Embryo quality is the ability of an
embryo to perform successfully in terms of conferring a high
pregnancy rate Pregnancy rate is the success rate for getting pregnancy, pregnant. It is the percentage of all attempts that leads to pregnancy, with attempts generally referring to menstrual cycles where insemination or any artificial equivalent is used, which ma ...
and/or resulting in a healthy person. Embryo profiling is the estimation of embryo quality by qualification and/or quantification of various parameters. Estimations of embryo quality guides the choice in
embryo selection in ''in vitro'' fertilization.
In general, embryo profiling for prediction of pregnancy rates focuses mainly on visual profiles and short-term
biomarkers
In biomedical contexts, a biomarker, or biological marker, is a measurable indicator of some biological state or condition. Biomarkers are often measured and evaluated using blood, urine, or soft tissues to examine normal biological processes, pa ...
including expression of
RNA and
protein
Proteins are large biomolecules and macromolecules that comprise one or more long chains of amino acid residues. Proteins perform a vast array of functions within organisms, including catalysing metabolic reactions, DNA replication, res ...
s, preferably in the surroundings of embryos to avoid any damage to them. On the other hand, embryo profiling for health prediction puts more focus on the
genome
In the fields of molecular biology and genetics, a genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding g ...
, and where there is a risk of a
genetic disorder
A genetic disorder is a health problem caused by one or more abnormalities in the genome. It can be caused by a mutation in a single gene (monogenic) or multiple genes (polygenic) or by a chromosomal abnormality. Although polygenic disorders ...
it more often involves cell sampling from the embryo for
preimplantation genetic diagnosis
Preimplantation genetic diagnosis (PGD or PIGD) is the genetic profiling of embryos prior to implantation (as a form of embryo profiling), and sometimes even of oocytes prior to fertilization. PGD is considered in a similar fashion to prenatal ...
.
Prediction of pregnancy rates
Microscopy
Embryo quality is mainly evaluated by microscopy at certain time points using a morphological scoring system. This has shown to significantly improve
pregnancy rates Pregnancy rate is the success rate for getting pregnancy, pregnant. It is the percentage of all attempts that leads to pregnancy, with attempts generally referring to menstrual cycles where insemination or any artificial equivalent is used, which ma ...
.
Assessment of morphological feathers as a reliable non-invasive method that provides valuable information in prediction of IVF/intra cytoplasmic sperm injection (ICSI) outcome has been frequently used as an soring system of the embryo quality. The parameters for evaluation at day 2-3:
Number of cells and division rhythm: The optimal number of cells is 4 at day 2 and 8 at day 3 (A quality). In day 3 9-10 cells is B, >=10 is C (suboptimal) and <=4 is D (barely implant). A normal division rate is to double cell number each 24 hours. A higher rate implies chromosomal abnormalities and a lower rate entails possible embryo arrest (it is dying).
Fragmentation: happens due to cell apoptosis and can be quantified by the % of the embryo total volume eccupied by fragments. Fragments are cytoplasm fractions without nuclei.
Cells symmetry and size: it is normal that all blastomeres had same or similar size in embryos with 2, 4 or 8 cells, while for the rest of embryos, a certain variety in cells size is normal. When the number of cells is impaired, if all of them have the same size, it is considered asymmetrical. Those embryos with one big size blasomere is considered abnormal and is associated with high rate of polyploidy.
Multinucleation: multinucleated blastomeres on day 2 and day 3 is associated to a lower implantation rate. These embryos often are mosaics or with aneuploidy. It is more related to abnormalities on day 2 than on day 3.
Cytoplasm aspect: the presence of vesicles on day 3 is considered a sign of embryo genome activation and, therefore, of good prognosis. The presence of vacuoles is a sign of bad prognosis.
Time-lapse microscopy is an expansion of microscopy wherein the morphology of embryos is studied over time. As of 2014, time-lapse microscopy for embryo quality assessment is emerging from the experimental stage to something with enough evidence for broader clinical use.
Studies using the EmbryoScope (tm) time-lapse incubator have used several indicators for embryo quality, such as direct cleavage from 1 to 3 cells,
as well as the initiation of compaction and start of blastulation.
Also,
two-pronuclear zygotes (2PN) transitioning through 1PN or 3PN states tend to develop into poorer-quality embryos than those that constantly remain 2PN.
Molecular analysis
Molecular analysis can be performed by taking one of the cells from an embryo. The analysis can vary in extent from a single target
biomarker
In biomedical contexts, a biomarker, or biological marker, is a measurable indicator of some biological state or condition. Biomarkers are often measured and evaluated using blood, urine, or soft tissues to examine normal biological processes, p ...
to entire
genome
In the fields of molecular biology and genetics, a genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding g ...
s,
transcriptome
The transcriptome is the set of all RNA transcripts, including coding and non-coding, in an individual or a population of cells. The term can also sometimes be used to refer to all RNAs, or just mRNA, depending on the particular experiment. The t ...
s,
proteome
The proteome is the entire set of proteins that is, or can be, expressed by a genome, cell, tissue, or organism at a certain time. It is the set of expressed proteins in a given type of cell or organism, at a given time, under defined conditions. ...
s and
metabolome
The metabolome refers to the complete set of Small molecule, small-molecule chemicals found within a biological sample. The biological sample can be a Cell (biology), cell, a cellular organelle, an Organ (anatomy), organ, a Tissue (biology), tiss ...
s. The results may be used to score embryos by comparing the patterns with ones that have previously been found among embryos in successful versus unsuccessful pregnancies:
Transcriptome profiling
In transcriptome evaluation,
gene expression profiling
In the field of molecular biology, gene expression profiling is the measurement of the activity (the expression) of thousands of genes at once, to create a global picture of cellular function. These profiles can, for example, distinguish between c ...
studies of human embryos are limited due to legal and ethical issues.
[
]Gene expression profiling of cumulus cells
The cumulus oophorus, (discus proligerus), is a cluster of cells that surround the oocyte both in the ovarian follicle and after ovulation. In the antral follicle, it may be regarded as an extension of the membrana granulosa. The innermost layer ...
surrounding the oocyte and early embryo, or on granulosa cell
A granulosa cell or follicular cell is a somatic cell of the sex cord that is closely associated with the developing female gamete (called an oocyte or egg) in the ovary of mammals.
Structure and function
In the primordial ovarian follicle, and la ...
s, provides an alternative that does not involve sampling from the embryo itself.[ Profiling of cumulus cells can give valuable information regarding the efficiency of an ovarian hyperstimulation protocol, and may indirectly predict oocyte aneuploidy, embryo development and pregnancy outcomes, without having to perform any invasive procedure directly in the embryo.][
In addition, ]microRNA
MicroRNA (miRNA) are small, single-stranded, non-coding RNA molecules containing 21 to 23 nucleotides. Found in plants, animals and some viruses, miRNAs are involved in RNA silencing and post-transcriptional regulation of gene expression. mi ...
(miRNA) and cell-free DNA (cfDNA) can be sampled from the vicinity of embryos, functioning as transcriptome-level-markers of embryo quality.
Proteome profiling
Proteome profiling of embryos can indirectly be evaluated by sampling of proteins found in the vicinity of embryos, thereby providing a non-invasive method of embryo profiling.[ Examples of protein markers evaluated in such profiling include ]CXCL13
Chemokine (C-X-C motif) ligand 13 (CXCL13), also known as B lymphocyte chemoattractant (BLC) or B cell-attracting chemokine 1 (BCA-1), is a protein ligand that in humans is encoded by the ''CXCL13'' gene.
Function
CXCL13 is a small chemokine ...
and granulocyte-macrophage colony-stimulating factor
Granulocyte-macrophage colony-stimulating factor (GM-CSF), also known as colony-stimulating factor 2 (CSF2), is a monomeric glycoprotein secreted by macrophages, T cells, mast cells, natural killer cells, endothelial cells and fibroblasts that ...
, where lower protein amounts are associated with higher implantation rates.[ The presence of soluble ]HLA-G
HLA-G histocompatibility antigen, class I, G, also known as human leukocyte antigen G (HLA-G), is a protein that in humans is encoded by the ''HLA-G'' gene.
HLA-G belongs to the HLA nonclassical class I heavy chain paralogues. Classical HLA I pro ...
might be considered as another parameter if a choice has to be made between embryos of equal visible quality.[
Another level of opportunity can be achieved by having the evaluation of the embryo profile tailored to the maternal status in regard to, for example health or immune status, potentially further detailed by similar profiling of the maternal genome, transcriptome, proteome and metabolome. Two examples of proteins that may be included in maternal profiling are endometrium-derived ]stathmin
Stathmin, also known as metablastin and oncoprotein 18 is a protein that in humans is encoded by the ''STMN1'' gene.
Stathmin is a highly conserved 17 kDa protein that is crucial for the regulation of the cell cytoskeleton. Changes in the cytoske ...
and annexin A2, whose down- and up-regulation, respectively, are associated with higher rates of successful implantation.[
]
Genome profiling
A systematic review and meta-analysis of existing randomized controlled trial
A randomized controlled trial (or randomized control trial; RCT) is a form of scientific experiment used to control factors not under direct experimental control. Examples of RCTs are clinical trials that compare the effects of drugs, surgical te ...
s came to the result that there is no evidence of a beneficial effect of PGP as measured by live birth rate Pregnancy rate is the success rate for getting pregnant. It is the percentage of all attempts that leads to pregnancy, with attempts generally referring to menstrual cycles where insemination or any artificial equivalent is used, which may be simpl ...
.[ On the contrary, for women of advanced maternal age, PGP significantly lowers the live birth rate.][ Technical drawbacks, such as the invasiveness of the biopsy, and chromosomal mosaicism are the major underlying factors for inefficacy of PGP.][
A major drawback of genomic profiling for embryo quality is that the results generally rely on the assessment of a single cell, PGP has inherent limitations as the tested cell may not be representative of the embryo because of ]mosaicism
Mosaicism or genetic mosaicism is a condition in multicellular organisms in which a single organism possesses more than one genetic line as the result of genetic mutation. This means that various genetic lines resulted from a single fertilized ...
.
When used for women of advanced maternal age and for patients with repetitive IVF failure, PGP is mainly carried out as a screening for detection of chromosomal abnormalities such as aneuploidy
Aneuploidy is the presence of an abnormal number of chromosomes in a cell, for example a human cell having 45 or 47 chromosomes instead of the usual 46. It does not include a difference of one or more complete sets of chromosomes. A cell with any ...
, reciprocal and Robertsonian translocations, and few cases for other abnormalities such as chromosomal inversions or deletions. The principle behind it is that, since it is known that numerical chromosomal abnormalities explain most of the cases of pregnancy loss, and a large proportion of the human embryos are aneuploid, the selective replacement of euploid embryos should increase the chances of a successful IVF treatment. Comprehensive chromosome analysis methods include array-comparative genomic hybridization (aCGH), quantitative PCR
A real-time polymerase chain reaction (real-time PCR, or qPCR) is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR (i.e., in real ...
and SNP array
In molecular biology, SNP array is a type of DNA microarray which is used to detect polymorphisms within a population. A single nucleotide polymorphism (SNP), a variation at a single site in DNA, is the most frequent type of variation in the ge ...
s.[ Combined with single blastomere biopsy on day-3 embryos, aCGH is very robust with 2.9% of tested embryos with no results, and associated with low error rates (1.9%).][ There is no evidence that testing the embryo for abnormal number of chromosomes increases the number of live births.]
In addition to screening for specific abnormalities, techniques are in development that can avail for up to full genome sequencing
Whole genome sequencing (WGS), also known as full genome sequencing, complete genome sequencing, or entire genome sequencing, is the process of determining the entirety, or nearly the entirety, of the DNA sequence of an organism's genome at a ...
, from which genetic profiling can score the DNA patterns by comparing with ones that have previously been found among embryos in successful or unsuccessful pregnancies.
Health prediction
The main method currently used to predict the health of a resultant person of an embryo is preimplantation genetic diagnosis
Preimplantation genetic diagnosis (PGD or PIGD) is the genetic profiling of embryos prior to implantation (as a form of embryo profiling), and sometimes even of oocytes prior to fertilization. PGD is considered in a similar fashion to prenatal ...
(also called ''preimplantation genetic screening'', ''preimplantation genetic profiling'' or PGP), in order to determine whether the resultant person will inherit a specific disease or not. On the other hand, a systematic review and meta-analysis of existing randomized controlled trial
A randomized controlled trial (or randomized control trial; RCT) is a form of scientific experiment used to control factors not under direct experimental control. Examples of RCTs are clinical trials that compare the effects of drugs, surgical te ...
s came to the result that there is no evidence of a beneficial effect of PGP as measured by live birth rate Pregnancy rate is the success rate for getting pregnant. It is the percentage of all attempts that leads to pregnancy, with attempts generally referring to menstrual cycles where insemination or any artificial equivalent is used, which may be simpl ...
. On the contrary, for women of advanced maternal age, PGP significantly lowers the live birth rate.[ Technical drawbacks, such as the invasiveness of the biopsy, and chromosomal mosaicism are the major underlying factors for inefficacy of PGP.][
]
References
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Assisted reproductive technology