It is well-known that luteinizing hormone (LH) may have an effect on oocyte quality. As a result, the process of hypothalamic/pituitary downregulation is crucial for the clinical outcomes of patients undergoing controlled ovarian hyper-stimulation (COH) for in vitro fertilization (IVF). For the downregulation protocol, the majority of physicians focus on gonadotropin-releasing hormone-analogs (GnRH-a)/GnRH antagonists. In addition, the time point of GnRH treatment is relatively fixed, and it is administered by subcutaneous injection on the 2nd to 5th day of the menstrual cycle. The down-regulation of GnRH-a traditionally makes developmental synchronization of the antral follicle. However, there are some shortcomings. On the one hand, it is due to the discomfort caused by the subcutaneous injection of drugs; on the other hand, the cost of the drug is high. More importantly, some patients will inevitably develop ovarian hyperstimulation syndrome (OHSS) [1]. Therefore, it is particularly important to find a drug that is cost-effective, safe, and convenient to use.

Ethinylestradiol/cyproterone acetate (EE/CPA), named as Diane-35, has dual effects of antiandrogenic and inhibiting the pituitary gland. It is particularly effective in reducing LH surges and stabilizing ovarian responses. The premature LH-induced meiosis may disrupt follicular development, and the intervention aims to prevent the impairment [2]. Unlike other combined oral contraceptives (COCs), EE/CPA has been proven effective for people with polycystic ovary syndrome (PCOS). Hwang et al. [3] proposed a stimulation protocol for women with PCOS, using EE/CPA pretreatment and cetrorelix. The pregnancy outcome they obtained was similar to that of the GnRH agonist protocol used for fresh embryo transfer [3]. This indicates that EE/CPA may have wider applicability in the COH protocols. Although other COCs mainly inhibit ovulation through the synergistic effect of estrogen and progesterone (P), the cyproterone component of EE/CPA directly targets androgen excess and may improve follicular synchronization in non-PCOS populations.

The present study was conducted to investigate the effects of the EE/CPA downregulation stimulation protocol in IVF, compared with GnRH-a. The primary outcome was clinical pregnancy rate, and the secondary outcome was quality of oocytes, including the meiosis stage II (MII) rate, fertilization rate, the number of good-quality embryos, fertilization rate, cleavage rate, viable embryo rate, good-quality embryos rate, and implantation rate. In addition, the economic cost was analyzed.

During the procedure of IVF, excessive doses of GnRH-a prolong the recovery of the pituitary gland and require higher doses of Gns to overcome the inhibition of follicular development [9]. EE/CPA, an oral contraceptive pill (OCP), can inhibit the hypothalamus and pituitary gland to decrease LH and androgen levels. Currently, studies have only reported that EE/CPA can improve biochemical hyperandrogenemia, acne, sebum, hirsuteness and other adverse clinical symptoms [10]. As early as 2004, researchers had proposed that the Diane/cetrorelix protocol has a similar pregnancy outcome to the GnRH agonist long protocol in PCOS; however, the drawback was the 3-month-long administration of EE/CPA [3]. Thus, OCPs can replace GnRH-a for downregulation and can reduce the side-effects [11]. In 2010, Tehraninejad et al. [12]. found that the agonist protocol after OCP pretreatment in patients with PCOS achieved similar clinical outcomes as the antagonist protocol triggered by GnRH, with a reduction in the incidence of OHSS. The present study compared the cycle characteristics and clinical efficiency of the EE/CPA downregulation with the standard GnRH-a long protocol in IVF/ICSI cycles.

The present study demonstrated that the oral administration of EE/CPA for downregulation in a COH regimen may be a better option for patients. First, it can be used at any time and flexible hours of the menstrual cycle, which provides great convenience for patients. Second, the number of oocytes retrieved, MII oocytes, fertilized oocytes, viable embryos and good-quality embryos were comparative to those of the control group. To a certain extent, the higher cleavage rate of EE/CPA group might reflect improved cytoplasmic maturation due to prolonged LH suppression, minimizing premature luteinization of granulosa cells. A high cleavage rate is one of the main aims of such protocols for clinicians and patients. In addition, oral administration markedly alleviates the pain of intramuscular injections for the patients.

The study indicated that viable embryos were significantly fewer in the EE/CPA group, but good-quality embryo counts showed no difference. This phenomenon might be interpreted that the hyperandrogenic environment induced apoptosis of granulosa cells, leading to developmental arrest of the metaphase embryo. EE/CPA pretreatment significantly reduced serum free testosterone, and decreased the proportion of moderate-quality embryos, but there was no difference in the proportion of good-quality embryos. It suggested an elimination effect on low-potential embryos, which was related to the selective optimization of embryo development. Meanwhile, estrogen pretreatment enhanced oocyte maturation synchromia, and further optimized the distribution of embryo quality. Although the total number of viable embryos decreased, the retained good-quality embryos maintained the overall pregnancy potential, which was consistent with the non-inferior clinical outcome.

In addition, in the present study, Gn duration and total dose were comparable between groups, with adjustments made for individual ovarian response. The higher Gn requirement in the EE/CPA group may reflect prolonged pituitary suppression, necessitating higher doses to overcome follicular dormancy. This does not imply ovarian resistance, as oocyte yield remained comparable. The clinical pregnancy rate also did not differ significantly between the groups. Of note, the incidence of OHSS was significantly lower in the EE/CPA group compared to the control group. Thus, this suggests that the EE/CPA oral contraceptive may effectively inhibit the serum LH level, without affecting the clinical outcome.

With the impact of Corona Virus Disease (COVID)-19 in recent years, the economic capacity of individuals has also been greatly affected. The cost of IVF is becoming a matter of concern. Thus, clinicians aim to identify methods which may be used to achieve the most satisfactory results with the lowest costs. The cost of the use of EE/CPA is lower than that of conventional COH protocols. This is greatly appealing for patients.

With the development of vitrification, the survival rate of frozen-thawed embryos has markedly improved [13]. The oral administration of EE/CPA, which affects hormone levels, has negative effects on endometrial receptivity. Embryos obtained from the patients treated with EE/CPA thus require total freezing instead of fresh transfer. Therefore, the EE/CPA downregulation protocol can only be used with freeze-thaw embryo transfer. In the present study, freeze-thaw embryo transplantation was selected in all transplantation cycles. The clinical indicators demonstrated that there was no significant difference in the implantation and clinical pregnancy rates between the EE/CPA and control group. Above all, the use of EE/CPA markedly reduced the occurrence of severe OHSS. Perhaps EE/CPA can also help to reduce the adverse effects on endometrial receptivity in a high steroid environment [14], during the COH process, as well as when applying GnRH-a for ovarian stimulation.

Of note, the present study has several limitations which should be mentioned. First of all, a small percentage of patients experienced gastrointestinal adverse reactions following the administration of EE/CPA and they thus had to be administered other drugs for the downregulation protocol. Secondly, as of each patient responds differently to the drug, the dose of EE/CPA needs to be flexibly adjusted according to each individual. Thirdly, excessive downregulation may lead to an increase in the use of ovulation induction. Fourth, while embryologists were blinded, patients and clinicians were not blinded due to differing drug administration routes (oral vs. injection). This may introduce performance bias in subjective outcomes such as OHSS assessment. Finally, for the consistency, the study investigated the effect of EE/CPA down-regulation protocol on the clinical outcomes of IVF/FET cycles. The freeze-all protocol, while standardizing endometrial receptivity, may obscure potential advantages of GnRH-a in fresh transfers. Future studies should evaluate EE/CPA in fresh cycles and compare specific agonist regimens (short-acting and long-acting).

In the present study, the oral administration of EE/CPA as a downregulation protocol produced equivalent clinical outcomes compared with the control GnRH-a downregulation protocol. It may thus prove to be an effective strategy in ovarian stimulation protocols.

The datasets generated during this study are available from the corresponding authors upon reasonable request.

XB, FM and XW designed the research study. YW, SX and PZ collected materials and interpreted the patient data. XB and HW analyzed the data and wrote the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

The study was carried out in accordance with the guidelines of the Declaration of Helsinki, and the protocol was approved by the Ethics Committee of Children’s Hospital of Shanxi and Women Health Center of Shanxi (approval number: IRB-KYYN-2021-00478). All patients or their families/legal guardians gave their informed consent for inclusion before they participated in the study.

Not applicable.

The study was supported by the Innovation plan of Medical Science and Technology of “Four in a batch” (2020TD19).

The authors declare no conflict of interest.