not comment regarding the effect of LZ on the implantation and pregnancy rates. Based on the results obtained from previous studies in patients with dif - ferent causes of infertility, it is likely that LZ through the mentioned mechanisms can have a positive effect on reducing the duration of stimulation or the total dose of consumed gonadotropin and in some cases has accompanied by increasing the number of MII oocytes and/or high quality of embryos. However, the conclu - sion about its effect on the pregnancy rate requires RCT studies with larger sample sizes. The study has a strong point in that it is designed as a randomized clinical trial. The present study had limi - tations that should be mentioned, considering that the eligible population had a low prevalence, the number of subjects collected in the time frame considered for the study was less than the estimated sample size, so the study was terminated as a pilot trial. On the other hand, the number of cases of all-freeze embryos in both groups was high due to various reasons, including OHSS at risk, the spread of COVID-19 disease, or the personal preference of the patients, so we have no data to report about the effect of letrozole on the implanta - tion rate in fresh embryo transfer cycles. We suggest that future studies be designed with the primary aim of investigating the rates of implantation and clinical pregnancy in patients with endometriosis. The current pilot study indicated that the co-treat - ment of letrozole with gonadotropins during the antag - onist protocol was associated with a reduction in the total dose of gonadotropins, although it had no effect on the oocyte or embryo yield, more studies are nec - essary to determine its impact on the rate of implan - tation in fresh embryo transfer cycles in patients with endometriosis. Abbreviations ART Assisted reproduction technologies AMH Anti-müllerian hormone AIs Aromatase inhibitors COS Ovarian stimulation protocol E2: Estradiol FSH Follicle stimulating hormone GnRH Gonadotropin-releasing hormone hCG Human chorionic gonadotropin IVF In-vitro fertilization ICSI Intracytoplasmic sperm injection LZ Letrozole RCT Randomized clinical trial LH Luteinizing hormone SD Standard deviation

We would like to thank all the participants and co-workers at Arash Women’s Hospital for their assistance in this study. Authors’ contributions M.FM. and A.M. designed the research. M.FM and L.K. contributed to patient selection, data collection, interpretation of data, and manuscript editing. A.M. and M.FM wrote the manuscript. T.M. assisted in the analysis of the data. All authors read and approved the final manuscript. Funding The study did not have any funding support. Page 7 of 8 Mojtahedi et al. Middle East Fertility Society Journal (2023) 28:26 Availability of data and materials The datasets used or analyzed during the current study are available from the corresponding authors on reasonable request. Declarations Ethics approval and consent to participate The Institutional Review Boards and the Ethics Committees of Tehran University of Medical Sciences approved this study (approval code: IR.TUMS. MEDICINE.REC.1398.186). All procedures performed in studies involving human participants were in accordance with the ethical standards of the 1964 Helsinki Declaration and its later amendments. The eligible patients signed written informed consent forms prior to participation in the study. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. Author details 1 Department of Gynecology and Obstetrics, Infertility Ward, Arash Women’s Hospital, Tehran University of Medical Sciences, Tehran, Iran. 2 Breast Disease Research Center (BDRC), Tehran University of Medical Sciences, Tehran, Iran. 3 Department of Endocrinology and Female Infertility, Reproductive Biomedi- cine Research Center, Royan Institute for Reproductive Biomedicine, ACECR, Tehran, Iran. 4 Department of Obstetrics and Gynecology, Alavi Hospital, Ardabil University of Medical Sciences, Ardabil, Iran. Received: 14 December 2022 Accepted: 17 October 2023

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