Dr. Luca Gianaroli, S.I.S.Me.R. Reproductive Medicine Unit, Bologna Italy
Tuesday 25th April, 1.00 p.m., Stacey Lecture Theatre 1
Since the beginning of IVF, embryologists have directed major efforts to identify key pathways of normal embryo development and factors that contribute to embryo viability. Morphological evaluation remains the primary approach of embryo assessment despite its limitations, due not only to the subjectivity of the embryologist, but also because of the evaluation system itself, which does not provide any information whatsoever about embryo ploidy and metabolic activity.
Recent advances in genetic screening technologies greatly improved our capacity to investigate the embryonic DNA, thus allowing the identification and transfer of euploid embryos. This approach represented a great achievement, since aneuploidy is the major cause of failed implantation and spontaneous abortion. For this reason, the testing of preimplantation embryos focused primarily on nuclear DNA to detect numerical abnormalities and deselect the corresponding embryos. More recently, attention was diverted to the investigation of mitochondrial DNA (mtDNA) as a potential marker of embryo viability and implantation potential. This approach stemmed from the consideration that mitochondria are the highly specialized organelles principally charged with the production of cellular energy.
It is well known that the number of mitochondria differs from cell to cell. In gametes and embryos, this figure is extremely variable during the different stages of development. It has been demonstrated that defects in their distribution and function can negatively affect the embryo ploidy condition and viability (Carrol, 2012; Jessberger, 2012).
In the attempt to establish a possible correlation between mtDNA copy number and clinical outcome, two studies have tried to relate the embryonic mtDNA content to maternal age, chromosomal complement, implantation potential and blastocyst development. In both cases, the mtDNA content was calculated as a ratio to nuclear DNA. Despite some differences mainly due to the diverse size and cell type of the analyzed samples, both groups demonstrated that the quantity of mtDNA represents a marker for embryo viability. This led to define a threshold, above which the probability of embryo implantation is extremely low (Diez-Juan et al., 2015; Fragouli et al., 2015).
A criticism to these studies was made by another group based on the argument that when calculating the quantity of mtDNA in relation to nuclear DNA, the denominator of this ratio needs to be adjusted for each embryo according to the effective content of nuclear DNA. Variations in the nuclear genome are actually brought by sex (the Y chromosomes has a lower DNA content compared to the X chromosome) and by numerical aneuploidy (a monosomic embryo has a lower DNA content compared to either a disomic or a trisomic embryo). Therefore, after adding a corresponding correction factor to avoid extra/over estimation of mtDNA, no correlation was found between the mtDNA content and maternal age, chromosomal status, and implantation rate (Victor et al., 2015).
More research is needed in this direction to obtain robust data that can corroborate the presented results. Meanwhile, an approach other than the quantitative methodology can be investigated.
During oogenesis, a great amount of energy is requested to accomplish all the involved steps, including meiosis. It is well known that female oogenesis is a very peculiar process, during which chromosome segregation is particularly prone to errors in a modality that is strictly related to age. A correlation has been established between aneuploidy and defective energy supply, leading to conclude that mitochondria in the mature oocyte (they are maternally inherited) are crucial for the following steps of fertilization and early embryonic development.
To better understand the process of oogenesis, we studied the segregation of mitochondria at the first and second meiotic division. Therefore, we sequenced mtDNA in a series of triads composed by oocyte and corresponding polar bodies, and compared the results with the blood.
After sequencing the mitochondrial non-coding region (D-Loop) of 17 oocytes, full correspondence was found with the respective blood samples. However, the concordance with polar bodies dropped to 89.6%, suggesting that oocytes could have an active mechanism to preserve a condition of normality, similar to what has been observed for chromosome segregation during meiosis (compensated aneuploidy). Accordingly, some haplogroups were shown to be more prone than others to generate aneuploidy, with a significantly higher incidence of chromosomal errors in some of them (Gianaroli et al., 2015). The same trend was observed when sequencing the coding region (Gianaroli et al., 2014). When comparing the two polar bodies, there were no differences in the quantity of mutations between them, but a higher pathogenic score was detected in the first polar body. Also in this case, the presence of a selection process operating during meiosis was postulated. As the mitochondrial condition of the second polar body is closer to that of the oocyte, the by-product of the second meiotic division could be a good predictor of the genetic status of the oocyte (De Fanti et al., 2017).
In conclusion, the study of mitochondria in oocytes and embryos could represent an important tool in the definition of the most efficient approach towards the selection of viable concepti. More information is needed, but the preliminary data obtained so far point to the relevance of energy supply in determining the fate of the embryo.