stage of development limits the time to perform the genetic diagnosis, making it difficult to redo a second round of pcr or to rehybridize fish probes before the embryos should be transferred back to the patient. cumulus cell sampling sampling of cumulus cells can be performed in addition to a sampling of polar bodies or cells from the embryo. because of the molecular interactions between cumulus cells and the oocyte, gene expression profiling of cumulus cells can be performed to estimate oocyte quality and the efficiency of an ovarian hyperstimulation protocol, and may indirectly predict aneuploidy, embryo development and pregnancy outcomes. non-invasive preimplantation genetic screening methods (nipgs) traditional embryo biopsy can be invasive and costly. therefore, researchers have an ongoing quest to find a less invasive methods for preimplantation genetic testing. studies on new non-invasive preimplantation genetics screening methods (nipgs) such as blastocoel fluid and spent embryo media have recently been published as an alternative to traditional methods preimplantation genetic screening testing using blastocoel fluid (bf) during a normal ivf process, good practice to vitrify embryos increases the chance of a healthy pregnancy. during the process of vitrification a developed blast is dehydrated and it and its blastocoel cavity collapses for the freezing process. there are many methods that have been used to facilitate the collapse including laser-pulse, repeated micropipetting, microneedle puncture or microsuction normally this fluid would then be discarded, however with preimplantation genetic testing of bl, this fluid is saved and then tested for dna. this dna is thought to be from cells that have gone through apoptosis found in the developing embryo preimplantation genetic testing using blastocyst culture conditioned medium (bccm) another method for less invasive preimplantation genetic testing involves testing the culture media the embryo has developed in. it has been noted that the embryo releases dna fragments from the cells that have died within the incubation period. with this knowledge, scientists have reasoned that they could isolate this dna and use it for preimplantation genetic testing the benefits and consequences of less invasive pre implantation genetic testing while there's conflicting evidence as to whether or not the more traditional methods of preimplantation genetic testing are harmful to the embryo there is newer methods for less invasive and equally effective testing methods. to that effect we have turned to preimplantation genetic testing using blastocoel fluid and spent embryo media. one problem to these alternatives are the minimal amount of dna there is to work with. another very important question is whether or not this technology is accurate. both of these concerns were recently addressed by kuznyetsov. kuznyetsov decided to use both methods combining the amount of dna retrieved from both techniques. then once the dna was isolated it was used for preimplantation genetic testing. the results showed that when both methods blastocyst fluid and embryo spent media were used in combination they showed a cordance rate for the whole chromosome copy of 87.5% when compared to the trophectoderm, 96.4% when compared to the whole blastocyst (gold standard). additionally after amplification using this new method they were able to produce 25.0-54.0 ng/ul of dna per sample. with traditional methods such as trophectoderm they collected 10 to 44 ng/ul genetic analysis techniques fluorescent in situ hybridization (fish) and polymerase chain reaction (pcr) are the two commonly used, first-generation technologies in pgd. pcr is generally used to diagnose monogenic disorders and fish is used for the detection of chromosomal abnormalities (for instance, aneuploidy screening or chromosomal translocations). over the past few years, various advancements in pgd testing have allowed for an improvement in the comprehensiveness and accuracy of results available depending on the technology used. recently a method was developed allowing to fix metaphase plates from single blastomeres. this technique in conjunction with fish, m-fish can produce more reliable results, since analysis is done on whole metaphase plates in addition to fish and pcr, single cell genome sequencing is being tested as a method of preimplantation genetic diagnosis. this characterizes the complete dna sequence of the genome of the embryo. fish fish is the most commonly applied method to determine the chromosomal constitution of an embryo. in contrast to karyotyping, it can be used on interphase chromosomes, so that it can be used on pbs, blastomeres and te samples. the cells are fixated on glass microscope slides and hybridised with dna probes. each of these probes are specific for part of a chromosome, and are labelled with a fluorochrome. dual fish was considered to be an efficient technique for determination of the sex of human preimplantation embryos and the additional ability to detect abnormal chromosome copy numbers, which is not possible via the polymerase chain reaction (pcr). currently, a large panel of probes are available for different segments of all chromosomes, but the limited number of different fluorochromes confines the number of signals that can be analysed simultaneously. the type and number of probes that are used on a sample depends on the indication. for sex determination (used for instance when a pcr protocol for a given x-linked disorder is not available), probes for the x and y chromosomes are applied along with probes for one or more of the autosomes as an internal fish control. more probes can be added to check for aneuploidies, particularly those that could give rise to a viable pregnancy (such as a trisomy 21). the use of probes for chromosomes x, y, 13, 14, 15, 16, 18, 21 and 22 has the potential of detecting 70% of the aneuploidies found in spontaneous abortions. in order to be able to analyse more chromosomes on the same sample, up to three consecutive rounds of fish can be carried out. in the case of chromosome rearrangements, specific combinations of probes have to be chosen that flank the region of interest. the fish technique is considered to have an error rate of 5–10%. the main problem of the use of fish to study the chromosomal constitution of embryos is the elevated mosaicism rate observed at the human preimplantation stage. a meta-analysis of more than 800 embryos came to the result that approximately 75% of preimplantation embryos are mosaic, of which approximately 60% are diploid–aneuploid mosaic and approximately 15% aneuploid mosaic. li and co-workers found that 40% of the embryos diagnosed as aneuploid on day 3 turned out to have a euploid inner cell mass at day 6. staessen and collaborators found that 17.5% of the embryos diagnosed as abnormal during pgs, and subjected to post-pgd reanalysis, were found to also contain normal cells, and 8.4% were found grossly normal. as a consequence, it has been questioned whether the one or two cells studied from an embryo are actually representative of the complete embryo, and whether viable embryos are not being discarded due to the limitations of the technique. nevertheless, mosaic embryos can be transferred but only if there are not euploids available, having previously informed patients about the risks and doing a prenatal diagnose preferably. pcr kary mullis conceived pcr in 1985 as an in vitro simplified reproduction of the in vivo process of dna replication. taking advantage of the chemical properties of dna and the availability of thermostable dna polymerases, pcr allows for the enrichment of a dna sample for a certain sequence. pcr provides the possibility to obtain a large quantity of copies of a particular stretch of the genome, making further analysis possible. it is a highly sensitive and specific technology, which makes it suitable for all kinds of genetic diagnosis, including pgd. currently, many different variations exist on the pcr itself, as well as on the different methods for the posterior analysis of the pcr products. when using pcr in pgd, one is faced with a problem that is inexistent in routine genetic analysis: the minute amounts of available genomic dna. as pgd is performed on single cells, pcr has to be adapted and pushed to its physical limits, and use the minimum amount of template possible: which is one strand. this implies a long process of fine-tuning of the pcr conditions and a susceptibility to all the problems of conventional pcr, but several degrees intensified. the high number of needed pcr cycles and the limited amount of template makes single-cell pcr very sensitive to contamination. another problem specific to single-cell pcr is the allele drop out (ado) phenomenon. it consists of the random non-amplification of one of the alleles present in a heterozygous sample. ado seriously compromises the reliability of pgd as a heterozygous embryo could be diagnosed as affected or unaffected depending on which allele would fail to amplify. this is particularly concerning in pgd for autosomal dominant disorders, where ado of the affected allele could lead to the transfer of an affected embryo. several pcr-based assays have been developed for various diseases like the triplet repeat genes associated with myotonic dystrophy and fragile x in single human somatic cells, gametes and embryos. next-generation sequencing from 2014, next-generation sequencing (ngs) is being performed in the pgt. ngs, also known as massive parallel sequencing, is a group of techniques capable of sequencing great amounts of dna at a reasonable cost and time. it can give us a general perspective of the complete embryo genome, including the mitochondrial one. those techniques are based on sequencing short reads around 400 bases each and overlapping these reads with powerful alignment software. likewise, ngs also allows us to detect aneuploidies in the 24 chromosomes and single-gene defects when there is an indication from the carrier parents. the main advantage is that ngs can combine the detection of both aneuploidies and monogenic diseases with a single biopsy and has reduced affordable costs, making it more accessible. two examples of ngs are the pyrosequencing and the reversible dye terminator. establishing a diagnosis the establishment of a diagnosis in pgd is not always straightforward. the criteria used for choosing the embryos to be replaced after fish or pcr results are not equal in all centres. in the case of fish, in some centres only embryos are replaced that are found to be chromosomally normal (that is, showing two signals for the gonosomes and the analysed autosomes) after the analysis of one or two blastomeres, and when two blastomeres are analysed, the results should be concordant. other centres argue that embryos diagnosed as monosomic could be transferred, because the false monosomy (i.e. loss of one fish signal in a normal diploid cell) is the most frequently occurring misdiagnosis. in these cases, there is no risk for an aneuploid pregnancy, and normal diploid embryos are not lost for transfer because of a fish error. moreover, it has been shown that embryos diagnosed as monosomic on day 3 (except for chromosomes x and 21), never develop to blastocyst, which correlates with the fact that these monosomies are never observed in ongoing pregnancies. diagnosis and misdiagnosis in pgd using pcr have been mathematically modelled in the work of navidi and arnheim and of lewis and collaborators. the most important conclusion of these publications is that for the efficient and accurate diagnosis of an embryo, two genotypes are required. this can be based on a linked marker and disease genotypes from a single cell or on marker/disease genotypes of two cells. an interesting aspect explored in these papers is the detailed study of all possible combinations of alleles that may appear in the pcr results for a particular embryo. the authors indicate that some of the genotypes that can be obtained during diagnosis may not be concordant with the expected pattern of linked marker genotypes, but are still providing sufficient confidence about the unaffected genotype of the embryo. although these models are reassuring, they are based on a theoretical model, and generally the diagnosis is established on a more conservative basis, aiming to avoid the possibility of misdiagnosis. when unexpected alleles appear during the analysis of a cell, depending on the genotype observed, it is considered that either an abnormal cell has been analysed or that contamination has occurred, and that no diagnosis can be established. a case in which the abnormality of the analysed cell can be clearly identified is when, using a multiplex pcr for linked markers, only the alleles of one of the parents are found in the sample. in this case, the cell can be considered as carrying