Materials and Methods:
This study was conducted at a tertiary level infertility unit in the
United Kingdom (UK) performing approximately 1500 oocyte retrievals per
year. Data extraction was performed from the electronic database of all
oocyte retrievals performed from November 2015 to December 2017. This
period was chosen because at the start of November 2015 the culture
medium was changed from sequential media to a single step culture media
and then in January 2018 day three morphology checks were ceased. The
interim period therefore had consistent culture conditions for
comparison. Neither specific funding nor ethical approval was sought for
this study as it was a retrospective analysis of data. There was no
patient and public involvement in the design of this study. Only the
first cycle in this study period per woman was included for analysis.
The primary outcomes analysed were live birth and cumulative live birth
per oocyte retrieval. Details of the number of cycles included and the
exclusion criteria have been provided in figure 1.
Ovarian stimulation and oocyte
retrieval
Ovarian stimulation with gonadotrophins was performed using either a
conventional long agonist protocol or antagonist protocol to prevent a
premature Luteinising hormone (LH) surge. Follicular monitoring was
performed using transvaginal ultrasound with ovulation triggered using
either human chorionic gonadotrophin (hCG) or GnRH (gonadotropin
releasing hormone) agonist when at least three follicles were 17mm or
larger. Oocyte retrieval was performed 35 hours after ovulation trigger
under ultrasound guidance. IVF or ICSI was performed between 39 and 41
hours post ovulation trigger depending on the sperm parameters and
previous fertilisation results if applicable.
Embryo culture
The patients had the choice of two culture systems in our unit; SC (BT37
bench-top incubators, Planar, UK or Heracell 150 chamber incubators,
Thermo Scientific, UK) or TLI at an additional cost (EmbryoScope®,
Vitrolife, DK). Where IVF was performed and TLI was required, up to 12
fertilised zygotes were placed in individual wells of an EmbryoScope®
Slide at fertilisation check (at 16-19hpi) and placed into the TLI. For
TLI cases where ICSI was performed, injected oocytes were placed
immediately into TLI if there were 12 or fewer mature oocytes injected,
or alternatively fertilised zygotes were placed into TLI after
fertilisation check where there were more than 12 mature oocytes
injected. Where there were more than 12 zygotes for a patient the
additional zygotes were placed into SC. Cycles where both TLI and SC
incubators were used were excluded from the analysis.
A low oxygen (5%) culture environment with 6% carbon dioxide at a
temperature of 370C was used in both SC and TLI
culture systems. Quinn’s Advantage Fertilisation Medium (Cooper
Surgical, Denmark) was used from oocyte retrieval to fertilisation check
for IVF cases and until denudation for ICSI cases. This was followed by
Sage 1-Step™ (Cooper Surgical, Denmark), with both types of culture
medium having an oil overlay (Origio Liquid Paraffin, Cooper Surgical,
Denmark). In both TLI and SC incubators, single embryo culture was
performed in micro-drops. Embryos in SC incubators were removed from
culture on day 3 for morphology assessment, whereas the TLI embryos had
undisturbed culture until the day of embryo transfer. TLI embryos were
annotated throughout culture as has been described previously4.
Embryo transfer and follow
up
Morphological assessment and grading of embryos was performed according
to UK guidelines 8. Embryo transfer was performed on
either day 3 or day 5 depending on patient and embryo criteria
consistent with NICE guidelines 9). Patients who had
two or more good quality embryos on day 3 (at least 6 cells, grade 3 or
4 for both evenness and fragmentation) were offered extended culture to
day 5. The number of embryos transferred was based on patient age,
embryo quality, the number of previous transfers (if any), and
considering the unit’s multiple births minimisation protocol10. Morphology grading alone was used to select
embryo(s) to transfer in SC cycles. In TLI cycles, morphological grading
was the primary selection tool and in cases where there was more than
one morphologically good quality blastocyst, avoidance criteria and the
KIDScore D5 score (V2.0 algorithm, Vitrolife, Denmark) were used to
exclude embryos with lower implantation potential. The avoidance
criteria included unevenness at the two-cell stage, multinucleation at
four-cell, direct first cleavage and irregular or reverse cleavage as
described in detail previously 4 . Any surplus good
quality blastocysts on day 5 or 6 were cryopreserved by vitrification
(Cryotop® and KT801 vitrification media, Kitazato, Japan). Embryo
utilisation rate was defined as the number of embryos transferred or
cryopreserved per 100 zygotes.
Pregnancy was established using a urinary pregnancy test 18 days
following oocyte retrieval which was followed by a transvaginal
ultrasound scan at 7 weeks’ gestation. The International Glossary on
Infertility and Fertility Care and the Core Outcome Measure for
Infertility Trials (COMMIT) were used to define the outcomes assessed11,12. A clinical pregnancy was confirmed when cardiac
activity was visualised on ultrasound. A multiple pregnancy was
diagnosed where there was more than one foetal pole with cardiac
activity on ultrasound. Pregnancies which did not reach the stage of a
clinical pregnancy were defined as biochemical pregnancy loss and loss
of a clinical pregnancy prior to 22 completed weeks was classified as a
miscarriage. The birth of at least one viable infant after 22 weeks’
gestation was counted as a live birth.
Frozen embryo transfer
The electronic database was checked manually to ascertain the outcomes
of frozen embryo transfer (FET) cycles of women who did not have a live
birth after fresh embryo transfer but had surplus blastocysts
cryopreserved and women who had embryos electively cryopreserved after
oocyte retrieval (freeze-all embryos). FET cycles were either artificial
or natural and embryos were warmed rapidly following standard protocols
(VT802 warming media, Kitazato, Japan). In artificial cycles the
endometrium was primed with oestrogen following a long agonist
down-regulation protocol with progesterone supplementation when the
endometrium measured >7mm on ultrasound scan. In natural
cycles, blastocyst transfer was performed six days after an early
morning urine LH surge had been detected. If a live birth had occurred
after frozen embryo transfer, this was documented in order to calculate
the cumulative live birth rate. All live births reported until July 2020
were included as a reasonable time-frame for assessing the short term
cumulative live birth rate given that the study period encompassed
November 2015 to December 2017.
Statistical analysis:
The number of oocyte retrievals was taken as the unit of denominator for
comparison between TLI and SC incubators. As only the first cycle per
woman was included in this study, the per-oocyte retrieval data is
identical to per-woman data. Continuous variables were described by mean
and standard deviation and compared through mean difference (MD) with
95% confidence intervals (CI) and p values obtained through unpaired
t-tests. Categorical variables were described using percentages and
comparison performed with Chi-Square test and reporting utilised odds
ratio (OR) with 95% CI and p values. For the primary outcomes of live
birth and cumulative live birth, adjusting for confounding variables
(age and number of oocytes retrieved) was performed using binomial
logistic regression and adjusted OR reported. Statistical analyses were
performed using IBM SPSS Statistics for Windows, Version 21.0.