Study design and patient selection
A total of 4143 women who underwent vitrified-warmed blastocyst transfer cycles from October 2014 to December 2015 were included in this study. The blastocyst transfer cycles of women who underwent preimplantation genetic screening were excluded from this study.
Patients who underwent FET cycles were divided into AH group and non-AH group. Assisted hatching was performed on the warmed blastocyst every other day on the basis of even dates for warming . The study was double-blinded. In the AH group, all blastocysts that survived after warming underwent laser AH (or laser partial zona dissection). In the non-AH group, all the warmed blastocysts were cultured in G2 blastocyst culture media (Vitrolife, Göteborg, Sweden) without AH before the transfer. AH was performed by several expert embryologists. All AH blastocysts and non-AH blastocyst were transferred in this study.
This retrospective study was approved by the Ethics Committee of Reproductive Hospital Affiliated to Shandong University (Jinan, China). All patients signed written informed consent forms.
In vitro fertilization and embryo culture
All patients underwent in vitro fertilization (IVF) treatment. During IVF treatment, ovarian stimulation and natural cycles were performed according to the patients’ baseline data. Ovarian stimulation protocols included controlled ovarian hyperstimulation after gonadotropin-releasing hormone (GnRH) agonist down-regulation or antagonist protocol. Recombinant follicle-stimulating hormone (rFSH, PUREGON; MSD Organon, Oss, Netherlands) was started on day 1–3 of menstrual cycle. The dose adjustment of gonadotropin, monitoring of the ovarian response, and the timing for triggering the final oocyte maturation during ovarian stimulation was performed at the discretion of the supervising physician. Oocyte retrieval was performed 34–36 h after the administration of human chorionic gonadotropin (hCG) at a dose of 4000–10 000 IU. Based on the sperm quality, oocytes were inseminated approximately 3–6 h after follicular aspiration using a conventional insemination method or intracytoplasmic sperm injection. Embryos were cultured separately in pre-equlibrated culture media overaid with mineral oil. The culture dish were housed in 37°C tri-gas table top incubators (K-system, Denmark) containing 5% O2 and 6% CO2, balanced with N2. Two high-quality embryos were picked out for fresh transfer on day 3. For the patients who can only accept a single embryo transfer, a single blastocyst was selected and transferred on day 5. Supernumerary embryos were cultured for blastocyst cryopreservation. On day 3, the embryos were removed from the cleavage media and placed in blastocyst media. Morphologic criteria were used for day 3 embryo scoring based on the amount of anucleate fragments expelled during early cleavage and on developmental speed. Embryo scores on days 5 to 7 were assessed according to Gardner morphological criteria  and based on the degree of expansion and the development of the inner cell mass and trophectoderm. The selection of the vitrification blastocyst gave priority to the score of the inner cell mass, and the score of trophectoderm was also considered—ie, the rank of blastocyst grade from top to high quality was AA, AB, BA, BB, AC, and BC. All the virtrified blastocysts had developed to the stage of thinning zona pellucida. Concerns that the high pressure to which blastocysts are exposed during pipetting might rupture the trophectoderm and induce blastocoelic fluid leakage have been raised. A fully hatched embryo is expected to be more fragile. The time for vitrification performed on day 6 or day 7 was at morning time as early as possible to avoid the hatching of the blastocyst.
Vitrifying and warming procedures
Vitrifying and warming procedures for blastocysts were performed at 37°C. All blastocysts were collapsed by laser-assisted artificial shinkage before vitrification to avoid ice crystal formation by reducing the fluid content of the blastocoel . Vitrification was performed using the Mukaida protocol with a CryoLoop . The blastocysts were rinsed using a base solution that contained 5 mg/mL human serum albumin in HEPES-buffered modified human tubal fluid medium (Irvine Scientific, CA, USA) and subsequently placed in an equilibration solution containing 7.5% (v/v) dimethyl sulfoxide (DMSO, Sigma-Aldrich, St. Louis, MO, USA) and 7.5% (v/v) ethylene glycol (EG, Sigma-Aldrich, St. Louis, MO, USA). After about 2 min, the blastocysts were transferred to the vitrification solution containing 15% (v/v) DMSO, 15% (v/v) EG, 10 mg/mL Ficoll 70 (Pharmacia Biotech Inc., Sweden), and 0.65 mol/L sucrose and vitrified for <30 seconds. Finally, the vitrified blastocysts were placed on a CryoLoop (Hampton Research Corp., Laguna Niguel, CA, USA) and immediately immersed in liquid nitrogen.
Warming was performed in a four-well multi-dish based on the Mukaida protocol. Briefly, blastocysts were incubated in warming solution I containing 0.33 mol/L sucrose; warming solution II containing 0.2 mol/L sucrose; and base solution at 37°C for 2, 3, and 5 min, respectively. Survival of blastocysts was assessed based on the integrity of the inner cell mass and trophectoderm cells, with absent or partial degeneration and fully or partial re-expansion. After warming, the blastocysts were kept in blastocyst culture media and incubated at 37°C with 6% CO2 before they were transferred to the uterus. Warming was performed in the morning and transferred in the afternoon at regular time everyday. The delay between warming and transfer was no less than 4 hours and no more than 6 hours. The warmed blastocyst would be evaluated before transfer, and all the survived blastocysts would be transferred.
In the AH group, the survived blastocysts underwent laser AH (or laser partial zona dissection) immediately after warming. Laser AH was performed under a Nikon inverted microscope (Nikon, Tokyo, Japan) that was equipped with an RI laser system (Saturn Laser System Research Instruments Ltd., Basel, Switzerland). Laser dissection was performed at the largest perivitelline space, and exposure time and the number of consecutive irradiations were dependent on the thickness of the ZP and perivitelline space. Laser power, exposure time, number of laser shots were also different between blastocysts with different degrees of expansion. The size of the laser hole should not exceed the thickness of the ZP. AH was usually performed at a position away from the inner cell mass and the trophoblastic cells to minimize the risk of thermal damage to the embryos. For most of the blastocysts warmed, the perivitelline space was large enough to perform laser AH easily. Usually, the Laser was set as follows. Hole size was from 4.0 μm to 6.9 μm in diameter. Pulse width was from 0.38 ms to 0.574 ms or so. Approximately one-eighth to one-sixth of the blastocyst ZP circumference was cut before the transfer to G2 blastocyst culture media (Fig. 1).
Preparation for FET cycles and embryo transfer
Endometrial preparation for vitrified-warmed blastocyst transfer was performed during natural cycles, hormonal replacement therapy (HRT) cycles, controlled ovarian hyperstimulation cycles, or hormonal replacement. During HRT cycles, the endometrium was prepared with 4 mg oral estradiol valerate (Progynova; Bayer AG, Leverkusen, Germany) supplementation daily for 5 days starting on day 2 or 3 of the menstrual cycle, which was followed by 6 mg estradiol for an additional 5 days. If necessary, the dose of estradiol was increased to 8 mg for another 4–5 days. When endometrial thickness exceeded 8 mm, dydrogesterone (Duphaston 20 mg; Abbott Biologicals, Amsterdam, the Netherlands) twice daily and vaginal micronized progesterone (200 mg) once daily were initiated. During natural cycles, ovulation was assessed and documented based on the disappearance or typical change in the shape of the dominant follicle. Ovulation was induced by a bolus of 8000–10,000 IU of hCG when at least one dominant follicle reached ≥18 mm in diameter, and the thickness of the endometrium was at least 8 mm. Dydrogesterone (10 mg) was initiated twice daily. Blastocysts were warmed and transferred on day 5 after the initiation of progesterone treatment. Survived blastocyst was transferred after being cultured for 4~6 hours in blastocyst culture media with partial, fully re-expansion or different levels of hatching (Fig. 2).
Pregnancy was confirmed by an increase in serum hCG concentrations (>10 mIU/mL) at 12 days after blastocyst transfer. Clinical pregnancy was determined by ultrasonographic observation of a gestational sac at week 7. Early abortion was defined as natural abortion before 12 weeks of gestation. The live birth rate was calculated as the number of live births per transfer cycle. Live birth was defined as the delivery of a live-born infant after ≥28 weeks of gestation. The monozygotic twin pregnancy rate was calculated as the number of monozygotic twin cycles per clinical pregnancy cycle. Patient ages, endometrial preparation protocols, numbers of embryos to be transferred, embryonic periods of development, clinical pregnancy rates, early abortion rates, live birth rates, and birth defect rates were recorded and compared between the AH and the non-AH groups.
All analyses were performed using SAS 9.4 software (SAS Institute Inc., Cary, NC, USA). Statistical analyses were conducted using the t-test and chi-square test. The mean age was compared by the t test. The difference in the primary outcome between the two groups was analysed by the Pearson chi-square test. Logistic regression analyses were performed to assess the association of clinical features with blastocyst transfer results. P <0.05 was considered statistically significant.