Background: Previous studies have shown that androgens, in addition to serving as precursors for ovarian estrogen synthesis, also have a fundamental role in primate ovarian follicular development by augmentation of FSH receptor expression on granulosa cells. Recent studies have shown that aromatase inhibitor, letrozole, improves ovarian response to FSH in normal and poor responder patients, possibly by increasing intraovarian androgen levels. Studies in mice also showed an effect of letrozole to increase pigment epithelium-derived factor (PEDF) and to lower vascular epithelial growth factor (VEGF), which might be expected to reduce the risk of ovarian hyperstimulation syndrome (OHSS) with stimulation. The aim of this study was to compare the VEGF and PEDF levels in the follicular fluids of normal responders treated with letrozole and gonadotropins during the ovarian stimulation with patients treated with gonadotropins only. Methods: A single center, prospective clinical trial. We collected follicular fluid from 26 patients, on a GnRH antagonist protocol, dual triggered with hCG and GnRH agonist. The patients in one group were co-treated with letrozole and gonadotropins during the ovarian stimulation and the patients in the other group were treated with gonadotropins only. VEGF, PEDF, estrogen, progesterone and testosterone levels were measured by ELISA kits. Results: The age of the patients, the total dose of gonadotropinsand the numberofoocytes were comparable between the two groups. In the follicular fluid, the estrogen levels (2209 nmol/l vs. 3280 nmol/l, p = 0.02) were significantly decreased, and the testosterone levels (246.5 nmol/l vs. 40.7 nmol/l, p < 0.001) were significantly increased in the letrozole group compared to the gonadotropin only group. The progesterone levels (21.4 μmol/l vs. 17.5 p =NS) were comparable between the two groups. The VEGF levels (2992 pg/ml vs. 1812 pg/ml p = 0.02) were significantly increased and the PEDF levels (9.7 ng/ml vs 17.3 ng/ml p < 0.001) were significantly decreased in the letrozole group. Conclusions: Opposite to observations in the mouse, we found that VEGF levels were increased and PEDF levels were decreased in the follicular fluid in patients treated with letrozole during the stimulation cycles. Further investigation is required to determine if patients treated with letrozole during the IVF stimulation protocol are at increased risk for developing OHSS as a result of these findings. Keywords: Letrozole, VEGF. PEDF * Correspondence: email@example.com Division of Reproductive Sciences, University of Toronto, Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, Canada TRIO fertility partners, 655 Bay St 11th floor, Toronto, ON M5G 2K4, Canada Full list of author information is available at the end of the article © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Haas et al. Reproductive Biology and Endocrinology (2018) 16:54 Page 2 of 5 Background to sign a written informed consent after the provision of The addition of the aromatase inhibitor, letrozole, to go- complete information. nadotropin stimulation has been an accepted treatment This study included a total of 26 IVF cycles performed for oocyte retrieval in women with breast cancer and in 26 normal responders treated at our institution between has been demonstrated to result in lower serum estrogen June, 2016 and March, 2017. Patients with PCOS or POR levels [1, 2] an endocrine response that is considered to by Bologna criteria were excluded from the study. be favourable in these women who may have estrogen The study group included patients on a GnRH antag- sensitive tumors. In normal women undergoing IVF, onist protocol treated with daily letrozole 5 mg together lowering serum and follicular estrogen levels could also with gonadotropins, from the first day of ovarian stimu- be potentially beneficial by lowering the risk of ovarian lation until the trigger day. The control group included hyperstimulation syndrome (OHSS) since a correlation patients matched by age, infertility diagnosis and starting has been observed between administration of letrozole gonadotropin dose, on the same GnRH antagonist and lowering the incidence of OHSS . protocol, treated with gonadotropins only during the A previous study in a rat model of OHSS  demon- stimulation. All the patients included in the study had a strated that treatment with a single dose of letrozole on dual trigger with hCG and GnRH agonist for final oocyte the hCG trigger day, reduced ovarian diameter, reduced maturation. The study was not randomized and the deci- VEGF levels and increased the levels of pigment epithe- sion whether to co-treat with letrozole was made by lium derived factor (PEDF). VEGF has been identified as each treating physician independently. one of the prime causative factors in OHSS while PEDF The decision to administer letrozole was not made for has been shown to decrease the angiogenic activity of the sake of this study. The treating physicians made this VEGF . These combined results might be expected to treatment decision independently of the study. reduce the risk of OHSS. In addition, supraphysiologic levels of estrogen are be- Stimulation protocols lieved to have a negative effect on oocyte quality  and Gonadotropin treatment (with or without letrozole) was embryo implantation  with subsequent adverse preg- initiated on the 3rd day of menses with the use of re- nancy outcomes [8, 9] and therefore, lowering estrogen combinant FSH (Gonal F, EMD Serono). Once the lead- levels by adding letrozole could be beneficial in normal ing follicle had reached a size of 13 mm, or E2 levels women undergoing IVF by increasing implantation and exceeded 1200 pmol/L, co-treatment with a GnRH an- reducing pregnancy complications. tagonist 0.25 mg/day (Orgalutran, Merck) and recom- More recently, it has also been observed that breast binant LH (Luveris, Serono) or highly purified human cancer patients undergoing IVF with the addition of menopausal gonadotropin (Menopur, Ferring) was com- letrozole have an increase in the number of oocytes re- menced. Follicle growth and hormone levels were covered compared to controls [1, 2]. Several studies have serially monitored by ultrasound and blood tests until shown that letrozole, improves ovarian response to FSH the dominant follicles reached an average diameter of in normal and poor responder patients, possibly by in- 18–20 mm. At that point human chorionic gonado- creasing intraovarian androgen levels (1–3, 11–13). tropin (10,000 IU Pregnyl; Merck, Kirkland, Quebec) Previous studies have shown that androgens, in addition and GnRH agonist (0.5 mg Suprefact; Sanofi-Aventis, to serving as precursors for ovarian estrogen synthesis, Canada) were administered subcutaneously to trigger also have a fundamental role in primate ovarian follicu- ovulation. Thirty-six hours later oocyte retrieval was per- lar development by augmentation of FSH receptor ex- formed under transvaginal guided ultrasound and needle pression on granulosa cells . aspiration. The objective of this study was to measure VEGF and The follicular fluid (FF) was obtained at the time of PEDF levels as well as estrogen and testosterone levels oocyte retrieval for the IVF procedure. Only the first fol- in the follicular fluids of normal responder women licle from each ovary was collected avoiding blood clots, treated with letrozole and gonadotropins throughout the and only FF from mature full sized follicles (≥16 mm) entire ovarian stimulation for IVF and to compare the was included in the study. Only one single follicular fluid results with the follicular fluid levels in patients treated sample per patient was included in the study. with gonadotropins only. After isolation of the oocyte, the clear follicular fluid was centrifuged at 500 g for 10 min at room temperature to separate out cellular contents and debris Methods and was then transferred to sterile tubes and frozen at − Ethical approval 80 °C until analysis. The follicular fluid was analyzed for The study was approved by the Mount Sinai Hospital testosterone, estradiol, progesterone by an automated ethics review committee, and all couples were required assay (Vitros), and VEGF (Cedarlane; CL76149K) and Haas et al. Reproductive Biology and Endocrinology (2018) 16:54 Page 3 of 5 PEDF (Chemicon;CYT 420) concentrations were mea- Table 2 The hormone levels in the follicular fluid from patients co-treated with letrozole and gonadotropins vs. gonadotropins only sured by commercial ELISA using an ELISA kits accord- ing to the manufacturer’s instructions. We measured Letrozole group (13) Control group (13) P follicular fluid progesterone concentrations as a possible Estrogen(nmol/l) 2009 ± 1034 3280 ± 1371 0.01 way to ensure similar ovarian stimulation in each group. Testosterone(nmol/l) 246.5 ± 153.2 40.7 ± 14.3 < 0.001 Comparisons were performed using the paired two- Progesterone(μmol/l) 21.4 ± 8.3 17.5 ± 10.3 0.3 tailed student’s t-test. A P < 0.05 was considered statisti- cally significant. murine model, letrozole was administered only at the Results trigger day and not during the ovarian stimulation The age of the patients, the total dose of gonadotropins whereas in our current study, the patients were treated and the number of oocytes were comparable between during the entire ovarian stimulation, which might ex- the two groups (Table 1). In the follicular fluid, the mean plain the differences between the VEGF and PEDF levels estrogen level (2209 nmol/l vs. 3280 nmol/l, p = 0.02) observed. was significantly decreased, and the mean testosterone Similarly to the murine findings, He et al. demon- level (246.5 nmol/l vs. 40.7 nmol/l, p < 0.001) was signifi- strated a decrease in the VEGF serum levels after treat- cantly increased in the letrozole co-treated group ment with letrozole in the luteal phase. He found a dose compared to the gonadotropin only group. The mean fol- dependent decrease in the levels of VEGF with increas- licular fluid progesterone level (21.4 μmol/l vs. 17.5 μmol, ing doses of letrozole administered in the luteal phase p = NS) was comparable between the two groups . The findings of He et al. suggested that letrozole (Table 2). could decrease the risk of OHSS although it is not clear The mean VEGF level (2992 pg/ml vs. 1812 pg/ml if the effect on VEGF and PEDF secretion was a direct p = 0.02) was significantly increased and the mean PEDF action of letrozole or an indirect effect through a reduc- level (9.7 ng/ml vs 17.3 ng/ml p < 0.001) was significantly tion in estradiol levels. decreased in the letrozole group (Table 3). A randomized controlled study in hyper-responder pa- None of the patients in the study group or in the con- tients which aimed to compare the efficacy of letrozole trol group developed early or late OHSS. to aspirin during the luteal phase in primary prevention of early ovarian hyperstimulation syndrome showed a Discussion lower incidence of OHSS in women receiving letrozole In contrast to observations in mice, we found that VEGF compared with aspirin . In contrast to previous stud- levels were increased and PEDF levels were decreased in ies, the patients treated with letrozole had higher levels of the follicular fluids of patients treated with letrozole dur- VEGF in the serum compared to the patients not treated ing the stimulation cycles, despite a significant suppres- with letrozole. The authors hypothesized that the mechan- sion of estradiol concentration in follicular fluid. In the ism of lower incidence of OHSS was independent of Table 1 Characteristics of the IVF cycles for patients co-treated with letrozole compared to the control group Letrozole group (n = 13) Without letrozole (n = 13) P value Age (years) 36.3 ± 3.9 35.8 ± 3.7 NS AMH (pmol/l) 14.26 ± 7.7 16.4 ± 6.7 NS FSH 7.3 ± 1.6 6.6 ± 1.9 NS Etiology for infertility Unexplained-8 Unexplained-7 NS Male factor-3 Male factor-3 Mechanical-0 Mechanical-1 Fertility preservation-2 Fertility preservation- 2 Length of stimulation (days) 9.4 ± 1.8 10.7 ± 1.7 NS Dosage of gonadotropins 3085 ± 633 3294 ± 917 NS Oocytes (n) 11.7 ± 5.7 12.1 ± 6.1 NS 2PN(n) 6.6 ± 5.1 7.6 ± 4.4 NS Blastocysts (n) 3.1 ± 2.2 2.9 ± 1.9 NS Blastocyst rate (blast/2PN) 46.9% 38.1% NS E2 levels (pmol/l) 1032 ± 375 8069 ± 3068 0.001 Ongoing Pregnancy rate 5/11 (45.4%) 4/11(36.3%) NS Haas et al. Reproductive Biology and Endocrinology (2018) 16:54 Page 4 of 5 Table 3 The VEGF and PEDF levels in the follicular fluid from VEGF binds to specific receptors located in endothelial patients co-treated with letrozole and gonadotropins vs. cells called VEGF-R1 (Flt-1) and VEGF-R2. Soluble gonadotropins only VEGF-R1 is a naturally produced receptor capable of Letrozole group (13) Control group (13) p binding and sequestering VEGF and is able to reduce VEGF (pg/ml) 2992 ± 431.7 1812 ± 462.4 0.02 the level of free, active VEGF . Jakimiuk et al.  demonstrated that VEGF/ sFlt-1 ra- PEDF (ng/ml) 9.7 ± 5.7 17.3 ± 8.4 < 0.001 tio in FF on the day of oocyte retrieval in women undergo- ing IVF procedure, regardless of the type of stimulation VEGF but rather due to the induction of a luteolytic effect protocol, might predict the risk of developing OHSS. and lower estradiol concentrations which reduced the risk The sFlt-1 contribute to the amount of free, biological of early-onset OHSS (5). active VEGF in FF and later in serum by binding VEGF Although we didn’t measure the VEGF or PEDF levels and thereby depleting the amount of free circulating bio- in the serum, we found increased VEGF and PEDF levels logical active VEGF. in the follicular fluid of letrozole treated patients at the In our study we didn’t measure sFlt-1 levels in the FF time of oocyte retrieval. In the follicular phase, letrozole and therefore it’s still speculative whether free circulat- reduces serum estrogen levels which results in reduced ing VEGF levels were different between the groups. negative feedback on gonadotrophin secretion from the Tropea et al. , cultured human luteal phase with hypothalamus-pituitary axis [12–14]. By lowering serum androgens and demonstrated that different doses of estrogen concentrations in the early follicular phase, androgens significantly increased VEGF secretion. By letrozole causes secretion of more FSH and LH, which culturing the cells with aromatase inhibitor, VEGF levels acts directly on the granulosa cells and may be respon- decreased. We think it’s difficult to compare those re- sible for the increased secretion of VEGF. In addition, sults with our current study because in our study the pa- we found higher intrafollicular levels of testosterone in tients were treated with letrozole which causes secretion the letrozole group. We believe that the androgen of LH and FSH from the pituitary gland in contrast to increase may have a positive effect on follicular develop- the cultured granulosa cells which are not affected by ment, oocyte maturation and implantation. Since andro- those changes. Another major difference is that the gens have been shown to increase FSH receptor granulosa cells were cultured in the luteal phase whereas expression in both murine  and primate models [16, in our study the patients were treated with letrozole in 17] it is possible that the increased VEGF level could the follicular phase and the granulosa cells in the follicu- also be influenced by the impact of increased androgen lar phase may respond differently than in the luteal levels on the granulosa cell responsiveness to FSH in the phase to androgens in terms of VEGF production. letrozole treated group. In the letrozole group the estrogen levels in the follicular Previous studies demonstrated [18, 19] that women who fluid were significantly lower and more similar to the estro- did not conceive had higher FF VEGF concentrations than gen levels in the natural cycle compared to estrogen levels women achieving a clinical pregnancy. A negative correl- in the gonadotropins only group . Previous studies have ation was observed between FF VEGF concentrations, demonstrated a reduced pregnancy rate with increasing E2/ peak estradiol levels and number of oocytes retrieved. oocyte ratio [6, 26] and therefore we assume that treatment Friedman et al.  showed increased VEGF levels in the with letrozole which reduces the serum and intrafollicular FF from patients with advanced age compared with youn- estrogen concentrations, may have a positive effect on the ger women. They hypothesized that the higher VEGF con- oocyte quality and embryo development. centrations resulted from relative follicular hypoxia which Although none of the patients in our study group de- is a stimulant for VEGF production. veloped OHSS, we think that further investigation is re- As publishedpreviouslybyour group, we found quired to determine if patients treated with letrozole a higher number of oocytes, zygotes and blastocysts in during the IVF stimulation protocol are at increased risk women co-treated with letrozole compared to matched for developing OHSS as a result of our new findings patients treated with gonadotropins only. We demonstrating increased VEGF levels and decreased hypothesize that in normal responders co treated with PEDF levels after treatment with letrozole. letrozole the pathophysiology increasing VEGF levels is We conclude that co-treatment with gonadotropins different and not related to follicular hypoxia. We be- and letrozole during the follicular phase increase the lieve that by lowering serum estrogen concentrations in VEGF levels and decrease the PEDF levels in the follicu- the early follicular phase, letrozole causes secretion of lar fluid. Whether co-treatment with letrozole during more FSH and LH, which acts directly on the granulosa the follicular phase may increase the incidence of OHSS cells and may be responsible for the increased secretion is still unknown and further studies should be performed of VEGF. to evaluate this risk. Haas et al. Reproductive Biology and Endocrinology (2018) 16:54 Page 5 of 5 Abbreviations outcome: an analysis of 277,042 single-embryo transfer cycles from 2008 to ET: Embryo transfer; FET: Frozen embryo transfer; FSH: Follicle stimulating 2010 in Japan. Fertil Steril. 2014;101:128–33. hormone; IVF: In vitro fertilization; OHSS: Ovarian hyperstimulation; 9. Wennerholm UB, Henningsen AK, Romundstad LB, Bergh C, Pinborg A, PEDF: Pigment epithelium-derived factor; VEGF: Vascular epithelial growth factor Skjaerven R, Forman J, Gissler M, Nygren KG, Tiitinen A. Perinatal outcomes of children born after frozen-thawed embryo transfer: a Nordic cohort study Availability of data and materials from the CoNARTaS group. Hum Reprod. 2013;28:2545–53. The datasets supporting the conclusions of this article are included within 10. Weil S, Vendola K, Zhou J, Bondy CA. Androgen and follicle-stimulating the article. hormone interactions in primate ovarian follicle development. J Clin Endocrinol Metab. 1999;84:2951–6. Authors’ contributions 11. He Q, Liang L, Zhang C, Li H, Ge Z, Wang L, Cui S. Effects of different doses JH designed and conducted the study and wrote the initial draft of the of letrozole on the incidence of early-onset ovarian hyperstimulation manuscript. 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Published: May 29, 2018
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