Progestin primed ovarian stimulation using corifollitropin alfa in PCOS women effectively prevents LH surge and reduces injection burden compared to GnRH antagonist protocol

Utilizing corifollitropin alfa in GnRH antagonist (GnRHant) protocol in conjunction with GnRH agonist trigger/freeze-all strategy (corifollitropin alfa/GnRHant protocol) was reported to have satisfactory outcomes in women with polycystic ovary syndrome (PCOS). Although lessening in gonadotropin injections, GnRHant were still needed. In addition to using corifollitropin alfa, GnRHant was replaced with an oral progestin as in progestin primed ovarian stimulation (PPOS) to further reduce the injection burden in this study. We try to investigate whether this regimen (corifollitropin alfa/PPOS protocol) could effectively reduce GnRHant injections and prevent premature LH surge in PCOS patients undergoing IVF/ICSI cycles. This is a retrospective cohort study recruiting 333 women with PCOS, with body weight between 50 and 70 kg, undergoing first IVF/ICSI cycle between August 2015 and July 2018. We used corifollitropin alfa/GnRHant protocol prior to Jan 2017 (n = 160), then changed to corifollitropin alfa/PPOS protocol (n = 173). All patients received corifollitropin alfa 100 μg on menstruation day 2/3 (S1). Additional rFSH was administered daily from S8. In corifollitropin alfa/GnRHant group, cetrorelix 0.25 mg/day was administered from S5 till the trigger day. In corifollitropin alfa/PPOS group, dydrogesterone 20 mg/day was given from S1 till the trigger day. GnRH agonist was used to trigger maturation of oocyte. All good quality day 5/6 embryos were frozen, and frozen-thawed embryo transfer (FET) was performed on subsequent cycle. A comparison of clinical outcomes was made between the two protocols. The primary endpoint was the incidence of premature LH surge and none of the patients occurred. Dydrogesterone successfully replace GnRHant to block LH surge while an average of 6.8 days of GnRHant injections were needed in the corifollitropin alfa/GnRHant group. No patients suffered from ovarian hyperstimulation syndrome (OHSS). The other clinical outcomes including additional duration/dose of daily gonadotropin administration, number of oocytes retrieved, and fertilization rate were similar between the two groups. The implantation rate, clinical pregnancy rate, and live birth rate in the first FET cycle were also similar between the two groups. In women with PCOS undergoing IVF/ICSI treatment, corifollitropin alfa/PPOS protocol could minimize the injections burden with comparable outcomes to corifollitropin alfa/GnRHant protocol.

Participants. The present study recruited infertile women with PCOS, with a body weight between 50 and 70 kg, undergoing their first IVF/ICSI cycle. PCOS was diagnosed according to the Rotterdam consensus criteria (two out of three of the following criteria: oligo-or anovulation, clinical and/or biochemical signs of hyperandrogenism and polycystic ovaries) 16 . A diagnosis of congenital adrenal hyperplasia, Cushing's syndrome, androgen-producing tumours, hyperprolactinemia and thyroid dysfunction were all rulled out. Exclusion criteria were as follows: age > 38 years, basal FSH level > 12 mIU/mL, previous ovarian surgery, congenital uterine anomaly, intrauterine adhesion and male partner with non-obstructive azoospermia.
Ovarian stimulation in the corifollitropin alfa/GnRHant protocol. In the corifollitropin alfa/ GnRHant protocol, patients received oral pills (Diane; Bayer Weimar GmbH, Weimar, Germany) in the previous cycle to induce menstruation 17 . A single dose of corifollitropin alfa 100 μg, was administered on induced menstrual cycle day 2 or day 3 (defined as stimulation day 1, S1) in the afternoon (16:00-18:00). From stimulation day 8 (S8) onward, ovarian stimulation was continued with daily rFSH injections and the dose was adjusted according to ovarian response until the day of ovulation trigger. Ovarian response was monitored using folliculometry, and serum estradiol (E2), LH, progesterone (P4) measurement. Serial serum E2, LH and P4 levels were measured on the morning of S1, S5, S8, and the day of ovulation trigger. Between S9 and the day of ovulation trigger, folliculometry and hormonal measurement were performed every 1-3 days according to ovarian response.
To prevent a premature LH surge, 0.25 mg cetrorelix (cetrotide; Merck Serono, Halle, Germany) was administered subcutaneously once daily from S5 (16:00-18:00) until the day before ovulation trigger. When more than 3 follicles had reached 18 mm in diameter, 1 mg leuprolide acetate (Lupro; Nang Kuang, Tainan, Taiwan) was administered subcutaneously to trigger final oocyte maturation, and oocyte retrieval was performed 36 h later. Vitrification of good blastocysts was performed on day 5 or day 6 after oocyte retrieval 18 . Our working routine including ultrasound and hormonal monitoring, oocyte retrieval and embryo transfer was 6 days per week.
Ovarian stimulation in the corifollitropin alfa/PPOS protocol. Dydrogesterone replaced cetrorelix for the prevention of premature LH surge in the corifollitropin alfa/PPOS protocol. Dydrogesterone 10 mg two times per day was prescribed from S1 until the day of ovulation trigger. Other clinical management including gonadotropins stimulation and dosage tailoring, monitoring schedule of ovarian response, criteria for ovulation trigger, GnRHa triggering final oocyte maturation and oocyte retrieval was the same as that in the corifollitropin alfa/GnRHant protocol.
Follow-up after oocyte retrieval. Patients were assessed for signs and symptoms of OHSS at 3 and 6 days after oocyte retrieval according to history taking, physical examination, and ultrasound scan. The severity of OHSS was assessed according to the classification described by Golan et al. 19 . If moderate clinical features of OHSS were present, complete blood count, coagulation profile, serum creatinine, serum electrolyte and liver function were assessed. follow up was performed every 48 h to assess the progression of moderate OHSS.
Embryo vitrification/thawing and replacement. All blastocysts were vitrified on day 5 or 6. Good blastocysts were defined as blastocyst expansion and hatching status 3-6, inner cell mass and trophectoderm grade A or B 20 . The vitrification/thawing protocol was cryotop method (Kitayazo, Japan) based on the method described by Kuwayama 21 . FET was performed in the subsequent cycle, as described previously 18 . Outcome variable. The primary outcome measure was the duration of reduction in GnRHant injections. Secondary outcome measures included the incidence of premature LH surge, incidence of OHSS, additional duration/dose of daily gonadotropin administration, number of oocytes retrieved, fertilization rate, number of embryos frozen, implantation rate, clinical pregnancy rate, and live birth rate in the first FET cycle. The incidence of premature LH surge was defined as a serum LH level of ≥ 10 IU/L and P4 level of ≥ 1.0 ng/L before reaching the ovulation trigger criteria 22 . Implantation rate was calculated as the number of fetal cardiac activity detected via transvaginal ultrasound at 7 weeks of gestation divided by the number of embryos transferred. Clinical pregnancy was defined as the presence of fetal cardiac activity by transvaginal ultrasound at 7 weeks of gestation. Live birth was defined as delivery of a live child after 24 weeks of gestation.
Statistical analysis. Statistical analysis was performed using Statistical Package for the Social Sciences (Release 10.0; SPSS). Continuous variables are expressed as mean and standard deviation (SD). The independent-sample t test or paired t test was used for continuous variables as appropriate. Categorical variables, are expressed as raw frequencies with corresponding percentages, and the between-group differences were assessed using either the chi-square test with Yates correction if required, or the Fisher exact test. A P value less 0.05 was considered to be statistically significant.
Ethical approval. The study was approved by the Ethics Committee of National Taiwan University Hospital (201811078RINB). All patients who entered the IVF/ICSI cycles at the beginning had signed the informed consent. All methods were performed in accordance with the relevant guidelines and regulations of the Institution.

Results
A total of 333 cycles met the inclusion criteria for analysis: 173 in the study group (corifollitropin alfa/PPOS group); and 160 in the control group (corifollitropin alfa/GnRHant group). All patients underwent their first IVF/ICSI cycles. No significant differences in terms of demographic data and baseline characteristics, including age, body weight, body mass index (BMI), proportion of primary infertility, duration of infertility, anti-Mullerian hormone (AMH) and baseline hormonal levels were observed between the two groups ( Table 1).
As shown in Table 2, no patients in either group experienced premature LH surge and OHSS. There was a significantly longer duration of GnRHant injections in the corifollitropin alfa/GnRHant group compared with the Scientific Reports | (2021) 11:22732 | https://doi.org/10.1038/s41598-021-02227-w www.nature.com/scientificreports/ corifollitropin alfa/PPOS group. An average of 3.9 and 3.8 days of additional daily rFSH injections were required in the corifollitropin alfa/PPOS group and corifollitropin alfa/GnRHant group, respectively (P > 0.05). The additional total and average daily amount of rFSH consumption were also similar in both groups. No patients in either group experienced premature LH surge and OHSS. Other clinical outcomes with regards to serum hormonal levels on ovulation trigger day, numbers of oocyte retrieved, rate of metaphase II (MII) oocytes, fertilization rate, number of blastocyst frozen and number of good blastocysts are summarized in Table 2. Figure 1 showed the frequency distribution of number of oocytes retrieved in the two groups of patients. The clinical outcomes of the first FET cycle are presented on  Fig. 2. In the corifollitropin alfa/PPOS group, dydrogesterone administration began from S1. Serum LH level decreased gradually from S1 to S9-11, then stabilized till the day of ovulation trigger. The corifollitropin alfa/PPOS group had a significantly lower serum LH level on S5 compared with the corifollitropin alfa/GnRHant group, whose GnRHant had not begun in the morning of S5. In corifollitropin alfa/GnRHant group, the first dose of GnRHant was administered in the afternoon of S5 and the serum LH level on S8 was significantly decreased as compared to the serum LH levels on S1 or S5. On S8, GnRHant also resulted in a significantly lower serum LH level in corifollitropin alfa/GnRHant group compared with the corifollitropin alfa/PPOS group. However, the serum LH level became similar between the two protocols on S9-11 and the day of ovulation trigger. There were no significant difference of the serum E2 and P4 profiles during COS between the two groups.

Discussion
This study demonstrated that the corifollitropin alfa/PPOS protocol could be a simplified, patient friendly regimen for women with PCOS undergoing IVF/ICSI treatment. No patients experienced premature LH surge. The injection burden of GnRHant and gonadotropins was decreased. The risk for OHSS was minimized and the clinical outcomes appeared to be satisfactory.
The COS during IVF/ICSI treatment typically requires daily injection of gonadotropin to recruit more oocytes. This causes physical burden and psychological stress in patients, and also increases the risk for injection errors 23 . A dropout rate of > 50% has been reported by several studies even though the treatment costs could be reimbursed [24][25][26] . Schroder et al. reported a dropout rate of 39.9% after failure of the first cycle, which then increased to 62.2% after the fourth cycle, indicating the high level of distress and frustration experienced by patients during the IVF/ICSI treatment 24 . Substantial psychological stress and physical burden associated with conventional ovarian stimulation are the major causes for dropout before achieving pregnancy, which in turn   31 . Therefore, it is crucial to develop a simple treatment regimen that mitigates the physical burden and psychological stress in women with PCOS 13 . Corifollitropin alfa is a feasible option to reduce the injection burden of gonadotropins. Corifollitropin alfa was introduced into clinical practice before the popularity of the GnRHa trigger/freezeall strategy and, hence, was not recommended to be used in women with PCOS in whom an excessive ovarian response was a concern. Currently, it is well recognized that the GnRHa trigger/freeze-all strategy substantially eliminates OHSS in high responders 32,33 . Thus, long-acting FSH is a reasonable alternative to conventional daily gonadotropin injections for women with PCOS because the risk for OHSS can be minimized. Corifollitropin alfa combined with a GnRHa trigger/freeze-all strategy has seldom been studied in women with PCOS or other high responders. We described a corifollitropin alfa/GnRHant protocol in PCOS patients undergoing IVF/ICSI treatment with satisfactory clinical outcomes and low risk for OHSS. Nevertheless, an average of 6.7 days of GnRHant injections was still needed to prevent premature LH surge 13 .
La Marca and Capuzzo reported that PPOS is shown to effectively prevented premature LH surge, with similar clinical outcomes compared with conventional ovarian stimulation 15 . A PPOS approach is especially suitable for high responders because a freeze-all strategy is mandatory 15 . Several oral progestins have been reported to be successfully used in women with PCOS using a PPOS protocol, including MPA 34-36 , MIP 37 , and dydrogesterone [38][39][40] . Daily injections of human menopausal gonadotropin (hMG) or rFSH, rather than corifollitropin alfa, was used in these studies. Our study demonstrated that co-administration of dydrogesterone with corifollitropin alfa could also effectively prevent premature LH surge in women with PCOS to further reduce the injection burden of GnRHant, meanwhile obtaining comparable clinical outcomes to those achieved using a corifollitropin alfa/ GnRHant protocol. The number of additional injections needed for this protocol during COS were an average of 3.9 days of daily gonadotropin and 1 dose of GnRHa to trigger ovulation. There were no studies comparing early and late start of dydrogesterone on IVF outcomes. According to Kuang's study, if MPA was started during the midfollicular phase in patients with multiple growing follicles and elevated serum E2 levels, the blockade of LH surge could fail 14 . In our study, we started dydrogeterone on stimulation day 1 for fear of failing to prevent a premature LH surge.
Two previous studies compared dydrogesterone primed ovarian stimulation with GnRHant protocols for women with PCOS undergoing IVF/ICSI treatment 38,40 . Eftekhar et al. conducted an RCT that recruited 60 patients receiving dydrogesterone primed ovarian stimulation and 60 patients receiving the GnRHant protocol. The number of MII oocytes, fertilized oocytes and the trigger day serum E2 levels were significantly lower in the PPOS group than in GnRHant group. The trigger day serum LH levels was significantly higher in the PPOS group 38 . Gurbuz et al. retrospectively analyzed 525 patients, of whom 258 were treated using a dydrogesterone primed ovarian stimulation protocol and 267 treated using a GnRHant protocol 40 . Similar clinical outcomes, including the incidence of premature LH surge, were reported, except for a significantly higher serum LH levels on the ovulation trigger day in the PPOS group. Although the two studies reported that dydrogesterone had a www.nature.com/scientificreports/ lower pituitary suppression effect compared to GnRHant, we found that both medications resulted in similar serum LH levels on the ovulation trigger day. In our study, as shown in Fig. 2, co-administration of dydrogesterone with corifollitropin alfa resulted in a steadily decrease in serum LH levels from S1 to the trigger day, while a rapidly decrease in LH levels was observed one day after GnRHant administration in the GnRHant group. On the trigger day, both regimens resulted in similar LH levels. In short, all of these studies suggest that dydrogesterone is an effective oral medication for the prevention of premature LH surge in women with PCOS undergoing IVF/ICSI treatment. The starting dose of gonadotropin in women with PCOS undergoing IVF/ICSI treatment has not been extensively studied. To our knowledge, there have been no RCTs investigating the optimal starting doses of gonadotropins for patients at high risk for developing OHSS 41 . Thakre and Homberg proposed that gonadotropin doses should be lower than conventional doses (no greater than 150 IU), at least in the first cycle, to lower the risk for excessive response 9 . We used corifollitropin alfa 100 μg in this study because the potency was equivalent to 150 IU rFSH according to the ENSURE trial 42 . In the present study, an average of 20.7 oocytes were retrieved, and no patients experienced OHSS or other major complications, such as internal bleeding or ovarian torsion. It indicated that corifollitropin alfa 100 μg was suitable for this group of women with PCOS. In a systematic review addressing the topic of COS for freeze-all cycles, Mizrachi et al. concluded that there was strong evidence showing that the cumulative live birth rate increased with the number of oocytes retrieved, and a goal for the recovery of 15-20 oocytes in freeze-all cycles would be an acceptable balance between safety and efficacy 43 . Patients with a body weight < 50 kg or > 70 kg were excluded from the present study because we usually prescribed 112.5 IU and 200 IU rFSH, respectively, to these patients during the first 4 days of COS, then adjusted according to ovarian response. Further studies exploring the optimal corifollitropin alfa dose for these patients, therefore, are warranted.
Dydrogesterone was chosen to prevent premature LH surge in this study because it has been used for decades in pregnant women who experienced recurrent pregnancy loss and threatened miscarriage 44 . Besides, a recent systematic review and individual participant data meta-analysis comparing dydrogesterone and vaginal progesterone for IVF luteal phase support showed similar safety parameters for the mother and the fetus between the two treatments 45 . Recently, a retrospective cohort study reported similar neonatal outcomes and incidence of major congenital malformation in 1429 live-born infants after dydrogesterone primed ovarian stimulation, compared with 2127 live-born infants after a GnRHa short protocol for IVF 46 . Compared with MIP, dydrogesterone will not elevate the serum progesterone levels and interfere with the interpretation of premature luteinization during COS 47 .
There were four studies comparing IVF outcome data among different progestin formulations undergoing PPOS protocol 39 48,49 . In a recent systemic review including meta-analysis, similar clinical outcomes including effectiveness of preventing premature LH surge in PPOS protocol using MPA, MIP and dydrogesterone were reported 50 .
The major drawback to our study was its retrospective design. However, no changes in staff, laboratory protocols, and clinical practice occurred during the study period. All patients received their first IVF/ICSI treatment cycle. The results of this study may have some implications for daily clinical practice and further research. This protocol also could be applied to non-PCOS high responders, in whom a freeze-all strategy has been reported to result in significantly higher live birth rates and lower risk for OHSS as compared to fresh embryo transfer 51,52 .
In summary, results of the present study demonstrated that a corifollitropin alfa/PPOS protocol could effectively prevent premature LH surge while reducing the injection burden of GnRHant and gonadotropins. It has the potential to be a simplified, patient friendly protocol for women with PCOS undergoing IVF/ICSI treatment. The clinical outcomes appeared to be promising. Further RCTs are needed to compare clinical outcomes, including OHSS prevention and cumulative live birth rate with the conventional GnRHant protocol using daily administration of gonadotropins.