Noninvasive preimplantation diagnosis

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The goal of in vitro fertilization, in the context of couple infertility treatment, is to achieve a healthy baby, possibly in the shortest possible time. The causes of failure to implant embryos are many, but genetic abnormality of the product of conception plays a major role. It is estimated that 20-80% of human embryos are aneuploid [1]. Over the past 30 years, several avenues have been attempted for genetic evaluation of the embryo prior to implantation, either to avoid transmission of monogenic diseases (so-called PGT-M) or to exclude genetically abnormal embryos from implantation, which usually leads to failed implantation or early abortion (PGT-A).

All methods used to assess the genetic status of the embryo to date have been based on direct sampling of genetic material, such as polar blood cell biopsy for oocytes or zygotes, or such as biopsy of blastomeres or trophoectoderm cells for embryos at the cleavage or blastocyst stage .

However, these retrieval methods involve some critical issues. First, micromanipulation techniques are not without risk of embryo damage. It is estimated that the implantation rates of each biopsied embryo are reduced, even if euploid, and this naturally raises questions about the positivity of the balance between risks and benefits [4]. Second, biopsy techniques involve special equipment, particularly lasers, which increase operating costs. In addition, experienced personnel who have had adequate training are needed.

Preimplantation diagnosis has undergone periods of exaltation and periods of fierce criticism over time. Polar blood cell diagnosis does not allow assessment of paternal contribution, and this is a major limitation. Biopsy performed on day 3, at the cleavage stage, has the major limitation related to the important genetic rehoming activity, so biopsy of 1-2 cells is exposed to a high risk of error [5]. Moreover, it is possible that cell removal can reduce embryonic viability up to more than 30% [6]. Biopsy at the blastocyst stage is the only one performed to date, with the removal of some cells from the trophoectoderm, although the potential for embryo implantation is still reduced to some extent, depending also on the skill of the operator [4].

In view of the problems brought about by biopsies, the discovery of fetal DNA in the culture medium and blastocele has created interest related to the possibility of noninvasive diagnosis (niPGT) [7].

During blastocyst formation, embryonic cells differentiate into two different lineages, those of the inner cell mass, which will give rise to the embryo proper, and those of the trophoectoderm, which will give rise to the placenta. The blastocyst within it sees the formation of a space with fluid, the so-called blastocele. In the course of blastocentesis, which is the practice of taking fluid from the blastocele, an ICSI pipette goes through the layer of the trofoectoderm on the side opposite the inner cell mass, and the fluid is carefully aspirated until the blastocyst is collapsed. Blastocyst collapse, by the way, is an event that occurs near the exit of the blastocyst from the zona pellucida (Hatching). Blastocele aspiration is not harmful to the embryo, and is also performed in many Centers before vitrification to reduce the formation of ice crystals during cryopreservation [8].

There are two important requirements to be able to obtain a result applicable to clinical practice when using DNA sources for preimplantation diagnosis. The first is that DNA analysis be consistently and reproducibly obtained, that is, that the method be able to provide adequate sensitivity and specificity. The second is that the DNA is indeed representative of the state of the embryo. With regard to PGT-M, blastocele analysis has so far encountered significant difficulties in isolating and amplifying DNA, due to the low amount of DNA it contains, and also the alterations it exhibits [9]. Concordance with the results of trophoectoderm biopsy has also been limited so far, suggesting that blastocele DNA may be partially degraded [10].

PGT-A can be performed using different methods, such as PCR, CGH, SNP and NGS [11]. In this case, the difficulty in obtaining sufficient DNA to perform an analysis remains, but the concordance of the results with respect to trophoectoderm biopsy is between 94 and 96 percent for individual chromosomes, falling, however, to 66 percent for the whole genome according to Gianaroli’s group [12]. Other groups, however, have found much lower concordances, such as Capalbo [13] and Tobler [14].

Blastocentesis is certainly attractive conceptually, but there are several difficulties yet to be overcome. First, the amount of DNA present in the blastocele varies significantly between embryo and embryo, and in several cases it is not detectable in sufficient quantity for amplification. Second, it is critical to identify the correct mode of fluid extraction and the amount to be analyzed, because if you go from 0.3 to 1 microliter there may be differences in amplification efficiency [15]. Therefore, blastocentesis still needs to be standardized in terms of both the methodology of obtaining the sample and the mode of amplification and analysis.

One potential alternative that is receiving much attention is the analysis of the medium used for culture. The culture medium used for blastocysts appears to provide more DNA than blastocele fluid, thus becoming an even less invasive source of material than the blastocele, as it does not involve any micromanipulation [16]. DNA of mitochondrial and genomic origin can be found in the culture medium as early as day 2 to 3 of embryonic development, but increases significantly by day 4 and 5, also excluding any contamination [17]. The zona pellucida of preimplantation embryos is permeable to various macromolecules, and it is possible that DNA may also cross it. In any case, genomic DNA is little and often degraded, suggesting that it may result from cell death mechanisms [18].

Cell apoptosis is common at the blastocyst stage, much less so at earlier stages, but an interesting finding is that the concentration of double-helix DNA in the culture medium in some work appears to be higher in samples derived from lower quality embryos than that found in samples from embryos of excellent quality. Similarly, the mitochondrial DNA found is higher in samples from embryos with high fragmentation or from older women [19]. In a large multicenter study involving more than 1300 blastocysts, Simon’s group showed high concordance between culture medium analysis and trophoectoderm biopsy [20].

It is relevant to understand whether the DNA found in the blastocele or medium is derived from physiological or pathological processes, that is, whether DNA is more likely to be found in samples from embryos of good morphology, which therefore have more cells, or from poorly viable embryos, which are more likely to undergo cell death. At present, studies have yielded mixed results. Zhang and his group [9] found no association between morphology and DNA quantity, while other groups, such as Capalbo’s, reported better detection efficiency in samples from good-quality blastocysts [13]. In contrast, Magli’s group reported higher DNA concentrations in samples from blastocysts diagnosed with aneuploidies at trophoectoderm biopsy [12]. The same group when transferring embryos in utero showed a higher percentage of pregnancies with embryos in which DNA determination had failed than those with higher amplification success. This therefore represents a still open question.

Precise standardization of both culture conditions and sample collection is also needed before niPGT can be used clinically. Studies that have indicated that DNA accumulates with prolonged culture have suggested the possibility of collecting the culture medium on day 6 or 7. On the other hand, prolonged culture, especially at day 7, could be detrimental to embryo survival and create more difficulties for cryopreservation. Moreover, in clinical practice, there are culture media that are changed on day 3 and others that are maintained for all 5 days. It needs to be understood whether the loss of DNA from the first three days, which occurs by changing the culture medium, is an advantage or a disadvantage. In addition, exogenous DNA contamination, whether derived from the mother or operators, should be carefully avoided [21].

Certainly, the elimination of embryo biopsy would bring practical and economic advantages, with reduced risks to embryos. However, the reliability of niPGT strategies, in relation to amplification possibilities and concordance with results obtained by classical diagnosis, varies greatly among studies. It is still unclear which method of amplification and investigation may be the most suitable. Probably, techniques that preserve more DNA fragment length are the most suitable, as further fragmentation of already damaged DNA may make the investigation inconclusive. It will also be necessary to make the insemination, culture and collection process very precise so as to avoid contamination of DNA not belonging to the embryo [22].

In conclusion, the use of niPGT at present may not be of clinical utility, further studies and optimization of its efficacy will be needed before leaving the experimental stage.

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