To maximize the chance of refractory infertile patients to achieve pregnancy, we designed the time-sensitive SF course to consist of 3 months of consecutive OR. The aim of this treatment course is to acquire as many oocytes as possible in a limited period of time as early as possible in the patient’s life. After fertilization and embryo culture, the acquired competent embryos, blastocysts in general, were vitrified for subsequent transfer. This process was performed quickly to minimize the amount of time lost as a result of futile embryo transfer or miscarriage. To participate in the SF course, patients had to meet at least one of the three following criteria: (1) maternal age ≥40 years, (2) three or fewer oocytes retrieved after previous stimulation, and (3) reduced ovarian reserve (anti-Müllerian hormone [AMH] <1.0 ng/mL). Between June 2017 and October 2018, we implemented 101 treatment courses and performed 485 OR cycles.
This was a retrospective cohort study conducted in a private fertility center in Japan. The study was approved by our clinic’s Institutional Review Board and carried out in accordance with the Code of Ethics of the World Medical Association (Declaration of Helsinki) for experiments involving humans. All the candidates provided informed consent after receiving individual counseling about the time significance and merit and demerit of multiple consecutive stimulations. This study’s main objective was to confirm the efficacy of the SF strategy for patients who are refractory to treatment and who fulfill the Bologna criteria . We also examined whether more than three consecutive ORs and luteal-phase ORs could obtain competent embryos more efficiently, ultimately increasing the chance of childbearing.
Ovarian stimulation protocol
Ovarian stimulation was considered during any phase of the menstrual cycle, when one or more antral follicles was recognized by transvaginal ultrasound, to squeeze out all oocytes. In the first attempt, ovarian stimulation began on cycle day 2 to 4, as is the normal convention, and a short, antagonist, or mild stimulation protocol was selected for each patient. Gonadotropin-releasing hormone (GnRH) agonist nasal spray (Buserelin acetate; Buserecur®, Fuji Pharm, Japan) and/or clomiphene citrate (CC; Clomid®, Fuji Pharm, Japan)/aromatase inhibitor (AI; Femara®, Novartis, Basel, Switzerland) and/or gonadotropin injection were administered. Follicular monitoring was performed at the beginning, 5–7 days after the start and then as needed, continuing every 2–3 days until the follicle reached approximately 18 mm in diameter. Serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), estradiol, and progesterone levels were measured on ultrasound days using the analyzer Cobas e 411 plus (Roche, Basel, Switzerland), which uses electrochemiluminescence technology for immunoassay analysis. A GnRH antagonist was administered when the serum LH level was elevated or the leading follicle reached 16 mm in diameter. Final oocyte maturation was triggered by a GnRH agonist nasal spray and/or human chorionic gonadotropin (rec-hCG 250 μg; Ovidrel®, Merck Biopharma, Darmstadt, Germany). Thirty-six hours after the trigger, ORs were performed via transvaginal ultrasound, in which all the follicles with a diameter greater than 10 mm were aspirated. The next trial began when the preceding trial resulted in a smaller number of retrieved oocytes (e.g., fewer than five), and at least one antral follicle was recognized by transvaginal ultrasound examination. In general, CC began the day after retrieval, when there were one or two visible antral follicles. When more than three antral follicles were visible, daily administration of 150 IU human menopausal gonadotropin (Ferring®, Ferring Pharma, Saint-Prex, Switzerland) was started following the same timing. After one week or longer, we attempted to retrieve the newly matured follicles in the luteal phase after administering the rec-hCG maturation trigger. Follicle monitoring was sometimes more difficult in the luteal phase because of the presence of corpora lutea and hormones; thus, we used serial ultrasound examinations and serum hormone measurements to help determine the timing. The same was true for the luteal-phase ORs, and careful examination helped distinguish the new follicles. When no follicle growth was detected after the seventh day, ovarian stimulation was stopped until the next menstruation. Thus, we attempted to repeat the OR as many times as possible for 3 months, regardless of the menstrual cycle.
Embryo culture and assessment, and cryopreservation
Sperm was collected after liquefaction and centrifugation and swim-up and then provided for conventional in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI), depending on the semen condition. IVF was performed by inseminating each oocyte with 105 motile spermatozoa/mL, and ICSI was performed after 2–4 h of incubation, after the cumulus and corona radiata cells were removed by hyaluronidase and pipetting. Embryos were cultured in the medium NX (Continuous Single Culture NX Complete Medium: IS Japan, Co. Ltd.) or S1 (SAGE 1-Step Medium: Origio Japan, Co. Ltd.) at 37°C in 6% carbon dioxide and 5% oxygen tension in a time-lapse incubator (Embryo Scope plus; Vitrolife, Co. Ltd., Göteborg, Sweden). Embryos that developed to the blastocyst stage were graded according to Gardner’s criteria. A blastocyst with a score better than grade 3BB was considered fair. Embryos that scored greater than grade 3CC on day 4 to 7 were vitrified.
To preserve the value of the concept, a robust cryopreservation program is mandatory. The vitrification and warming procedures were performed according to the Cryotop Safety Kit manual (Kitazato Co, Japan) at room temperature. In the vitrification procedures, blastocysts were equilibrated for 12–15 min in an equilibration solution and then washed and floated for 45–60 s in a vitrification solution (VS). Subsequently, blastocysts were placed on the Cryotop sheet with a minimum volume of VS. Immediately, the Cryotop sheets were submerged into liquid nitrogen. In the warming procedures, the Cryotop sheets were placed into a thawing solution and incubated at 37℃ within 1 s. After 1 min, the blastocysts were placed in a diluent solution for 3 min. Finally, the blastocysts were placed in a washing solution for 5 min. The blastocysts were cultured at 37℃ for at least 3 h before ET.
Endometrial preparation and frozen embryo transfer
Thawed embryo transfers were performed in hormone replacement cycles. Endometrial preparation was undertaken by incremental doses of transdermal estradiol (ESTRANA® Tapes 0.72 mg; Hisamitsu Pharmaceutical Co., Inc., Tokyo, Japan). After conﬁrming that an endometrial thickness greater than 8 mm by ultrasonography, oral progestin (chlormadinone acetate; Lutoral®, Fuji Pharm, Tokyo, Japan) and transvaginal natural progesterone (LUTINUS®, Ferring Pharma; or OneCrinone®, Merck Biopharm) were administered daily. Blastocyst transfer was performed on day 5 or 6 of progesterone administration.
Clinical pregnancy was deﬁned as a pregnancy when a gestational sac was observed in the uterus by ultrasound. Ongoing pregnancy was deﬁned as a pregnancy in which fetal cardiac activity was detected by ultrasound beyond 9 weeks.
Chi-square test and t tests were performed to compare the differences between the two groups. A statistically significant difference was defined as P < 0.05.