Clinical Trial Details
— Status: Completed
Administrative data
| NCT number |
NCT03354858 |
| Other study ID # |
Follicular flushing |
| Secondary ID |
|
| Status |
Completed |
| Phase |
|
| First received |
|
| Last updated |
|
| Start date |
December 1, 2017 |
| Est. completion date |
June 1, 2018 |
Study information
| Verified date |
July 2018 |
| Source |
Eugonia |
| Contact |
n/a |
| Is FDA regulated |
No |
| Health authority |
|
| Study type |
Observational
|
Clinical Trial Summary
The purpose of this study is to investigate the impact of follicular flushing on the oocyte
recovery rate, oocyte maturity, fertilization rate, and embryo development and utilization as
compared to direct aspiration (no flushing) in women undergoing IVF treatment
Description:
This will be a prospective observational paired study on the effect of follicular flushing
oocyte recovery rate, oocyte maturity, fertilization rate, and embryo development and
utilization as compared to direct aspiration (no flushing) in women undergoing IVF treatment.
In each woman enrolled, one ovary (right or left) will be randomly assigned to be aspirated
with either follicular flushing or direct aspiration without flushing. The primary endpoint
will be oocyte recovery rate (oocytes retrieved per follicle aspirated). Secondary outcomes
will be oocyte maturity, fertilization rate, and embryo development and utilization.
Pregnancy rates will be reported as a surrogate outcome.
TRANSVAGINAL OOCYTE COLLECTION
Following controlled ovarian stimulation (COS), final oocyte maturation is triggered using
hCG or triptorelin. Oocyte collection is performed 35-36 hours post triggering. The procedure
involves transvaginal ultradound-guided aspiration of follicles via a double lumen aspiration
needle (16G, Casmed),. An interdisciplinary team is required for the procedure, including
Gynaecologists, Embryologists Midwives/Nurses and Anaesthetists. It is performed in a
gynaecological operating theatre, adjacent to an embryology laboratory, with the patient
being sedated and under aseptic conditions and strict regulation of environmental conditions
to ensure oocyte viability and patient safety.
Before the procedure The day before the procedure, the embryologist prepares culture dishes
(Falcon Central Well dishes) containing bicarbonate buffered media specifically designed for
oocyte culture (Fert, Origio) overlaid with mineral oil (Fertipro). The dishes are placed in
a 5-7% CO2 incubator at 37oC to equilibrate overnight. Aliquots of HEPES buffered media
(Flushing medium, Origio) which achieves pH regulation outside of the incubator is pre-heated
to 37oC. Finally, 160 ml flasks of DPBS (phosphate buffered saline) for use in follicular
flushing are placed in the theatre's incubator for pre-warming. All the embryological
preparation is performed aseptically under a laminar flow hood.
The procedure utilises surgical equipment which is autoclaved and packagedd in sterile
surgical cloths. All materials that come into contact with follicular fluid, such as oocyte
needles, 20ml syringes (non toxic, BD) and collection tubes (14ml IVF tubes, NUNC), are IVF
tested via MEA testing (mouse blastocyst survival), pre-sterilised and single use. These are
stored prior to use in a 37oC warming chamber.
We use a 16G double lumen needle (Casmed, UK) which allows continuous aspiration and flushing
of follicles. The needle has a length of 30 cm and echogenic markings near the tip for easy
recognition under transvaginal ultrasound guidance. The length of the tubing is 60cm and
includes a bung for connection to a standard test tube and separate tubing with a Luer-lock
for connection to a syringe for flushing. The needle and its connections are all manufactured
from high grade surgical stainless steel. The tubing is manufactured from translucent PTFE
(Polytetrafluoroethylene).
All products are manufactured in accordance with quality standards ISO 9001:2008, ISO 13485
and CE marking.
Preparing for the procedure Patient Preparation Before entering the operating theatre, the
anaesthesiologist confirms the patient's medical history pertinent to her imminent
anaesthesia.
The patient is escorted to the operating theatre and her identity is confirmed by two
persons, who sign the witnessing document. In patients with very few expected oocytes (poor
responders, mild IVF or natural cycles) a transvaginal ultrasound is performed just prior to
the procedure by the treating physician in order to confirm the presence and location of
mature follicles. In cases of high risk for ovarian hyperstimulation syndrome, a blood sample
is also taken, according to our standard clinical practice as previously described (Lainas et
al., 2012, 2013, 2014). The procedure followed for the preparation of the patient is the same
as that of most gynaecological surgeries. The patient is laid on the operating table in the
lithotomy position and her external genitalia are washed with hibitane solution (which is
considered milder than using betadine scrub) and then rinsed with saline. After performing a
standard hand and arm scrub, the surgeon's assistant wears surgical gloves and opens the
sterilised tool packages, including pre-warmed and sterilised tube blocks, which is placed on
sterilised surgical paper towels within a heating stage. Sterilised collection tubes are
placed in the heating block, for use during the procedure. If during the procedure more tubes
are required the assisting nurse should aseptically place more tubes in the heating block.
The patient is then covered with sterilised surgical towels on the body and legs.The probe of
the ultrasound is disinfected with CIDEX for 5 minutes and then washed with copious amounts
of water for injection. Following insertion of a vaginal dilator, the vagina is washed with
DPBS up to the cervical os. The assistant then wears new surgical gloves and gown and covers
the patient with additional sterilised surgical paper drapes The pre-sterilised oocyte needle
guide is opened and it and the flushing syringes are washed with DPBS. One syringe is
carefully filled with DPBS, which will be used to flush the needle and then discarded. The
ultrasound probe is then aseptically placed on the patient's drapes, the needle guide is
attached to it and the probe is finally connected to the ultrasound central unit. A
pre-warmed 16 G double lumen oocyte collection needle is opened and the aspiration pump is
set to 250 mmHG. The needle's various tubing connectors are connected to: a) the aspiration
pump, b) the flushing syringe, c) the first oocyte collection tube.
The surgeon performs a standard hand and arm scrub, wears a surgical gown and surgical gloves
before assuming the surgical position at the surgical table. The needle and its tubing are
washed by flushing with warm DPBS before use and the first collection tube is discarded. This
ensures that the entirety of the needle/tubing is filled with DPBS before puncturing the
follicle. The anaesthesiologist sedates the patient and monitors her breathing and heart
function throughout the procedure.
Embryological Laboratory Preparation The entire embryological procedure is performed under a
laminar flow hood, with a heated stage set at 40oC in order to endure a dish temperature of
37+/-0.2 oC. The embryologist prepares central well culture dishes with HEPES media. Empty
pre-heated round dishes, heat-polished glass Pasteur pipettes and an empty container for
discarded follicular fluid are all placed on the heated workbench. All of the above should be
clearly labelled with the patients details.
During the procedure
The physician places the oocyte collection needle into the guide and then attaches it to the
transvaginal probe of the ultrasound. He then performs an ultrasound estimation of the
ovaries and Douglas pouch. After he has located the follicles, he punctures the vaginal wall
with the needle at a point adjacent to the first ovary and proceeds to aspirate all antral
follicles. larger than (...mm).
As each follicle is aspirated, the fluid is collected in the tube and the surgeon's assistant
replaces it with a new tube from the heated stage. A nurse takes the filled tube and
estimates its volume based on a volumetrically marked "reference" tube. The volumetric
estimation is based on a series of tubes of known volume. The tube is then quickly placed in
the heating block within the embryological laboratory (via the connecting window). Tubes are
placed from left to right on the heating block, with the leftmost spot being reserved for
fluids from newly aspirated follicles and the rightmost positions for tubes containing
flushed follicular fluid.
The embryologist takes the tube and carefully pours the contents of each tube into the dish.
He then searches for the Cumulus-Oocyte Complex (COC) through his microscope. The results of
this search are communicated to the physician and fall into three categories: a) COC found,
b) COC not found but follicle cells present, c) COC not found and no follicle cells present.
When the COC is found, the physician moves on to the next follicle, while the embryologist
transfers it to the moat of the collection dish and thoroughly washes it, before moving it to
the central well. If the COC is not found, the physician then proceeds to flush DPBS into the
follicle via the syringe. The volume flushed should not exceed 50% of the initial volume of
the follicle to avoid rupturing it, which may cause haemorrhaging of the follicle. The
flushed fluid is then aspirated and the procedure is repeated as described above. This
process is repeated until the COC is found or 4-5 flushes are performed. When the flushing
syringe runs out of DPBS it is replaced with a new one which has been carefully and
aseptically filled just prior to use (no more than 2 minutes prior to use, in order for the
temperature to not drop significantly).
As the procedure continues, COCs are placed by the embryologist in the collection dish until
a maximum of 10 are collected. A new dish should be used for additional COCs. Following the
end of the oocyte collection, the oocytes are transferred to dishes containing Fert medium
and placed in the incubator. After the embryologist has ensured that all tubes have been
searched he informs the physician of the final number of oocytes retrieved. Before ending the
procedure the physician scans the ovaries and Douglas pouch one last time and if excess fluid
is detected, he aspirates as much as possible. Finally the physician removes the needle and
probe and ensures that there is no vaginal bleeding.
Factors contributing to successful oocyte retrieval
A transvaginal oocyte retrieval is a complicated procedure with many variables that need to
be accounted for in order to make it successful. Side effects from the operation can be
affected by the length of the procedure, the skill of the physician and the type of needle
used. The length of the procedure is primarily affected by the number of oocytes collected
and the ease that these were collected. Procedures that include flushing of follicles are on
average longer than those without flushing. The width of the needle (gauge) directly affects
the level of vaginal bleeding and the level of pain the patient experiences due to the
operation. The above variables (flushing and width of needle) have been proposed as major
factors for the effectiveness of the oocyte collection, i.e. the number of oocytes retrieved.
Follicular flushing Flushing has been proposed as a method that may increase the oocyte yield
due to the fact that oocytes that are not immediately retrieved from the follicle can be
collected after flushing. Double lumen needles allow for flushing via the use of two channels
within the needle, one for aspirating the follicles and one for flushing. However, there is
controversy regarding its routine use mainly due to the fact that the procedures tend to last
longer, but also due to the risk of haemorrhage and infection from flushing. Many studies
have been performed over the years comparing flushing (with double lumen needles) and no
flushing (with single lumen needles), with conflicting results.
A Cochrane review published in 2010, assessed the use of flushing, found no significant
benefit in the use of double-lumen needles. In a recent randomised study performed in EUGONIA
clinic, flushing was found to be associated with significantly higher retrieval rates
compared to no flushing using a 16G Casmed double lumen needle. In addition, around 97% of
oocytes collected were retrieved in the first 3 flushes and oocytes were found to have
similar maturation and fertilisation rates regardless of the number of flushes required,
suggesting that a small number of flushes may increase the final number of developmentally
competent oocytes retrieved.
Regarding the safety of flushing, adherence to the protocol presented above, especially with
regard to aseptic techniques, will minimise the risk of infection from the flushes. It should
be stressed that care should be taken to avoid rupturing follicles via too vigorous flushing.
The amount of DPBS flushed into the follicle should not exceed 50% of the volume of the
follicle and follicles with excess vascularisation should not be flushed at all. The
procedure should be performed by an experienced team to ensure safety of the patient while
also minimising the length of time of the surgery.
Aspiration Needles A significant parameter when comparing both needles and retrieval
techniques is the working diameter of the needle used to aspirate. This is especially true
when performing oocyte retrievals with the use of follicular flushing. Double lumen needles
have as a rule smaller working diameters compared to similar gauge single lumen needles. This
fact should be considered when setting the negative pressure on the aspiration pump, as
double-lumen needles will require significantly higher pressure in order to achieve a similar
flow rate as single-lumen needles. Even similar double-lumen needles from different
manufacturers can have dissimilar inner lumen diameters, significantly altering their
performance, since even small changes in the diameter have a large effect on the flow-rate.
Below is a comparison of the diameters of some commonly used aspiration needles. The inner
diameter in the double lumen category refers to the working diameter of the inner lumen. Note
the significant differences in diameters between similar gauge needles.
SINGLE LUMEN DOUBLE LUMEN 16G 16G 17G 17G 16G 16G 17G 17G OUTER INNER OUTER INNER OUTER INNER
OUTER INNER WALLACE 1,7 1,24 1,42 1 1,7 1,17 1,5 0,9 GYNETICS 1,6 1,3 1,4 1 1,65 1 1,5 0,8
VITROLIFE 1,6 1,1 1,4 1 1,6 1 1,5 0,9 CASMED n/a n/a n/a n/a 1,65 0,81 n/a n/a
