Fertility Clinical Trial
Official title:
A Randomized Double Blinded Controlled Study Comparing a Shorter Exposure of Oocyte to Spermatozoa Versus a Standard Incubation on the Live Birth Rate of In-vitro Fertilization Treatment
In vitro fertilization (IVF) treatment is now commonly used to treat infertile couples.
During IVF treatment, oocytes and sperm are routinely incubated overnight and this may lead
to suboptimal culture conditions because of increased reactive oxygen species (ROS) produced
by sperm in the standard incubation. High levels of ROS can adversely affect the quality of
the embryos, result in hardening of the zona pellucida and impair the implantation capacity
of embryos. Studies shows successful fertilization of an oocyte occurs 20mins after the
gametes are put together. Sperm can penetrate through the cumulus cells within 15min, and
80% of oocytes can be fertilized when they are exposed to a large number of spermatozoa
within 1hr. As an attempt to avoid possible detrimental effects on the oocytes from long
exposure to sperm, the brief incubation insemination protocol was developed. It implies that
prolonged incubation of oocytes and sperm may not be necessary and may even be harmful. Some
reports suggest that a sperm-oocyte exposure time of 1-6 h improves IVF outcomes. However,
other studies report no such advantage with a short insemination time .
A recent meta-analysis shows brief incubation of gametes was associated with significantly
higher rates of clinical pregnancy , ongoing pregnancy and higher rate of implantation than
standard incubation. But the rates of normal fertilization, good quality embryos and
polyspermy were not significantly different compared with standard incubation. In a Cochrane
meta-analysis, eight RCTs with 733 women were included, and showed similar results. But it
only reported clinical pregnancy rate and ongoing pregnancy rate which were significantly
higher in brief incubation group than standard incubation. However, the live birth rate,
which is the important outcome parameter, was not reported in all these studies. It is
uncertain whether brief incubation improves the life birth rate compared with standard
incubation.
The aim of this randomized double blinded study is to compare the live birth rate of IVF
treatment following brief incubation of oocytes and sperm versus standard incubation. The
hypothesis is that a brief incubation improves the live birth rate of IVF treatment.
1. Background
In vitro fertilization (IVF) technique is now commonly used for the treatment of
infertile couples. To obtain better outcomes of IVF, it is important to enhance embryo
quality by optimizing IVF techniques. In IVF procedures, oocytes and sperm are
routinely co-incubated overnight, which may expose oocytes and zygotes to suboptimal
culture conditions with increased reactive oxygen species (ROS) produced by sperm in
this long term culture[1]. High levels of reactive oxygen species (ROS) can adversely
affect the quality of the embryos, result in hardening of the zona pellucida and impair
the implantation capacity of embryos [2-4]. Studies shows successful fertilization of a
mamamal oocyte occurs 20mins after gametes meet [5]. Sperm can penetrate through the
cumulus cells within 15min, and 80% of oocytes can be fertilized when they are exposed
to a large number of spermatozoa within 1hrs[1]. As an attempt to avoid possible
detrimental effects on the oocytes from long exposure to sperm, the brief co-incubation
insemination protocol was developed. It implies that long-term co-incubation of oocytes
and sperm may not be necessary and may even be harmful. Some reports suggest that a
sperm-oocyte exposure time of 1-6 h improves IVF outcomes[1,2,6,7] However, other
studies report no such advantage with a short insemination time[8,9,10] .
Recent published Meta analysis shows[11] brief co-incubation of gametes was associated
with significantly higher rates of clinical pregnancy (RR: 1.84, 95% CI: 1.24-2.73),
ongoing pregnancy (RR: 1.73, 95%CI: 1.27-2.33) and higher rate of implantation (RR:
1.80, 95% CI: 1.43-2.26) than standard insemination. But the rates of normal
fertilization (RR: 0.98, 95% CI: 0.93-1.02), good quality embryos (RR: 1.24, 95% CI:
1.0-1.53) and polyspermy (RR: 0.84, 95% CI: 0.7-1.01) were not significantly different
compared with standard insemination. In another Cochrane meta-analysis[12] eight RCTs
with 733 women were included, and got the similar results. But it only reported
clinical pregnancy rate, ongoing pregnancy rate which was significantly higher in brief
co-incubation group than standard insemination. For the miscarriage rate, there were
six miscarriages in one trial including 167 women. This low quality evidence suggested
no significant difference in the odds of miscarriage between brief co-incubation and
standard insemination (OR 1.98, 95% CI 0.35 to 11.09; P = 0.44). And live birth was not
reported in the included studies, it is unclear whether brief co-incubation improves
the life birth rate compared with the standard overnight insemination protocol.
Drawbacks inherent to the quality of these studies, such as lack of allocation
concealment and no blinding, limit the quality of the available evidence. To better
evaluate these issues, more well designed RCTs are required to assess whether brief
co-incubation would contribute to a higher live birth rate and a lower miscarriage rate
compared to the standard overnight insemination protocol.
2. Objectives:
The objective of the present randomized, double blinded controlled study is to compare
clinical effectiveness (especially on live birth rate) of a shorter exposure of oocyte
to spermatozoa compared to a standard incubation in women undergoing IVF treatment.
3. Trial design:
A total of 280 infertile women undergoing IVF treatment will be randomized into one of the
following two groups by computer-generated random numbers that was placed in sealed
envelopes:
Shorter exposure of oocyte to spermatozoa group: oocytes will be exposed to spermatozoa for
2 hours Standard incubation group: oocytes will be exposed to spermatozoa for 20 hours
Treatment of subjects
All techniques of IVF including ovarian stimulation will be according to local protocols.
4.1 Ovarian stimulation and IVF
All women started their IVF treatment with ovarian stimulation using either the long agonist
or antagonist protocols. For long protocol, gonadotropin-releasing hormone analogue (GnRHa)
will be given for pituitary desensitization, on Day 2-3 of the menstrual cycle, they will
undergo transvaginal ultrasound examination and serum E2 measurement. Human menopausal
gonadotrophin (hMG) or recombinant FSH will started at 150-300 IU per day based on the
antral follicle count, age of women and previous ovarian response, according to the standard
operation procedures of the centre. Ovarian response was monitored by serial transvaginal
scanning with or without hormonal monitoring. Further dosage adjustments will base on the
ovarian response at the discretion of the clinicians in charge. For antagonist protocols,
antagonist (cetritide0.25mg daily) will be given on the 6th day of ovarian stimulation until
day of hcg priming.
When three leading follicles were ≥18 mm, hCG 10 000 IU or ovidrel 250 mg will be given to
trigger final maturation of oocytes. Oocyte retrievals will be arranged around 36 h later.
4.2 Insemination Procedures
All semen samples were prepared by the conventional swim-up procedure. At2hr after oocyte
retrieval, each matured oocyte will be inseminated with approximately 50,000-100,000 motile
spermatozoa.
4.3. Assignment and masking
On the day of OPU, patients will be randomized after occyte pick up by a laboratory
technician according to a computer generated randomization list in sealed envelopes into the
shorter incubation group or the control group. Until the completion of the whole study, both
the patients and the clinicians were blinded to the group assigned.
Control arm Women who allocated to the control arm, all the oocytes will be exposed to
spermatozoa for 20 hours, and checked for fertilization on day 1 (20 hours) after denuding
Intervention arm For those allocated to the intervention arm, all the oocytes will be
exposed to spermatozoa for 2 hours and gently wash through two organ dishes containing 1.5
ml of equilibrated medium, and then transferred to the corresponding microdroplet of
equilibrated fresh medium. Surrounding cumulus cell will be retained, not removed from the
ooctyes.
4.4 Fertilization and Embryo Evaluation Both groups of oocytes will be decoronated and
checked for the presence of two pronuclei and two polar bodies to confirm fertilization. All
other outcomes (i.e., no fertilization, one pronucleus, polyspermia, degeneration) were also
recorded.
Embryo morphology will be assessed every day of culture. Embryos will be scored according to
the following criteria. Briefly, embryos are classified as: gradeA, no cellular
fragmentation; grade B, <20% fragmentation; grade C, 20% to 50% fragmentation; and grade D,
>50% fragmentation. The number of blastomeres per embryo was also recorded. Grade A and B
embryos constituted the "good quality embryo".
The transfer is performed by the team clinician with a maximum of 2 embryos being replaced
according to the standard protocol under transabdominal ultrasound guidance. Luteal phase
support is given at the discretion of the physician.
4.5 Follow up strategy:
A pregnancy test will be carried out 2 weeks after embryo transfer in both arms. All women
who have a positive pregnancy test 2 weeks after a fresh embryo transfer will undergo a
transvaginal ultrasound scan after a further 5 weeks to identify the presence of a gestation
sac with a fetal heart signifying an ongoing pregnancy.
Data on all pregnancy outcomes including early pregnancy losses such as miscarriage or
ectopic pregnancy will be collected.
In order to achieve consistency with respect to the collection of outcome, standardised case
report forms (CRF) will be completed for each woman at each centre. These CRFs will include
details on treatment received, pregnancy outcomes, complications in pregnancy, mode of
delivery and birth outcomes.
Statistics
5.1 Analysis plan: Demographic factors and clinical characteristics collected as part of
baseline data collection will be summarised with counts (percentages) for categorical
variables, mean (standard deviation [SD]) for normally distributed continuous variables, or
median (interquartile [IQR] or entire range) for other continuous variables.
Outcomes for participants will be analysed in the groups to which they are assigned
regardless of deviation from the protocol or treatment received (intention-to-treat).
Comparative statistical analysis will entail calculating the relative risk (RR) (95% CI) for
the primary outcome (99% CIs for all dichotomous secondary outcomes), the mean difference
(99% CI) for normally distributed continuous secondary outcomes, or the median difference
(99% CI) for skewed continuous variables.
All analyses will adjust for the minimisation factors to account for the correlation between
treatment groups introduced by balancing the randomisation (which forces outcomes between
treatment arms to be similar apart from any treatment effect).
Adjusted risk ratios will be estimated using a log binomial regression model, or using a log
Poisson regression model with a robust variance estimator if the binomial model fails to
converge. Linear regression will be used for normally distributed outcomes and quantile
regression for skewed continuous variables.
Pre-specified subgroup analyses will include Cleavage versus blastocyst stage transfer.
6. Sample size estimation:
The average live birth rate in our center from 2010-2012 was 30% per transfer. According to
recent published Meta analysis[11],brief co-incubation of gametes was associated with
significantly higher rates of ongoing pregnancy (RR: 1.73, 95%CI: 1.27-2.33) than standard
insemination.
Assuming live birth in the treatment group =50% (increase by 70%); with 90% power and a
two-sided 5% level of statistical significance, we will need to recruit 124 women in each
arm. To account for 10% loss to follow up, we will recruit 140 subjects in each group or 280
subjects for the whole study.
7. Assessment of safety
Recults from recent published Meta analysis[11,12] showed the rates of normal fertilization,
good quality embryos and polyspermy were not significantly different with short time
incubation of gametes compared with standard insemination. Thus, there should be no major
safety concern in this study.
8. Direct access to source data / documents:
Trial-related monitoring, audits, IRB review and regulatory inspections are allowed.
9. Quality control and quality assurance
Patients will be managed by the listed investigators who have adequate experience in
managing assisted reproduction treatments. A Trial steering committee will be formed which
will oversee the conduct of the trial.
10. Ethics
Written consent will be obtained from all patients. The patient information sheet and
consent forms in English and Chinese are enclosed. Ethics approval will be obtained from the
Institutional Review Board, Shanghai first Maternity and Infant health hospital Hospital.
11. Data Handling and record keeping
All data will be stored for three years. A designated research nurse will be responsible for
data management including data coding, monitoring and verification.
12. Financing and insurance
The study will be funded by MerckSerono China Research Fund for Fertility Experts.
13. Publication policy
The findings of this study will be submitted for consideration for publication in
peer-reviewed scientific journal.
14. Supplements
The study will be conducted in compliance with the Declaration of Helsinki and Good Clinical
Practice (ICH-GCP)
;
Allocation: Randomized, Endpoint Classification: Safety/Efficacy Study, Intervention Model: Parallel Assignment, Masking: Double Blind (Subject, Caregiver, Investigator, Outcomes Assessor), Primary Purpose: Treatment
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