P-677 Letrozole inhibits ovulation via reducing responsiveness to luteinizing hormone in in vitro-grown mouse follicles
Authors: Kobayashi T , Ishikawa H , Hirokazu T , Ishii K , Mitsuhashi A , Shozu M , Usui H , Koga K 
Published: June 2023

Abstract

Study question
Does letrozole reduce responsiveness to luteinizing hormone (LH) in in vitro-grown mouse follicles?

Summary answer
We found that letrozole reduced responsiveness to LH via reducing of luteinizing hormone/chorionic gonadotropin receptor (Lhcgr) transcription in in vitro.

What is known already
The continuous dosing of letrozole, the third-generation aromatase inhibitor, is used for the controlled ovarian stimulation (COS) of fertility preservation cycles in women with hormone-sensitive breast cancer to prevent the transient rise of serum estrogen levels. Recently, lower oocyte maturation rates and higher abnormal fertilization rates in COS with letrozole compared to standard COS have been reported. The studies using aromatase knockout mice and estrogen receptor beta knockout mice showed that intra-follicular estrogen is essential for responsiveness to LH of granulosa cells. However, whether letrozole reduces responsiveness to LH of follicles remains unknown.

Study design, size, duration
We evaluated the effect of letrozole on responsiveness to LH using in vitro mouse follicle culture system. Preantral follicles (150-200 µm) with a small number of stromal cells were isolated from 3 weeks old C57BL/6jjcl mouse ovaries and cultured in the presence of letrozole. We analyzed in vitro ovulation ability, and expression levels of ovulation-related genes to clarify the effect of letrozole on responsiveness to LH of follicles.

Participants/materials, setting, methods
The follicles were cultured with letrozole (0.01µM, 0.1µM, or 1µM) or vehicle (0.1% DMSO) for 5 days followed by 5 IU/ml hCG and 5 ng/ml hEGF stimulation as ovulation induction. The diameter and survival rate of follicles were assessed. After16 hours after ovulation induction, we assessed the ovulation and measured the transcription of Lhcgr, Ptgs2, Runx1 and Actb (internal control) in the follicles by RT-qPCR. We conducted a collateral experiment in the presence of estradiol.

Main results and the role of chance
The follicle growth and survival rate did not change by letrozole in vitro. However, in vitro ovulation was reduced by the addition of letrozole in a dose-dependent manner (Cochran-Armitage trend test, P &lt; 0.0001): 41.2% in the 0.01µM letrozole treated group (n = 23), 10.5% in the 0.1µM letrozole treated-group (n = 17), and no ovulation in the 1µM letrozole treated-group (n = 19) compared with 69.6% in the vehicle-group (n = 18). Exogenous estrogen addition restored the ovulation ability of 1µM letrozole-treated follicle to the same levels as the vehicle group. RT-qPCR revealed that 0.1 µM letrozole significantly reduce the transcription levels of Lhcgr (P = 0.0027), Runx1 (P = 0.0124), and Ptgs2 (P = 0.0067) compared with vehicle-treated follicles. Exogenous estrogen addition restored the transcription of these genes to levels observed in vehicle-treated follicles. We also observed a positive correlation between the transcription of Lhcgr and Ptgs2 (R = 0.55, P = 0.0003), Lhcgr and Runx1 (R = 0.71, P &lt; 0.0001), and Ptgs2 and Runx1 (R = 0.77, P &lt; 0.0001). Our results suggested that letrozole-treated follicles impaired ovulation due to dysregulation of Lhcgr transcription and its downstream cascade caused by estrogen deficiency.

Limitations, reasons for caution
This is an in vitro experiment using a mouse model. Further research is needed to determine whether our findings are applicable to humans.

Wider implications of the findings
Our findings imply that letrozole may have disadvantages in not only ovulation, but also subsequent reproductive processes affected by the effectiveness of the LH surge, such as oocyte maturation, fertilization, and preimplantation embryo development. When evaluating oocyte cryopreservation for fertility preservation, it may be necessary to consider the COS protocol.

Trial registration number
Not applicable