Clinical Protocols
An initial assessment was carried out, which included a blood test for AMH (Roche Assay), FSH, E2 and ultrasound for AFC performed between days 1-4 of the menstrual cycle. The ovarian stimulation protocol was individualised depending on age, AFC, AMH and BMI. Ovarian stimulation using an antagonist protocol was commenced on the third day of the menstrual cycle. From day 6 onwards, the gonadotrophin dose was adjusted according to E2 levels and ultrasound evidence of stimulation. On day 7 of the cycle and/or once the leading follicle was ≥14 mm in diameter, Cetrorelix (Cetrotide) 0.25 mg (Merck Serono, Germany) was commenced. The choice of trigger was individualised after assessment of age, baseline AFC and AMH, number of follicles and E2 levels on the day of trigger. Women considered low responders were given either Pregnynl a human chorionic gonadotrophin (hCG) trigger, or dual trigger with Pregnyl 1,500 IU (Organon, Netherlands) and Suprefact 1ml (Sanofi-Aventis, Germany), a gonadotrophin releasing hormone analogue (GnRH). Women considered normal or high responders were given Suprefact only. Oocyte maturation was triggered once the mean diameter of ≥3 follicles ≥18mm. In the event women were at high risk of ovarian hyperstimulation syndrome (OHSS), Suprefact 1ml (Sanofi-Aventis, Germany) was administered and LH level was measured 8-12 hours later. Oocyte retrieval was scheduled 37 hours after ovulation trigger. Denudation was carried out 39 hours post the trigger injection and metaphase II oocytes were vitrified immediately after.
All thaw cycles included within our study refer to the thawing of oocytes which were frozen initially at CRGH, undergoing the vitrification method. Vitrification at room temperature (24-26°C) took place in two steps. Firstly, once oocytes were denuded they were moved for 12-15 minutes within a solution containing 7.5% Dimethyl Sulfoxide-D6 (DMSO) and 7.5% Ethylene Glycerol (EG). Oocytes achieving full recovery to normal size were then transferred to a vitrification solution consisting of 15% DMSO and EG plus 0.5-m sucrose for 60 seconds. Secondly, oocytes were then transferred via Cryotop straws at fast rate into liquid nitrogen for storage, within a minimum volume of vitrification solution. Six welled dishes adjacent to three welled plates from Cryotech were used containing solutions warmed at 37° with various concentrations of sucrose (1.0. 0.5 and 0.0m). The vitrified oocytes within the Cryotop straws were then removed from storage and moved to liquid nitrogen, within 0.7ml 1.0 M of sucrose, followed by 3-5 minutes within 50 μl of varying decreasing sucrose concentrations at room temperature. Oocytes with full recovery were then transferred for 2 hours into culture before undergoing intracytoplasmic sperm injection (ICSI).
The oocytes were assessed 16-18 h post ICSI for the presence of pronuclei and were separated according to the number of pronuclei present. Oocytes displaying abnormal pronuclei numbers (zero, one and more than three) were either discarded or kept in a separate dish of cleavage medium (Vitrolife). Oocytes displaying 2 pronuclei were either moved to a fresh dish of cleavage medium (Vitrolife) while the two-step culture system was in effect, or single-step culture media (Sage/Origio). The embryos were either moved to Blastocyst medium (Vitrolife) or a fresh dish of single-step media (Sage/Origio) on day 3 in order to be cultured to the blastocyst stage, until day 5 or 6 post insemination. Any embryos that successfully formed a blastocyst on day 5 or 6 of culture (exhibiting the presence of clear inner cell mass and trophectoderm cell lines) were selected for vitrification.
Blastocyst vitrification took place at 37°C. Multiple 5-well Nunc dishes were used to warm 1 x 0.5ml wells of V1 per blastocyst, 1 x 0.5ml well of V2 and 1 x 1ml well of V3 media solutions (Blastocyst Vitrification Kit, Sydney IVF) to 37°C a minimum of 1 hour prior to use. Blastocysts were isolated into micro-drops of warm Hepes media under oil, assessed at 200x magnification and graded as per the CRGH in-house grading criteria (adapted Cornell). Blastocoelic cavity collapse was then initiated by a single laser shot to the junction of two outermost trophectodermal cells, before the blastocysts were transferred to individual wells of the warmed V1 media (up to 4 blastocysts per dish). Vitrification was conducted by moving a single blastocyst from V1 to V2 media for 2 minutes followed by thorough washing through 3 drops of V3 media for 30-35 seconds. The blastocyst was then loaded rapidly onto a Cryolock with minimal volume of vitrification solution and plunged into liquid nitrogen before placing a protective cap over the loading strip. Once blastocysts were frozen, women embarked on a single elective embryo transfer. A medicated frozen embryo transfer was performed.15 All patients were advised to perform a urinary pregnancy test 16 days later.