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Res Rep Gynaecol Obstet 2017 | Volume 1 Issue 4
November 02-03, 2017 | Chicago, USA
Embryology and In vitro Fertilization
World Congress on
Background:
Viability assessment prior embryo transfer is a
crucial question since the success rate of IVF experiments
is below expectations (approx. 30%). The practice of
multiple embryo transfer to overcome this limitation of IVF
is sometimes accompanied by multiple gestation adding
several new risk factors. The leading consensus is that the
best possible option is the practice of single embryo transfer.
The adoption of this policy however requires the best tools
to predict the embryo’s implantation potential during the
first couple of days of development. In parallel with the use
of the routinely used morphology based viability assessment
assay known as the Istanbul Consensus Scoring System
(ICCS), huge effort is made worldwide to find new markers
of embryo viability, preferably in a non-invasive way due to
ethical issues. The search for markers of embryo viability in
the embryo culture medium seems to be an ideal approach.
The aim of our work was to find any biomarker present in
the embryo culture medium using mass spectrometry, which
would qualitatively or quantitatively differ in the samples
of viable and non-viable embryos, and help predicting
implantation potential.
Methods:
Spent embryo culture medium samples (n=201)
weremeasured inaseriesof retrospective, blindexperiments,
all were suitable for transferation according to the ICCS. No
sample preparation was made, 15 µl of sample was directly
injected into the instrument after the addition of internal
standard solution. A Dionex Ultimate 3000 (Dionex Corp.,
USA) analytical HPLC equipped with an autosampler and a
column thermostat set at 30ºC was used. Separation was
carried out on a Kinetex C18 2.6 µm, 2.1 x 100 mm analytical
column (Phenomenex, USA) with a multi-step gradient
elution at a flow rate of 200 µL/min. The mass spectrometer
coupled was a Bruker micrOTOF accurate mass instrument
(Bruker Daltonik, Germany) equipped with an electrospray
ionization source (ESI) operated in the positive ion mode.
Results:
A protein marker was found which significantly
(p<0.001) differed in quantity between the samples of
embryos which did (clinical pregnancy), or did not (no
pregnancy) implant. Respective lots of unconditioned culture
media were used as controls. Deconvolution of the obtained
mass spectra revealed that this protein has a molecular mass
of 9186.4 Da. The protein was identified using tandem mass
spectrometry as the alpha-1 chain (HptA1) of the human
haptoglobin (Hpt) molecule. It was observed that Hpt was
present as a contaminant in the purified human serum
albumin used to supplement the culture medium therefore
it is not secreted by the embryo. A significant correlation
(p<0.001) was found when comparing the clinical outcome
(clinical pregnancy or no pregnancy) and the amount of
HptA1. The positive predictive value (PPV) of the biochemical
analysis was 51.2% while the negative predictive value (NPV)
was 100%. On the current material the ICCS assay performed
a PPV of 31.3%.
Discussion:
The increased amount of HptA1 in the media
of non-viable embryos compared viable embryos is
caused by the increased reduction of the intramolecular
disulphide bonds within Hpt. This reaction (leading to HptA1
liberation) is catalyzed in a higher extent by embryos with
failed pregnancy outcome. The biochemical evaluation can
select embryos having good morphological aspects but
low implantation potential due to visually (microscopically)
unnoticeable reasons. The increased PPV observed (51.2%vs.
31.3%) is due to the fact that the mass spectrometric analysis
theoretically decreased the number of false positive cases
of ICCS by 40% (n=78). Since the assay has an NPV of 100%
these 40% were all true negative cases. The results suggest
a possible contra selection tool, screening the embryos with
good morphological aspects, but no implantation potential.
e:
montsko.gergely@pte.huNon-invasivemass spectrometric viability assessment of
in vitro
fertilized embryos using the alpha-1 chain
of human haptoglobin
Gergely Montskó, Krisztina Gödöny, Ákos Várnagy, József Bódis
and
Gábor L Kovács
University of Pécs, Hungary