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.