infertility has been recognized as a public health issue worldwide ( 1 ) leading to an increasing need to the use of assisted reproductive technologies ( art ) , including in vitro fertilization ( ivf ) .
after the first reported case of ivf in 1978(2 ) art enabled millions of people to have their own children in cases when pregnancy did not occur under natural circumstances .
700,000 cycles a year in the usa and europe together ( 3,4 ) . despite of evolving intracytoplasmatic sperm injection ( icsi ) technique
a success rate of 25% and 28% has been reported in 2005(7 ) and 2008(8 ) , respectively .
nowadays , this rate went up to 32% ( 9 ) , which can not be considered as a significant development .
earlier clinical protocols preferred multiple embryo transfer , but multiple gestations can result in the increased risk of preterm delivery ( 10 - 16 ) .
other studies report that multiple gestations also increased the risk of low birth weight cerebral palsy ( 17 ) . in the us alone , preterm births resulting from multiple pregnancies during ivf cause a 1 billion usd extra cost to the social insurance ( 18 ) . in order to exclude the discussed risk factors ,
it is imperative , however , that accurate and economical methods should be developed to ensure that the most viable euploid embryo is selected for transfer .
ideally , such tests would be noninvasive , lessening the risks to the embryo and reducing costs and workload in the embryology laboratory ( 19 ) .
the biggest issue with pre - implantation viability assessment is that due to ethical reasons any assay should be completely non - invasive because no one can predict what kind of interference would be the unwanted result in the later embryonic development .
the most apparent  and routinely applied - way of the assessment of viability is the morphological evaluation of in vitro fertilized embryos using microscopy .
there are several morphological features described which could be used for viability assessment purposes , these are dependent on the time spent after fertilization . right after fertilization in the 1-cell embryo the size and symmetry of the two pronuclei can be examined .
the time of the first cell division is also a good predictor of later implantation potential , as zygotes that divide early tend to develop more frequently to the blastocyst stage .
criteria as cleavage rate and blastomere shape and symmetry , an adequate trophectoderm layer ( te ) and an inner cell mass ( icm ) is a morphological marker of the later stages ( 5,20 ) .
not only can the morphology of the fertilized embryo be used for further prediction of implantation potential , but morphological defects of the retrieved oocyte as well . fertilization and pregnancy rate correlates with the grade of cumulus - oocyte complexes , and embryos originating from dysmorphic oocytes show a larger grade of pregnancy loss ( 21 - 23 ) .
the cleavage stages of morulae and blastocysts or the symmetry and patterns of cell division are also notable and frequently used aspects , and are often examined during the prediction of embryo viability ( 23 ) .
the biggest issue of morphological assessment is that it is still a highly subjective method ( 20 ) .
the reason is partly due to the fact that the final decision is made by a clinician , and not by an objective test result , and secondly it is does matter how important are the individual morphological features in the final conclusion ( 24 - 26 ) . to overcome the different practice of laboratories worldwide in 2011 an international consensus ( istanbul consensus ) has been reached on embryo viability assessment ( 27 )
. the selected morphological markers of respective stage embryos , the weighing of individual features and a scoring system has been set up .
the limitations due to static time - point observation , is now solved with the use of time - lapse microscopy ( 28,29 ) .
time - lapse microscopy also enables the observation of dynamics of cytoplasmic movements and cytokinesis , reflecting the functionality of microtubule and actin cytoskeleton , which is critical for proper development . in our laboratory
, we aimed to improve the success rate of implantation by adopting and further optimizing the istanbul consensus .
this score has been called as the optimized criteria system ( ocs ) . according to this scoring
, 3-day old embryos were divided into two subgroups : the subgroup with low blastomere number ( less than 7 ) and with high blastomere number ( 7 or more ) .
symmetric position of blastomeres indicates the rate of symmetry of holoblastic cleavage along the embryo axis .
it was classified as good ( full symmetry ) ; fair ( light asymmetry ) ; or poor ( evident asymmetry ) .
the percent values of fragmentation are based on the ratio of fragmented to total cell numbers . as a further modification to the istanbul consensus ,
embryos were considered as good if the fragmentation rate was < 15% ( instead of the original 10% ) .
this shift from 10 to 15% was the result of our observation that a fairly high proportion of the embryos between 10 - 15% appeared to be viable . in summary the optimized criteria system ( ocs )
highlights 3 modified or new parameters : fragmentation ( with a more permissive criterion of < 15% in the  good  category ) ; symmetry and the blastomere number . in addition , the blastomere size was evaluated according to the original istanbul consensus .
a scoring - map was created to facilitate the evaluation ( table 1 ) as far as the 5-day old embryos are concerned , we modified the original istanbul consensus for blastocysts by leaving out the hatched stage from the evaluation .
the istanbul consensus for the 5-day old embryos has a shortcoming , i.e. it does not express the viability of embryos with a single category ( good , fair , poor ) .
we tried to overcome this by using a scoring map ( table 2 ) . in conclusion
, we constructed a composite score for day-3 , as well as day-5 old embryos , based on morphological parameters .
as it is evident from the results , this composite score is sensitive to evaluate viability ( figure 1 )
another possibility for non - invasive embryo viability assessment is the metabolomic examination of the culture medium surrounding the in vitro fertilized embryo .
metabolomic , ( proteomic ) profiling of spent embryo culture ( sec ) offers an exceptional , non - invasive opportunity for the assessment of embryo viability ( 30,31 ) .
the metabolomic profiling ( 32,33 ) of early embryo development might mean the analysis of the total metabolome by following the changes of several selected compounds , metabolomic analysis using unidentified , but significantly differing metabolomic changes , or by the analysis of a limited population of nutrients or end products .
the common feature in all three concepts is that they are concentrating on the metabolomic alterations caused by differently developing embryos in the culture medium .
very simple idea is the monitoring of glucose consumption or pyruvate formation , since this would directly indicate the metabolism of the developing embryo and it is an obvious conclusion that a metabolically active embryo would have higher implantation potential .
some authors reported that the identification of these parameters resulted in successful prediction of embryo implantation potential , but other research groups describe contradictory results ( 34 , 35 ) .
the amino acid profile of culture media is also used in the prediction of implantation potential , though not exclusively as an independent parameter , rather in combination with morphological features ( 36 ) .
the detection of unidentified metabolomic changes using near infra - red ( nir ) or raman spectroscopy ( 37 , 38 ) is a very interesting and challenging possibility .
more complicated is the concept when unknown , new biomarker molecules of embryo viability are searched for , assuming that these biomarkers were secreted by the embryo .
the difficulty of the concept is that only 4 - 8 cells are present in the culture medium ; thus a very sensitive analytical tool is required .
mass spectrometry ( ms ) has the potential of specific and sensitive quantification in a wide spectrum of molecular mass ranges and therefore suites well the needs of metabolomic or proteomic fingerprinting and quantification . in parallel to the spreading of mass spectrometry , proteomics is also an emerging field in the understanding of embryo development ( 39,40 ) .
the analysis of the embryonic secretome ( 41,42 ) provides information of the total transcriptome of the developing embryos .
mass spectrometry can be used both in targeted and discovery analysis with accurate quantification of identified biomarkers after molecular identification by bottom - up or top - down proteomics using tandem or multiple ms ( 43 - 46 ) . in a recent publication from our laboratory ( 47 ) using liquid chromatography coupled mass spectrometry ( lc - ms ) , a fragment of the human haptoglobin molecule was identified in the culture medium . rather than analyzing the embryonic secretome , the aim this experiment was to use preexisting molecules present in the cell culture media as biomarkers .
haptoglobin - which was detected in the culture medium - is not a product of the developing embryo ; the polypeptide is a contaminant of the human serum albumin standard used to supplement the culture medium ( 47,48 ) . during the first three days of embryo development the formation of a subunit ( alfa-1 ) of the human haptoglobin molecule was observed .
this subunit similar to the total haptoglobin molecule was detectable in the blank control medium samples as well .
the differentiation of the viable and non - viable embryos was done using the observation that compared to blank controls the samples of embryos which later did not resulted in pregnancy contained the alpha-1 subunit in a much larger quantity than the samples of embryos which did ( figure 2 ) .
clinical statistical analysis of the results revealed that the specificity of the diagnostic test was 64% , while the sensitivity was 100% .
it is more informative that the positive predictive value of the assay was 51% and  maybe more importantly  the negative predictive value was 100% .
receiver operating characteristic ( roc ) analysis provides tools to select possibly optimal models and to discard suboptimal ones .
roc analysis is related in a direct and natural way to cost / benefit analysis of diagnostic decision - making
. the roc curve of the morphological versus metabolomic approach in relation to the correct prediction of pregnancy outcome is illustrated in figure 3 .
it is obvious that our biochemical investigation method enables a selection of the embryos by sorting out the non - viable ones .
the test selected with 100% potential the embryos , which did not lead to successful implantation at all .
one of the areas of collaboration between clinicians , the clinical laboratory and the research laboratory at the university of pcs is related to the research of infertility . since the clinical background gives the beauty and the medical importance of laboratory research , it was of outstanding importance for us to receive the eflm - abbott diagnostics award for excellence in outcomes research in laboratory medicine ( paris , 2015 ) , the award given to the best published paper ( 47 ) , as judged by an independent panel of experts , which demonstrates improved outcomes arising out of the application or improved utilization of an in - vitro diagnostics test .
 human reproduction is a relatively inefficient process and therefore the number of infertile couples is high . 
 assisted reproductive technologies ( art ) have facilitated the birth of over five million children worldwide . 
 art , however , superimposes its own relative inefficiency on the preexisting inefficiency of normal reproduction . 
 the efficiency ( expressed as pregnancy rate ) is generally not more than 30% . 
 modern reproductive medicine is gradually moving from multiple embryo transfer to the transfer of a single embryo , mainly because of obvious and unwanted side effects of multiple embryo transfer ( e.g.  epidemic  multiple pregnancies ) . 
 this concept , however , requires a fast , professional selection of the most viable embryo during the first few days of art . 
 thus the aim of a modern art is the safe transfer of a healthy , viable , single embryo . 
 accurate and rapid methods of quantifying embryo viability are needed to reach this goal . 
 methodological advances have the potential to make an important contribution , and there has been a drive to develop alternative non - invasive methods to better meet clinical needs . 
 metabolic and genetic profiling of spent embryo culture ( sec ) media should offer an exceptional opportunity for the assessment of embryo viability . 
 the current review focuses on the latest non - invasive diagnostic approaches for pre - implantation viability assessment of in vitro fertilized embryos . 
INTRODUCTION
THE MORPHOLOGICAL APPROACH
THE BIOCHEMICAL APPROACH