Study setting and patients

We retrospectively examined 86 cycles of oocyte collection for fertility preservation at our hospital between January 2016 and April 2021. Breast cancer cases accounted for 73% of the cases, followed by hematological disorders at 15%. For patients with male partners either with or without legal marriage, oocyte and/or embryo cryopreservation were offered for fertility preservation. For patients without male partners, only oocyte cryopreservation was offered. Semen analysis for male partners was always performed before oocyte collection in couples with no history of pregnancy. The cases in this study did not have severe male infertility. The endpoints were the number of days of stimulation, total dose of gonadotropin, serum E2 concentrations on the day of ovulation trigger, oocyte maturation rate, fertilization rate in cases of embryo freezing, and good blastocyst rate. This research protocol was approved by the Saitama Medical Center, Saitama Medical University Ethics Review Board (approval number: 2575). This study was not funded, and it was performed retrospectively using electronic medical record information and ultrasound findings.

Controlled ovarian stimulation

For referred fertility-preserving cases, ovarian stimulation was promptly started regardless of the menstrual cycle if oocyte collection was desired after counseling. A random-start PPOS or GnRH-ant protocol was used depending on the practice of the attending physicians and the patient preference of ovarian stimulation method.

Both methods were initiated by subcutaneous injection of recombinant FSH (GONAL-f; Merck). All of the breast cancer patients were referred to our hospital along with the information of estrogen receptor status which had been tested either on the biopsy sample or on the surgical specimen. For estrogen receptor-positive breast cancer cases, 5 mg/day of letrozole was used in combination from the start of ovarian stimulation to the day of trigger.

In the GnRH-ant group, a flexible method was adopted wherein 0.25 mg of ganirelix acetate (Ganirest, MSD) was administered daily from the day when the main follicle diameter reached 14 mm by ovarian stimulation. Follicular diameter was defined as the average value of the major and minor axes. In contrast, in the PPOS group, 20 mg of dydrogesterone (Duphaston, Mylan) was administered each day from the start of ovarian stimulation to the day of trigger (Fig. 1). In both groups, follicular growth was monitored every 2–5 days using serum levels of FSH, LH, E2, and progesterone (P4), as well as transvaginal ultrasonography.

Fig. 1

Random-start progestin-primed ovarian stimulation regimens. Abbreviations: FSH, follicle-stimulating hormone; HMG, human menopausal gonadotropin; HCG, human chorionic gonadotropin

In both groups without letrozole, in general, when it was confirmed that there were ≥ 2 follicles of ≥18-mm diameter, dual trigger with four nasal sprays (150 μg each) of buserelin (Buserelin, Fuji) and an injection of 1500 IU of urine human chorionic gonadotropin (HCG) (HCG, Fuji) was administered to induce maturation of oocytes, and oocyte pickup was performed 35–37 h later. In the groups with letrozole, however, the dual trigger was delayed until when there were ≥ 1 follicles of ≥20-mm diameter. Also, even when the sizes of dominant follicles reach 18 or 20 mm, the time of triggering may be slightly delayed when the sizes of other nondominant follicles do not reach 16 mm in order to maximize the number of maturate oocytes.

Embryo culture and assessment

Patients with male partners who opted for embryo cryopreservation underwent intracytoplasmic sperm injection (ICSI), in vitro fertilization (IVF), or split insemination after oocyte collection. Although selection was based mainly on the preference of the patient, ICSI was actively recommended in cases with poor semen findings on the day of oocyte collection. The cases in this study did not include severe male infertility.


On day 0 of oocyte retrieval, the collected eggs were pipetted to remove the granulosa cell layer (PINU06-20FT, Prime Tech Ltd.) connected to a piezo-electric actuator (Prime Tech Ltd.). After injecting sperm, the cells were cultured using a single-step culture medium (SAGE 1-Step, CooperSurgical). Embryos were observed in the time-lapse culture system (Astec Co. Ltd.) on days 1, 2, 3, 5, and 6 after oocyte retrieval. Normal fertilization was determined if two pronuclei were observed on day 1 after oocyte retrieval. Blastocysts were evaluated according to the blastocyst scoring system developed by Gardner et al. [6], and blastocysts that reached Grade 3BB were frozen.


Within 2 h of oocyte collection, the final sperm concentration was adjusted to 300,000/mL, and insemination was performed. Fertilization was determined when the granulosa cell layer was removed, and the second polar body was confirmed approximately 5 h after fertilization.

Study outcomes

The primary outcome was numbers of oocytes and metaphase two oocytes per cycle. The secondary outcome was the number of vitrified blastocysts per cycle for embryo freezing cases. In addition, AMH levels, ovarian stimulation period, total dose of gonadotropin preparation, days of hospital visits, start of menstruation rate before oocyte collection, number of collected oocytes, number of mature oocytes, and number of normal fertilized oocytes were comparatively examined. Oocyte maturation was evaluated at the timing of oocyte vitrification and ICSI. For the oocytes with which standard IVF was performed, oocyte maturation was evaluated under the removal of cumulus cells 4 h after insemination.

Statistical analysis

All statistical analyses were performed using JMP version 16.0.0 software. Patient characteristics, ovarian stimulation characteristics, and endocrinological characteristics were expressed as mean (± standard deviation). For frequency/ratio comparisons, chi-squared test with Yates’ correction or Fisher’s exact test and the average ratio were used. The Student’s t-test was used for comparison. Statistical significance was set at P <  0.05.

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