Infertility Clinical Trial
Official title:
Examination of Gene Expression, Time-lapse and Near-infrared Spectroscopy (NIR) to Identify Differences in Embryo Viability
Infertility affects an increasing number of couples. For many, the choice of treatment is in
vitro fertilization (IVF) . Currently, there are no markers fully predictive of
developmental competence of IVF embryos. Present embryo selection is based on morphology
assessment, which produces implantation rates in the range of 20%-30 %. The overall purpose
of the present study is to investigate methods for selection of the best embryo. We aim to
examine the relationship between pregnancy outcome and the transcriptional profile of
selected genes, cleavage kinetics (time-lapse), and metabolic profile. We hypothesise that
the quality of the embryo is reflected by the transcription of selected genes, the cleavage
kinetics, and the metabolic profile. If so, these parameters can predict the success or
failure of a pregnancy. Furthermore, the interrelationship - if any - between these
parameters will be evaluated.
A secondary aim is to evaluate the effect of blastomere biopsy using time-lapse and
metabolic analysis
Introduction:
Infertility affects an increasing number of couples. For many, the choice of treatment is In
Vitro Fertilization (IVF). Currently, there are no markers fully predictive of developmental
competence of human IVF embryos. Present embryo selection is based on morphology assessment,
which produces implantation rates in the range of 20-30 %. To maximize the probability of
obtaining a pregnancy, multiple embryos are often transferred simultaneously at the risk of
multiple gestations. The high multiple pregnancy rates associated with IVF increases the
risk of neonatal complications and maternal pregnancy-related health problems. Single Embryo
Transfer (SET) is an effective way of minimizing the risk of multiple gestations, but an
accurate method of selecting the most viable embryo will optimise the use of SET. Today, the
quality of an embryo is assessed using microscopic evaluation of morphological parameters,
but the method has limited predictive value. Therefore, several areas are investigated in
search of additional markers of viability in order to reduce multiple gestation rates and
improve implantation rates, but all have yet to prove their value in a clinical setting.
Gene expression profiling is a novel strategy with potential to identify the most viable
embryo. Several publications suggest that differences in gene expression patterns are
related to embryo potential and quality. A bovine study identified specific genes related to
viability using microarray technology, proposing their use as markers of viability. A
similar study has been performed on human embryos, identifying 7000 transcripts associated
to successful IVF treatment.
Time-lapse analysis is another way of assessing embryo potential. Studying cleavage
kinetics, where milestones of early development, such as fertilization, cleavage, synchrony
and duration of cleavage, are correlated to differences in time intervals, might very well
be a useful marker of embryo viability. Recently, an EmbryoScope (Unisense Fertitech,
http://www.unisense.com/Default.aspx?ID=170) has been developed that allows series of photos
taking during incubation (Time-lapse). The pictures are taken every 30 minutes within a
controlled incubator housed with camera and microscope
Embryo metabolism is considered a critical determinant of viability, and it is likely that
viable embryos possess a unique metabolic profile, expressed by composition of metabolites
in the culture medium. Metabolomics studies the inventory of metabolites representing the
functional genotype. Near-Infrared (NIR) spectroscopy is an analytic technique based on
spectroscopy in the near infrared region suitable for rapid analysis of the metabolome.
Recent publications suggest that the metabolic profile can be used to identify high
potential embryos using NIR .
Aim and hypothesis. The purpose of the present study is to examine pregnancy
outcome/potential to implant and its relationship to 1) transcriptional profile of selected
genes,, 2) cleavage kinetics, and 3) metabolic profile. In particular we hypothesize that
the transcription of selected genes, the cleavage kinetics and the metabolic profile from
embryos with high developmental competence and implantation potential will differ from
embryos of lower quality and therefore that these parameters will be predictive of pregnancy
success. Furthermore, the interrelationship - if any - between these parameters will be
evaluated.
A secondary aim is to evaluate the effect of blastomere biopsy using time-lapse and
metabolic analysis
Perspective: Should the analysis of selected genes, Time-Lapse or metabolic profiles prove
predictive of viability, the methods could be used supplementary when assessing embryo
viability, refining the existing criteria. Apart from generating new knowledge concerning
the biology of the preimplantation embryo, the study aims to improve existing IVF
techniques.
1. Bioinformatical and comparative examination of homology and transcription in bovine and
human genes; identification of human candidate genes Purpose: To identify human
analogues to the 52 bovine candidate genes by comparing nucleotide sequences and gene
transcription between humans and cattle, using databases and bioinformational tools.
Based on the results and existing publications and knowledge concerning gene expression
and embryogenesis in humans and cattle 12 genes will be selected for further analysis.
After designing primers for RT-PCR, ultra sensitive, quantitative (q)-rt-PCR analyses
are developed. Before testing humane embryos, the analyses will be performed on human
cell-lines.
Materials and methods: In brief, cells are lysed and mRNA isolated, using an mRNA
extraction kit. cDNA is generated using reverse transcriptase PCR with sequence
specific primers. Quantitative analyses of embryo cDNA are performed with Real Time PCR
on Roche Light-cycler using fluorescence DNA labelling. Results are reported as
relative expressions to an endogenous control.
2. Quantification and variation of putative pregnancy-related genes in biopsies from human
blastocysts Purpose: Quantification of max. 12 selected genes on biopsies from humane
IVF blastocysts using RT-PCR and Real-Time PCR. The results will be used to select 5
genes for further analysis. The main purpose is to characterize and describe variations
in transcription of the selected genes in human preimplantation embryos.
Materials and methods Couples undergoing IVF treatment in order to permit preimplantation
genetic diagnosis (PGD) of the embryo will be requested permission to include embryos with
diagnosed gene or chromosomal disorders in the project, embryos which under normal
circumstances are discarded. The diagnosis healthy/diseased is made by PCR og FISH analysis
of a blastomere biopsied on day 3 (8-cell stage). The embryos are routinely cultured in the
EmbryoScope until day 5 after oocyte retrieval.
Embryo biopsy Embryos included in the project are cultured until day 5 after oocyte
retrieval. On day 3 the embryos are biopsied using a laser. The biopsies are marked and
frozen for later analysis.
Gene expression analysis The gene expression in cells from the biopsies are analysed using
RT-PCR and real-time PCR as described (project 1) with the purpose of quantifying 2-5 genes
from each individual cell, quantifying at maximum 12 genes. Each gene is analysed in
biopsies from 10 different embryos, to characterize inter-individual differences. From each
blastocyst are taken at least two biopsies, which are subjected to identical RT-PCR and
rt-PCR analyses to evaluate potential intra-individual differences and to validate the
method.
Time-lapse analysis: Embryos are cultured in the EmbryScope, images are recorded every 20
min.
3A, B and C: Implantation in relation to transcription of selected genes, Time-Lapse
parameters, and metabolic profile Purpose: To correlate result of the IVF treatment (+/-
clinical pregnancy) to A: the expression of 5 selected genes B: cleavage kinetics using
Time-Lapse analysis C: metabolic profile using Near-Infrared (NIR) Spectroscopy
Culturing of embryos In Denmark, IVF embryos are currently normally transferred 2-3 days
after oocyte retrieval.. Improvements of media techniques used for culturing have led to a
trend towards blastocyst transfer in many clinics. During the first week of preimplantation
development, gene transcription switch from maternal to embryonic control, and the energy
metabolism changes. Furthermore, the proportion of euploid embryos is higher when comparing
blastocysts with cleavage stage embryos. On the other hand, some embryos arrest their
development, leaving fewer embryos available for transfer. The latest Cochrane (2007)
analysis concludes that there is growing evidence in favour of blastocyst transfer for SET,
in particular when treating women with many retrieved oocytes. Genetic material from the
embryo can be obtained by performing biopsies on either the cleavage-stage embryo (8 cells),
where one or two blastomeres are removed, or from the blastocyst (about 100 cells), taking
2-10 cells from the trophectoderm (TE). Advantages of TE biopsy include obtaining more
genomic material (DNA/RNA), which improves the diagnostic reliability. The embryo is exposed
to less stressful conditions, since a relatively smaller proportion of cells is removed,
plus the biopsy is performed from extra-embryonic tissue, that is, cells forming the
placenta. Blastocyst biopsy does not appear to impair developmental competence and
implantation (13,17-19). In conclusion, performing TE biopsy on blastocysts seems to be less
invasive and stressful while yielding more information, thus making it our choice of method.
Biopsy On day 3 after oocyte retrieval, a small hole in zona pellucida is made with a laser,
allowing the TE to herniate through the hole during the next 2 days incubation. Early on day
5 embryos are assessed for blastocyst development according to the criteria listed below,
and one embryo is chosen for transfer. TE biopsy is performed on this embryo by aspirating
the herniating TE, approximately 2-20 cells, into an embryo biopsy pipette and cutting it
off with laser. The cells are immediately frozen for later analysis. The spent embryo medias
are collected individually and frozen at - 80 C for later NIR analysis. Models of spectral
regions are used to calculate an individual viability index for each embryo. Urine hCG test
and ultrasound at gestational ages 8 and 12 are used to determine pregnancy outcome.
Blastocyst score: Embryos are evaluated on day 5 using criteria described by Gardner et al,
where blastocyst are scored according to size. Briefly, the blastocysts were graded
according to size: [1] early blastocyst, the blastocoel is less than half the volume of the
embryo; [2] blastocyst, the blastocoel is greater than or equal to half of the volume of the
embryo; [3] full blastocyst, the blastocoel completely fills the embryo; [4] expanded
blastocyst, the blastocoel volume is larger than that of the early embryo and the zona is
thinning; [5] hatching blastocyst, the trophectoderm has started to herniate through the
zona; and [6] hatched blastocyst, the blastocyst has completely escaped from the zona. For
blastocysts graded as 3 to 6 (i.e., full blastocysts and onward) the development of the
inner cell mass (ICM) and trophectoderm can be assessed. The ICM grading is as follows: A.
tightly packed, many cells; B. loosely grouped, several cells; C. very few cells. The
trophectoderm grading is as follows: A. many cells forming a tightly knit epithelium; B. few
cells; C. very few cells forming a loose epithelium.
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