Embryo's Noninvasive Chromosome Screening: Main Issues. Perspectives

Noninvasive Chromosome Screening of Embryos Using Nuclear DNA and Mitochondrial DNA Content in Blastocoele Fluid and Embryo Culture Medium: Main Issues. Dilemmas. Breakthroughs. Perspectives.

Introduction

In Vitro Fertilization (IVF) therapy and Embryo Transfer (ET) is a well–established procedure to overcome infertility. In Vitro Fertilization (IVF) Treatments typically involve the production of multiple embryos.One of the most important and unsolved problems in In–Vitro Fertilization is to decide which embryos are more suitable to implant and therefore should be transferred and which embryos should be cryopreserved for further usage. Synchronically with top–quality Embryo Selection process, in the same time period, the use of sequential, stage–specific media combined with low–oxygen tension culture systems and the introduction of Vitrification strategy in IVF have permitted blastocyst culture and Cryopreservation to be accomplished with high efficiency [McArthur et al., 2005; Schoolcraft et al., 2010]. However, the viability of individual embryos is highly variable. Even among a cohort of sibling embryos competence can vary greatly. The great challenge for IVF clinics is to correctly identify the most viable embryos and prioritize them for further transfer to the uterus.

Currently, the decision of which embryo(s) to transfer is made on the basis of morphologic assessments conducted in the IVF laboratory. To date, embryos are routinely selected for transfer based on their morphology and time to first cleavage [Racowsky, 2002; Scott, 2003; Sakkas and Gardner, 2005]. Blastomere number, size and shape, and the presence or absence of extracellular fragments are the relevant characteristics that form the current basis for non–invasive evaluations of developmental competence [Giorgetto et al., 1995]. In In Vitro Fertilization (IVF) Treatment cycle, current methods of diagnosing chromosome abnormality and screening for viability of transfer require biopsy of embryos, which affects embryo quality, awaits long–term biosafety test, and requires specialized skills. Unfortunately, such examinations do not provide reliable information concerning chromosomal copy number, one of the most important aspects of embryo viability.

Embryos are prone to chromosomal abnormalities, mainly due to age–dependent chromosome segregation errors during meiosis I [Battaglia et al., 1996]. The consequences of chromosomal imbalance or, in other words, chromosomal abnormalities could cause miscarriage (early pregnancy loss) or severe chromosomal diseases [Munné 2006]. High embryonic losses occur during cleavage with, at best, only 50% in vitro fertilized oocytes competent to reach the blastocyst stage [Hardy et al., 2002]. Aneuploidy is the leading cause of implantation failure and early spontaneous abortion of the embryo [Macklon et al., 2002; Spandorfer et al., 2004; Menasha et al., 2005], but does not represent a strong negative selection barrier for embryo preimplantation development to the blastocyst stage [Alfarawati et al., 2011].

Preimplantation Genetic Diagnosis (PGD), following In Vitro Fertilization (IVF) Treatment cycles, preimplantation embryo biopsy, and Genetic Analysis of a single cell or small numbers of cells, is now clinically well established as an alternative to invasive methods of prenatal diagnosis for couples at risk of a range of Single–Gene Defects (SGDs) and chromosome abnormalities [Handyside and Xu 2012; Harper et al., 2012]. Preimplantation Genetic Screening (PGS) is currently applied to evaluate the presence of aneuploidies in embryos of couples at risk of chromosome abnormalities, i.e. Advanced Naternal Age (AMA), Recurrent Miscarriage (RM), Recurrent In Vitro Fertilization (IVF) failure or severe male factor [Donoso et al., 2007]. The weakness of correlation between conventional methods of embryo evaluation and chromosomal complement has lead the introduction of Preimplantation Genetic Screening (PGS) as a complementary Treatment during In Vitro Fertilization (IVF) cycles to avoid the transfer of aneuploid embryos and improve the delivery rate per transfer cycle [Munné et al., 1993]. It is of paramount importance that the method of Preimplantation Genetic Screening (PGS) is accurate and that the technique itself does not diminish the developmental potential of the individual embryo tested [Northrop et al., 2010; Treff et al., 2010a; Fragouli and Wells, 2012].

Chromosomal abnormalities can be prevented in In Vitro Fertilization (IVF) therapy by performing Preimplantation Genetic Screening (PGS) of all 24 chromosomes. Preimplantation Genetic Screening (PGS) is widely used to select In Vitro–Fertilized embryos free of chromosomal abnormalities and to improve the clinical outcome of In Vitro Fertilization (IVF) Treatment cycle. There are various Preimplantation Genetic Screening (PGS) methods for transparent and comprehensive chromosome screening currently in clinical use, including Comparative Genomic Hybridization (array–CGH) [Hellani et al., 2008; Gutiérrez–Mateo et al., 2011], Single–Nucleotide Polymorphism (SNP) arrays [Treff et al., 2011; Natesan et al., 2014a, 2014b; Thornhill et al., 2015], multiplex quantitative PCR [Treff et al., 2012], and Next–Generation Sequencing (NGS) [Martín et al., 2013; Hou et al., 2013]. Multiple clinical trials have confirmed the clinical efficacy of Preimplantation Genetic Screening (PGS), including increasing implantation and clinical pregnancy rates, as well as decreasing miscarriage rates [Yang et al., 2012; Forman et al., 2013; Keltz et al., 2013; Scott et al., 2013].

With the development of Comprehensive Chromosome Screening (CCS) platforms, the promise of aneuploidy screening is becoming a reality in Assisted Reproduction Technologies [Voullaire et al., 2000; Wells and Delhanty, 2000; Fishel et al., 2010; Harper and Harton, 2010; Schoolcraft et al., 2010; Treff et al., 2010b; Gutiérrez–Mateo et al., 2011]. The main disadvantage of Preimplantation Genetic Screening (PGS) is that it requires biopsy of the preimplantation embryo, which can limit the clinical applicability of Preimplantation Genetic Screening (PGS) due to the invasiveness and complexity of the process.

Comparing the above–mentioned Preimplantation Genetic Screening (PGS) methods for transparent and Comprehensive Chromosome Screening (CCS) currently in clinical use, what is essential to mention is although mosaicism has been reported to exist in preimplantational embryos, affecting the accuracy of Preimplantation Genetic Screening (PGS) [Munne et al., 1994; Bielanska et al., 2002b; Li et al., 2005], another reason why the implantation rate is not improved by Preimplantation Genetic Screening (PGS) using FISH is probably the fact that the whole set of chromosomes is not analyzed and aneuploidies in chromosomes that are not screened could be hampering the implantation or development of transferred embryos. Comparative Genomic Hybridization (CGH) is a well–established procedure which allows for the study of all chromosomes simultaneously. The main advantage of Comparative Genomic Hybridization (CGH) over FISH is that the whole chromosomal complement can be ascertained and this also allows for the detection of not only chromosomal imbalances generated by aberrant segregation but also structural imbalances of fragments larger than 10–20 Mb [Griffin et al., 1998; Malmgren et al., 2002]. However, Comparative Genomic Hybridization (CGH) is a rather labour–intensive technique and it takes ∼4 days to obtain results. For this reason, two strategies have been developed to enable its application in PGD. The first strategy is the use of the first Polar Gody (1PB) [Wells et al., 2002; Sher et al., 2007; Obradors et al., 2008, 2009] or the first and second Polar Bodies (1PB and 2PB) [Fragouli et al., 2010] as indirect indicators of the oocyte’s chromosomal complement, in which case, only maternal contribution of first or first and second meiotic division, respectively, is determined and paternal meiotic and post–zygotic errors cannot be detected. In order to obtain a more complete analysis, a second strategy has been developed, based on the Comparative Genomic Hybridization (CGH) analysis of one blastomere from Day–3 embryos [Wilton et al., 2001; Voullaire et al., 2002; Sher et al., 2009] or of several cells removed from the trophectoderm at the blastocyst stage [Fragouli et al., 2010; Schoolcraft et al., 2010]. Nevertheless, owing to the duration of Comparative Genomic Hybridization (CGH), these two approaches require the Cryopreservation of the biopsied embryos and the transfer of those diagnosed as euploid in a subsequent cycle.

High–quality embryos’ selection plays a significant role in Assisted Reproduction Technologies (ART). It is therefore essential to identify and select the most viable embryos for transfer to maintain pregnancy success rates. The common practice is to transfer embryos during early cleavage (day 2–3) after selection based upon morphology and timing of cleavage, although the use of blastocyst culture has increased in recent years [Gardner and Lane, 1997; Blake et al., 2005]. Blastocyst culture allows continued morphological evaluation of embryos and, where relevant, screening for aneuploidy or genetic disease after biopsy [Gardner and Lane, 1997; Gardner et al., 2002; Hardy et al., 2002; Racowsky, 2002]. The optimal biopsy stage for Preimplantation Genetic Screening (PGS) is highly debated theme among the embryology’s experts. Blastocyst stage biopsy coupled with Comprehensive Chromosome Screening (CCS) now represents the most promising way for Preimplantation Genetic Screening (PGS) to detect aneuploidies coming from both male and female meiosis, as well as clinically relevant mitotic errors fixed during preimplantation embryo development [Schoolcraft et al., 2010; van Echten–Arends et al., 2011; Forman et al., 2012; Capalbo et al., 2013]. Furthermore, no negative impact of Trophectoderm (TE) biopsy on subsequent embryo development was reported and the clinical predictive value of Trophectoderm Comprehensive Chromosome Screening (TE CCS) was shown to be very promising [Scott et al., 2012]. However, due to the still–limited experience obtained so far, several concerns, related to the incidence and impact of mosaicism, through testing of Trophectoderm (TE) clinical biopsies and the representativeness of Trophectoderm (TE) on the relative Inner Cell Mass (ICM) (i.e. if data obtained from Trophectoderm (TE) biopsies can be generally considered diagnostic of the ICM) still remain. Synchronically with concerning mosaicism at the blastocysts stage of embryo development, conflicting data have been reported in the scientific articles. Although based on small numbers, the first data describing the cytogenetic constitution of blastocysts using conventional cytogenetic methods, such as G–banding and Fluorescence In Situ Hybridization (FISH), showed that more than half of all the blastocysts assessed were found to carry varying degrees of mosaicism [Bielanska et al., 2002a,b, 2005; Derhaag et al., 2003; Coonen et al., 2004; Daphnis et al., 2005], whereas more recent findings obtained during the analysis of good–quality blastocysts by Comprehensive Chromosome Screening (CCS) methods showed a lower incidence of mosaicism [Johnson et al., 2010; Fragouli et al., 2011; Northrop et al., 2010].

Thus, the actual prevalence and impact of mosaicism on testing at the blastocyst stage remains still unclear.

A further open point of blastocyst cytogenetics is the representativeness of Trophectoderm (TE) on the Inner Cell Mass (ICM), id est, if data obtained from Trophectoderm (TE) biopsies can be generally considered diagnostic of the Inner Cell Mass (ICM). In In Vitro Fertilization embryos, no evidence for aneuploid cell confinement has been reported so far either by FISH [Evsikov and Verlinsky, 1998; Magli et al., 2000; Derhaag et al., 2003; Fragouli et al., 2008] or CCS–based studies [Johnson et al., 2010; Northrop et al., 2010]. However, all previous studies employed Inner Cell Mass (ICM) isolation procedures of unproven efficiency and/or a small sample size [Hovatta, 2006]. Moreover, testing the ability to generate Embryonic Stem Cells (ESCs) has usually indirectly assessed the efficiency of these methods and none of them have been characterized in terms of number of Inner Cell Mass (ICM) cells obtained and/or Trophectoderm (TE) contamination rate.

Additionally, it should be noted that embryos’ high–quality correlates with exclusion criterion – chromosomal mosaicism. At present, the exact threshold level of abnormal cells in a mosaic diploid/aneuploid blastocyst, above which there is self–elimination of the embryo during post–implantation development, is still not clear. Furthermore, whether different chromosomes involved in an abnormality may have a different effect on embryo viability is also unknown. However, emerging evidence suggests that the impact of mosaicism on post–implantation embryonic development might have been underestimated as well as its contribution to post–natal and adult pathological conditions [Kalousek and Dill, 1983; Youssoufian and Pyeritz, 2002; Vorsanova et al., 2005; Yurov, 2007]. In this view, the identification of mosaic aneuploidies may be seen as an opportunity to avoid the transfer of potentially compromised or affected embryos. Consequently, one further advantage of the blastocyst stage approach relies on being the only Preimplantation Genetic Screening (PGS) strategy able to reveal the presence and even the extent of chromosomal mosaicism in embryos.

All the methods described above are invasive because the usual screening procedure proposed for further examination is biopsy. Therefore, the ability to screen embryos for aneuploidy or inherited disorders in a minimally invasive manner may represent a major advancement for the future of embryo viability assessment.

An alternative method to select embryos’ viability is to monitor embryo metabolism non–invasively [Houghton et al., 2002; Brison et al., 2004; Houghton and Leese, 2004; Stokes et al., 2007]. It was found that embryos capable of developing to the blastocyst stage were metabolically more quiescent during early cleavage compared with embryos that arrested prior to blastocyst formation [Houghton et al., 2002]. Subsequently, it was shown that amino acid turnover of embryos measured from day 1 to day 2 of development was also capable of predicting pregnancy after transfer [Brison et al., 2004]. More recently, amino acid turnover has been shown to predict development to the blastocyst stage of cryopreserved embryos as well as being able to differentiate between the developmental capacity of embryos of the best morphological grade [Stokes et al., 2007].

It was established that fragmentation of one or more blastomeres prevails during the early cleavage stages of embryonic development in vitro. The complete or partial fragmentation of one or more blastomeres resulting in conversion to a pleiomorphic population of cytoplasts is a common occurrence during the early cleavage stages of embryonic development in vitro. Both apoptotic and necrotic processes have been suggested as causes of blastomere fragmentation in embryos [Juriscova et al., 1996], but a definitive aetiology has yet to be determined. In this regard, it remains unclear whether all forms and degrees of fragmentation are indications that the competence of an affected cell(s) or the entire embryo has been necessarily compromised. Outcome data from some clinical In–Vitro Fertilization (IVF) studies suggests that embryo developmental potential declines significantly as the number of cytoplasmic fragments increases [Giorgetto et al., 1995], while others have shown no significant correlation [Hoover et al., 1995].

In the majority of cleavage stage embryos that contained blastomeres with cytoplasmic fragments, immunofluorescent analysis by scanning laser confocal microscopy demonstrated that these structures frequently formed from the portion of the plasma membrane associated with the regulatory protein domains, and that as a result of the incorporation of portions of the affected domain(s) into the cytoplasmic extrusions, the apparent complement of these proteins was either reduced or undetectable in the affected blastomere(s). These findings suggest that the differential developmental potential exhibited by fragmented embryos may be associated with a blastomere–specific depletion of critical regulatory proteins that is determined both by the specific pattern of fragmentation and the specific portion of the plasma membrane and subjacent cytoplasm involved [Antczak and Blerkom, 1999].

The degree of fragmentation is presently one of the most important empirical criteria used in embryo assessments because the number and relative sizes of fragments can be readily estimated. Although some apparent differences in frequency have been reported, fragmentation has been observed to occur in the presence of different culture media and growth conditions, including the presence of a feeder layer [Van Blerkom, 1993, 1997; Morgan et al., 1995], suggesting that this phenomenon may be largely embryo–specific and not a function of a particular set of growth conditions. With respect to developmental competence, the prevailing hypothesis is one that suggests that developmental viability declines as the number of fragments increases [Giorgetto et al., 1995]. Although a precise aetiology is unknown, fragmentation has been suggested to be an accurate indicator of developmentally lethal defects at the blastomere level associated with an instability within the cortical microfilament network [Van Blerkom et al., 1995b], levels of ATP generation [Van Blerkom et al., 1995b], chromosomal abnormalities including aneuploidy and mosaicism [Munne et al., 1993] and apoptotic and necrotic processes [Juriscova et al., 1996]. In this regard, fragmentation may not have a common origin, but rather may be the overt manifestation of different underlying disorders. As noted here and by other investigators [Hoover et al., 1995], normal births are known to have resulted from fragmented embryos in which numerous extracellular fragments were left undisturbed and therefore free to influence the fate of the affected embryos. This indicates that fragmentation per se is not an absolute determinant of developmental potential or lack thereof, with the possible exception of those instances where the level of fragmentation is so extensive that few, if any, blastomeres remain unaffected. As discussed below, the results suggest that the relationship between fragmentation and developmental potential may be associated with the specific pattern of fragmentation, the stage at which fragmentation occurs, and the particular blastomere(s) involved. This notion could explain why embryos with the same apparent degree of fragmentation can have very different developmental fates [Antczak and Van Blerkom, 1999].

Morphological assessment at the blastocyst stage of development offers improved Embryo Selection; however, even an embryo of the highest blastocyst grade may still fail to implant or may result in a miscarriage [Capalbo et al., 2014]. This discrepancy is largely due to blastocyst grade being unable to identify aneuploidy, which significantly increases with female age, and is the main cause of implantation failure and miscarriage [Farfalli et al., 2007; Fragouli et al., 2013; Capalbo et al., 2014]. It has been reported that even top–quality blastocysts have a 56% euploidy rate, demonstrating the large discrepancy between morphology and the chromosomal complement of the developing embryo [Capalbo et al., 2014]. Recently, it has been suggested that elevated levels of mitochondrial DNA (mtDNA) at the blastocyst stage are associated with aneuploidy and decreased implantation potential for euploid embryos, and therefore the levels of this genome may reflect embryo viability [Diez–Juan et al., 2015; Fragouli et al., 2015].

Direct genetic assessment of embryos by removing a single cell or multiple cells of the preimplantation embryo is a valuable tool for Embryo Selection. Preimplantation Genetic Screening (PGS) can avoid the transfer of an aneuploid embryo, and although it is an invasive procedure, it has the potential to improve IVF success [Yang et al., 2012; Forman et al., 2013; Scott et al., 2013a; Chen et al., 2015; Dahdouh et al., 2015]. Following Single Embryo Transfer (SET), it has been reported that ongoing pregnancy rates may improve by almost 30% with the transfer of a known euploid blastocyst [Yang et al., 2012]. In addition to aneuploidy screening, Preimplantation Genetic Diagnosis (PGD) can be applied to prevent the transmission of single gene disorders such as cystic fibrosis and β–thalassemia [Gutierrez–Mateo et al., 2009].

Genetic assessment can be performed by the analysis of genetic material from a biopsy of either Polar Bodies following their extrusion from the oocyte [Montag et al., 2013], blastomeres during the cleavage stage of development [Handyside et al., 1989] or trophectoderm during the blastocyst stage [Schoolcraft et al., 2010]. Although Polar Body biopsy represents a minimally invasive approach for genetic analysis because these are naturally extruded from the oocyte, it is limited because it does not allow the genetics of the embryo to be directly assessed [Salvaggio et al., 2014]. For this reason, Polar Body assessment may only able to predict embryo aneuploidy due to mitotic errors that occur up to 70% of the time in the embryo post–fertilization [Capalbo et al., 2013; Salvaggio et al., 2014]. In contrast, blastomere biopsy does directly assess embryo genetics; however, the biopsy procedure is known to reduce the developmental potential of the embryo [Scott et al., 2013b]. Of the three sampling stages, trophectoderm biopsy at Day 5 or 6 of development produces the most reliable Preimplantation Genetic Screening (PGS) results because it is more representative of the complete chromosomal complement of the preimplantation embryo [Fragouli and Wells, 2011; Scott et al., 2013b]. While trophectoderm biopsy does not appear to be detrimental to the embryo, it still involves the invasive removal of cells from the embryo and is a technical procedure. Therefore, novel methodologies for the genetic assessment of embryos in a minimally invasive manner may represent a significant advance in our ability to screen embryos for aneuploidy and inherited disorders.

Very recently, accessing genetic material from blastocoele fluid has attracted interest following reports that it contains nuclear DNA [Palini et al., 2013; Gianaroli et al., 2014; Tobler et al., 2015; Magli et al., 2016]. Preliminary results suggest that the blastocoele fluid can be successfully isolated from the embryo using a minimally invasive approach, and the genetic material can be purified, amplified and undergo genetic analysis. Further evidence is needed however of the efficiency, accuracy and optimal methodologies of this approach. In addition, there have been recent reports that spent embryo culture medium contains both nuclear DNA and mtDNA, which can be obtained in a way that is completely non–invasive to the embryo [Stigliani et al., 2013, 2014; Assou et al., 2014; Wu et al., 2015]. The origin of the DNA in the culture medium has not been established, but it has been suggested that if it is possible to extract embryonic genetic material in this way, that it could be analyzed and may provide useful information about embryo quality [Stigliani et al., 2013, 2014]. Furthermore, some studies have suggested that the genetic material present in the culture medium has the potential to be used for Preimplantation Genetic Diagnosis (PGD), but this remains controversial [Assou et al., 2014; Wu et al., 2015]. In order to investigate this as a possibility, a full characterization of the genetic material, including an assessment of its origin, needs to be undertaken. While the genetic material may be an independent biomarker of embryo viability, the possibility that it may contain contaminating cells from either a maternal or paternal source could compromise its use for genetic analysis in Preimplantation Genetic Screening (PGS) and Preimplantation Genetic Diagnosis (PGD).

The development of the preimplantation embryo is a complex and dynamic process that is not completely understood, and the mechanisms behind the release of the genetic material from the developing embryo, or the identification of potential sources of contamination, needs to be addressed for both the blastocoele fluid and embryo culture medium. There is currently concern over the origin of the genetic material and the degree to which it reflects the genetics of the developing embryo, or possible contamination. This makes it difficult to interpret the genetic analyses reported by the studies to date and to assess the potential use of this genetic material in a clinical setting. Thus, more studies are needed to address this issue and provide definitive data to determine its utility for genetic analysis of the embryo and for viability assessment.

One of the most sophisticated and accurate scientific theories, which outlines the current evidence that suggests the presence of genetic material in the blastocoele fluid and embryo culture medium is represented by the scientists Elizabeth R. Hammond, Andrew N. Shelling and Lynsey M. Cree in their review “Nuclear and mitochondrial DNA in blastocoele fluid and embryo culture medium: evidence and potential clinical use” [Elizabeth R. Hammond et al., 2016]. Furthermore, this review describes potential mechanisms of DNA release from the embryo, discusses potential sources of contaminating DNA and addresses the technical considerations that currently limit the analysis and source of this low template DNA. Ultimately, given the current evidence, the potential for this genetic material to be used for genetic analysis and embryo quality assessment in a clinical setting will be assessed [Elizabeth R. Hammond et al., 2016].

1. The mitochondrion, the mitochondrion configuration; The mitochondrial DNA, the mitochondrial DNA configuration, The mitochondrial DNA nucleoid and the mitochondrial DNA replication, The mitochondrial DNA (mtDNA) replication in the pluripotent preimplantation embryo and the mitochondrial DNA (mtDNA) replication and embryonic differentation: intracellular dimension overwiew

Mitochondria play a vital role in embryo development. Mitochondria are the principal site of energy production and have various other critical cellular functions. Mitochondrial DNA (mtDNA) encodes key proteins associated with the process of oxidative phosphorylation. Defects to mitochondrial DNA (mtDNA) cause severe disease phenotypes that can affect offspring survival. Mitochondrial DNA (mtDNA) replication is strictly down–regulated from the fertilized oocyte through the preimplantation embryo. At the blastocyst stage, the onset of mitochondrial DNA (mtDNA) replication is specific to the trophectodermal cells. The inner cell mass cells restrict mitochondrial DNA (mtDNA) replication until they receive the key signals to commit to specific cell types. However, it is necessary to determine whether somatic cells reprogrammed through somatic cell nuclear transfer, induced pluripotency or fusion to an ESC are able to regulate mitochondrial DNA (mtDNA) replication so that they can be used for patient–specific cell therapies and to model disease.

Despite the importance of this organelle, little is known about the extent of variation in mitochondrial DNA (mtDNA) between individual embryos prior to implantation. Mitochondria are small membrane–enclosed structures and are found inside the cells of the body. Mitochondria actively participate in cellular life, and their main function is to generate energy which is used by the cell. For this reason mitochondria are considered as the powerhouses of cells. Unlike other cellular organelles, mitochondria contain their own DNA – mitochondrial DNA (mtDNA). Mitochondrial DNA (mtDNA) carries important genetic information concerning cellular metabolism and the generation of energy. It has been suggested that mitochondria and mtDNA could be of significance during early embryo development [Fragouli et al., 2015].

Mitochondria are unique compared to other organelles in cells in that they contain one or more copies of their own genome. The mitochondrial DNA (mtDNA) is circular and composed of 16.6 kb of double stranded DNA. Genes encoded by this DNA molecule have direct roles in cellular metabolism, producing subunits of several complexes with key roles within the Electron Transport Chain (ETC) [Anderson et al., 1981]. Complexes encoded by the mitochondrial genome, along with other ETC components, are situated in the inner mitochondrial membrane and are vital for the production of ATP in the cell. Additionally, mitochondrial DNA (mtDNA) encodes some of the components of the organelle’s transcriptional and translational machinery including 22 tRNAs and 2 rRNAs, with the remainder being encoded by the nuclear genome [Anderson et al., 1981]. It has been shown that cells are capable of redistributing their mitochondria so as to replace damaged organelles, and adjust to variation in intracellular energy requirements [Palmer et al., 2011].

Mitochondrial DNA (mtDNA) replication is strictly down–regulated from the fertilized oocyte through the preimplantation embryo. It is likely that there is an association between the fitness of mitochondria and their ability to support normal cellular function. Oocytes are greatly enriched in the number of mitochondria as they support essential developmental processes such as oocyte maturation and embryonic development, while their replication is deferred until gastrulation. At the blastocyst stage, the onset of mitochondrial DNA (mtDNA) replication is specific to the trophectodermal cells. The inner cell mass cells restrict mitochondrial DNA (mtDNA) replication until they receive the key signals to commit to specific cell types. However, it is necessary to determine whether somatic cells reprogrammed through somatic cell nuclear transfer, induced pluripotency or fusion to an ESC are able to regulate mtDNA replication so that they can be used for patient–specific cell therapies and to model disease [Justin C. St. John, 2010].

The mitochondrion exists within the cytoplasm of nearly all eukaryotic cells. Along with mediating processes such as steroidogenesis, apoptosis, homeostasis and cell division, mitochondria are also essential generators of ATP through the process of Oxidative Phosphorylation (OXPHOS) which takes place within the electron transfer chain (ETC). OXPHOS, unlike any other cellular process, is highly dependent on the expression of proteins encoded by the mitochondrial genome [mitochondrial DNA (mtDNA)] and chromosomally encoded genes. Although the transmission of mitochondrial DNA (mtDNA) is dependent on the mitochondrion as its vehicle, both the mitochondrion and mitochondrial DNA (mtDNA) should be considered as independent entities that co–exist, just as the mitochondrion co–exists within the cytoplasm of a cell but its origins are bacterial [Cavalier–Smith, 2009]. The role played by the mitochondrial genome in reproductive biology has been largely ignored. However, since the advent of more sophisticated reproductive technologies, such as nuclear transfer, its importance is becoming increasingly recognized, especially as mixed and diverse populations of mitochondrial DNA (mtDNA) co–existing together could severely affect cellular function and thus offspring survival and quality of life [Justin C. St. John, 2010].

The mitochondrion and the mitochondrion configuration

Mitochondria are involved in the regulation of multiple essential cellular processes, such as apoptosis, amino acid synthesis, calcium homeostasis, and the generation of energy in the form of ATP via the process of Oxidative Phosphorylation (OXPHOS) [May–Panloup et al., 2005; St John et al., 2010; Bentov and Casper, 2013; Tilly and Sinclair, 2013]. For this reason mitochondria are considered as the principal cellular power houses.

Mitochondria exist in various topographical forms, which reflect the type and functions of the cells they serve and thrive off. In maturing oocytes and early cleavage stage embryos, they tend to be oval and spherical while in the differentiating and mature cells, they adopt a more elongated configuration [Sathananthan et al., 2002]. Furthermore, the density of mitochondria per cell is often indicative of the cell’s requirements for ATP production [Moyes et al., 1998]. Consequently, cells such as neurons and muscles tend to have higher densities, though these are considerably fewer than the metaphase II stage oocyte, which possesses between 150 000 and 300 000 mitochondrial profiles [Jansen and De Boer, 1998]. The mtDNA genome encodes 13 polypeptides of the ETC.

What is Mitochondrial DNA (mtDNA)?

The circular, double–stranded mitochondrial DNA (mtDNA) genome is ∼16.6 kb in size and resides within the inner membrane of the mitochondrion. It consists of a Heavy (H) and a Light (L) strand, which encode 13 of the polypeptides contributing to Complexes I, III, IV and V of the Electron Transfer Chain (ETC). The remaining genes and those of Complex II are derived from chromosomal DNA [Anderson et al., 1981]. Specifically, mitochondrial DNA (mtDNA) codes seven subunits of NADH dehydrogenase (Complex I), one subunit of cytochrome c reductase (Complex III), three subunits of cytochrome c oxidase (Complex IV) and two subunits of the ATP synthase (Complex V; Fig. 1). mtDNA also encodes some of its own transcriptional and translational machinery, i.e. 22 tRNAs and 2 rRNAs, emphasizing the semi–autonomous nature of this genome as these processes are also dependent on chromosomally encoded genes. Furthermore, the chromosomally encoded transcription and replication factors interact with the only non–coding region of the genome, the displacement loop (D–loop). Otherwise, the mtDNA genome contains no introns interspersed between the coding regions with some of the coding regions even overlapping each other, namely ATPase 6, ATPase 8, ND4 and ND4L [Anderson et al., 1981]. Furthermore, some of the other genes do not possess termination codons, as these are generated through post–transcriptional polyadenylation [Ojala et al., 1981].

Mitochondrial DNA (mtDNA) nucleoid

Mitochondrial DNA (mtDNA) replication is mediated by several nuclear–encoded transcription and replication factors, which along with mitochondrial DNA (mtDNA) are packed within a space of ∼70 nm in diameter, forming the mitochondrial nucleoid [Chen and Butow, 2005]. There are ∼30 nucleoid proteins conserved between different species [Chen and Butow, 2005]. Some of these are responsible for the maintenance and packaging of mitochondrial DNA (mtDNA), whereas the others have, in addition to their role in mitochondrial DNA (mtDNA) maintenance, a metabolic function [Kucej and Butow, 2007]. A structural model has been suggested where the nucleoid is composed of a central core and a peripheral region where the central core proteins are involved in nucleic acid synthesis, whereas translation and protein assembly occur in the peripheral region [Bogenhagen et al., 2007]. The number of mitochondrial nucleoids per mitochondrion ranges from 1 to 10 [Satoh and Kuroiwa, 2004]. There is a dispute about mtDNA copy number as the estimated copies/mitochondrion ranged from 1 to 15 [Satoh and Kuroiwa, 2004], whereas others report that there are between 2 and 8 copies of mtDNA in each nucleoid [Legros et al., 2004].

Both mitochondrial transcription factor A [TFAM; Ekstrand et al., 2004] and ATAD3 [He et al., 2007] have been proposed to provide either the key–linking protein or the backbone to the nucleoid. ATAD3 (also known as TOB3) is an AAA domain protein, consisting of two subdomains, ATAD3f1 and ATAD3f2 [Wang and Bogenhagen 2006; He et al., 2007]. Although it is tightly associated with the inner mitochondrial membrane, its relationship with mtDNA is controversial. One study suggested that ATAD3 is attached to mitochondrial DNA (mtDNA) at the D–loop and it is involved in mitochondrial nucleoid formation and segregation [He et al., 2007]. However, immunofluorescence and protease susceptibility studies indicated that ATAD3 does not interact directly with mitochondrial DNA (mtDNA) [Bogenhagen et al., 2007]. On the other hand, TFAM belongs to the High Mobility Group (HMG) box family of genes, which consists of a subset of genes characteristically associated with chromosomal DNA packaging, such as histones [Bianchi and Agresti, 2005]. It has a molecular weight of ∼25 kDa [Parisi and Clayton, 1991] and has three functions, namely binding, unwinding and bending of mitochondrial DNA (mtDNA).

The first function suggests a direct relationship between TFAM and mtDNA levels whereby over-expression of TFAM leads to an increase in mitochondrial DNA (mtDNA) copy number [Ekstrand et al., 2004; Pohjoismaki et al., 2006]. Furthermore, the molar ratios of TFAM and mtDNA are ∼900–1000:1 [Ekstrand et al., 2004] again suggesting that mtDNA is extensively packaged with TFAM [Alam et al., 2003]. Indeed, the role of TFAM as an mtDNA–packaging molecule has been further supported by more recent studies demonstrating its co–localization to mitochondrial DNA (mtDNA) through immune–electron microscopy [Kaufman et al., 2007]. The latter two functions suggest an important role in mtDNA transcription further supporting it functional similarity to other HMG proteins [Fisher et al., 1992; Takamatsu et al., 2002; Bianchi and Agresti, 2005; Kaufman et al., 2007]. Furthermore, up–regulation of TFAM expression results in an increase in expression of the mitochondrial–specific polymerase gamma (Polg) [Ekstrand et al., 2004] implying a role in initiation of mitochondrial DNA (mtDNA) replication.

POLG mediates the replication, recombination and repair of mitochondrial DNA (mtDNA) [Grazieswicz et al., 2005], thus demonstrating its essential role in mitochondrial DNA (mtDNA) maintenance [Spelbrink et al., 2000]. Although it is capable of replicating mitochondrial DNA (mtDNA), it cannot replicate chromosomal DNA, perhaps resulting from its similarities to other members of the family A group of DNA polymerases. These include Escherichia coli DNA polymerase I and the T7 DNA polymerase [Fridlender and Weissbach 1971; Fridlender et al., 1972; Beese et al., 1993a, b]. In addition, a recent study has suggested that POLGB is the key factor that determines mitochondrial DNA (mtDNA) copy number within the nucleoid as it is required for D–loop synthesis [Di Re et al., 2009].

Two other factors play a key role in mitochondrial DNA (mtDNA) replication. The helicase, Twinkle, was originally identified in association with autosomal dominant progressive ophthalmoplegia, a mitochondrial disorder characterized by mitochondrial DNA (mtDNA) deletions [Spelbrink et al., 2001]. It shares a structural similarity to the C–terminal helicase part of bacteriophage T7 gene 4 protein which exhibits both helicase and primase activities [Bernstein and Richardson, 1989; Spelbrink et al., 2001]. The mitochondrial single-stranded DNA–binding protein (mtSSB) mediates the unwinding of mitochondrial DNA (mtDNA) through its physical interaction with Twinkle [Korhonen et al., 2003], which co–localizes with both TFAM and mtSSB. These proteins act not only as structural components but also stabilize mitochondrial DNA (mtDNA) during replication [Garrido et al., 2002].

Mitochondrial DNA (mtDNA) replication

Both the asymmetric and the coupled leading– and lagging–strand synthesis models have been described to explain mitochondrial DNA (mtDNA) replication. The asymmetric model has been the traditional approach to understanding how mitochondrial DNA (mtDNA) replication is mediated. It is initiated from the origin of H–strand replication (OH), located within the D–Loop region, where TFAM binds to the enhancer of the Light–Strand Promoter (LSP) and induces structural changes that expose the promoter region to the mitochondrial–specific RNA polymerase. This allows an RNA primer to be generated, which is used by POLGA to initiate mitochondrial DNA (mtDNA) replication. Mitochondrial DNA (mtDNA) replication then continues two–thirds round the genome to the origin of L–strand replication (OL). L–strand synthesis then proceeds in the opposite direction. The coupled leading– and lagging–strand synthesis model proposes that both H– and L–strands are replicated bidirectionally from the same initiation cluster site [Yasukawa et al., 2005]. This mechanism is thought to occur in addition to the asymmetric model, but is also typical of cells repopulating mitochondrial DNA (mtDNA). The coupled leading– and lagging–strand synthesis model has now been refined to include the presence of replication intermediates which allow gaps within the replicating mitochondrial DNA (mtDNA) on the lagging–stand to be filled to complete the replicon [Yasukawa et al., 2005]. It has more recently been proposed that replication is driven from several origins of replication within the vicinity of the OH. However, to date, there has been little resolution as to which model is the most appropriate with entrenched views being expressed by the two opposing groups [Bogenhagen and Clayton, 2003; Holt and Jacobs, 2003].

An important question that remains to be determined is how the expression of the key genes associated with mitochondrial DNA (mtDNA) replication is mediated. Recent analysis of the promoter region of TFAM produced inconclusive data as to whether its expression was regulated by DNA methylation [Choi et al., 2004; Gemma et al., 2009]. Nevertheless, more recent analysis of PolgA has however suggested that specific CpG islands within exon 2 of this gene are targeted [Oakes et al., 2007] and could thus account for changes in PolgA expression during spermatogenesis. This approach would certainly compliment the observation of an 8– to 10–fold decrease in the number of mtDNA molecules as these cells differentiate [Larsson et al., 1997]. This reduction in TFAM expression and mtDNA copy number would match other reports that indicate an association between TFAM and mtDNA copy number, most likely restricting it to a packaging role in this instance. This suggests that replication could then be regulated by either a conformational or a numerical change to TFAM that would result in exposure of OH or the broader initiation cluster coupled with a change in the DNA methylation status of PolgA.

The mitochondrial DNA (mtDNA) replication in the pluripotent preimplantation embryo and the mitochondrial DNA (mtDNA) replication and embryonic differentiation

The mature oocyte is among the cell types with the highest content for both mitochondria and mitochondrial DNA (mtDNA) [May–Panloup et al., 2005]. Oocyte mitochondrial replication begins during fetal development with cells of the oogonia containing approximately 200 mitochondria. Replication continues in synchrony with maturation, so that just before fertilization an oocyte arrested at metaphase II contains approximately 100,000 mitochondria and between 50,000 and 550,000 copies of the mitochondrial DNA (mtDNA) [May–Panloup et al., 2005; Chen et al., 1995; Motta et al., 2000; Cummins 2000; Steuerwald et al., 2000; Reynier et al., 2001].

Early embryo development consists of a series of mitotic divisions and other cellular events requiring a supply of energy, principally in the form of ATP generated by mitochondria [Van Blerkom 2001]. During the blastocyst stage, ATP production is up-regulated in order to satisfy the energetic requirements of further differentiation and development, and to support processes required for implantation.

It was established that the embryos inherit mitochondria (and thus mtDNA) exclusively from the population found in the oocyte just prior to fertilization. Data from quantification of mitochondrial DNA (mtDNA) in cleavage stage embryos suggests that amounts remain stable during the first three days of preimplantation development [May–Panloup et al., 2005; Steuerwald et al., 2000; Reynier et al., 2001; Barritt et al., 2002; Lin et al., 2004; Chan et al., 2005]. Significant replication of mitochondrial DNA (mtDNA) is not thought to be initiated until after the embryo has undergone the first cellular differentiation into Trophectoderm (TE) and Inner Cell Mass (ICM) and has become a blastocyst [St John et al., 2010; Eichenlaub–Ritter et al., 2011]. Preimplantation embryo development is a dynamic and energy demanding process. Early embryos require adequate energy levels so that they can successfully progress through each cell division. Existing data suggest that correct oocyte mitochondrial function and mitochondrial DNA (mtDNA) gene expression are essential during these early stages of life. Specifically, an association has been shown between the ATP content of oocytes, the developmental potential of an embryo, and the outcome of an In Vitro Fertilization (IVF) Treatment cycle [Van Blerkom et al., 1995].

Since mitochondrial functions are critical during the first few days of life, the embryologists were interested in carrying out a thorough investigation of mitochondrial DNA (mtDNA) in preimplantation embryos which had successfully reached the blastocyst stage of development. Specifically, the scientists examined the relationship between blastocyst mitochondrial DNA (mtDNA) content, female patient age, embryo chromosome status, viability and implantation potential. Additionally, the experts attempted to shed light on the stage of preimplantation development during which mitochondrial DNA (mtDNA) replication is first up–regulated, with the potential to increase the mtDNA content of individual cells. As well as relative quantification of mtDNA, a detailed analysis of the mitochondrial genome was undertaken, searching for mutations, deletions and polymorphisms [Fragouli et al., 2015].

2. The study “Nuclear and mitochondrial DNA in blastocoele fluid and embryo culture medium: evidence and potential clinical use”, written by Elizabeth R. Hammond, Andrew N. Shelling and Lynsey M. Cree in close focus.

2.1. Embryo development: accumulation of blastocoele fluid

Blastocyst biogenesis occurs over several cell cycles during the preimplantation period comprising the gradual expression and membrane assembly of junctional protein complexes which distinguish the outer Epithelial Trophectoderm (TE) cells from the Inner Cell Mass (ICM). Cavitation takes place at Day 4 of development. At this time, the cells of the embryo begin to differentiate into the inner cell mass and trophectoderm lineages. Ion pumps actively transport sodium ions to the blasolateral side of the trophectoderm epithelium leading to the accumulation of blastocoele fluid. This fluid coalesces and expands to occupy most of the volume of the blastocyst [Elizabeth R. Hammond et al., 2016]. The degree of blastocyst expansion, along with the number of cells and cohesion of the inner cell mass and trophectoderm, is an important parameter for the morphology assessment of the blastocyst on Day 5 or 6 of development [Gardner et al., 2000]. The blastocoele cavity is dynamic; however, often showing repeated shrinkage and expansion prior to hatching [Mio et al., 2011].

2.2. Pre–vitrification artificial blastocoele collapse performance

Artificial blastocoele collapse is routinely performed during Vitrification, which is a commonly used method of Cryopreservation to store surplus embryos for future use. The blastocoele cavity is collapsed prior to Vitrification to prevent ice crystal formation and improve embryo survival following Cryopreservation [Mukaida et al., 2006]. Artificial blastocoele collapse can be performed by microsuction, microneedle puncture, laser pulse or repeated pipetting of the embryo, and has produced good success rates in conjunction with Vitrification [Kenichiro Hiraoka, 2004; Li et al., 2014; Wong et al., 2014]. Following this artificial collapse, the blastocoele fluid disperses into the surrounding culture medium. The blastocoele fluid can be isolated from the embryo using a procedure termed blastocentesis, and can then undergo downstream analysis [Elizabeth R. Hammond et al., 2016]. Blastocentesis is a method where an ICSI injection pipette is used to firstly create a micropuncture though the mural trophectoderm opposite the inner cell mass, and is then used to aspirate the fluid until the blastocyst is fully collapsed around the pipette [D’Alessandro et al., 2012]. Around 0.01 µl of blastocoel fluid is typically isolated, highlighting the very small amount of fluid available for analysis [Magli et al., 2016].

Blastocentesis. Blastocoele fluid is isolated by blastocentesis. An Intracytoplasmic Sperm Injection (ICSI) injection pipette is used to puncture though the mural trophectoderm or opposite the inner cell mass. The blastocoele fluid should be aspirated until the blastocyst collapses around the pipette [Elizabeth R. Hammond et al., 2016].

There have been recent reports that the blastocoele fluid contains nuclear DNA. Following the isolation of blastocoele fluid by blastocentesis, DNA within the fluid can be successfully purified, amplified and undergo downstream genetic analysis for some embryos [Palini et al., 2013; Gianaroli et al., 2014; Tobler et al., 2015; Magli et al., 2016]. In a preliminary study of 29 embryos, multi–copy genes were targeted to improve the chances of successful amplification from this limited amount of genetic material. This study showed successful amplification of TBC1D3, a multi–copy gene on chromosome 17, by quantitative PCR in 90% of blastocoele fluid samples tested [Palini et al., 2013]. For the samples that successfully amplified TBC1D3, another gene called TSPY1, which is a multi–copy gene on the Y chromosome, was targeted for amplification. Initial results suggested that 65% of the blastocoele fluid samples were from male embryos, demonstrating that the genetic material may have the potential to be used for sex determination. Based on these promising early results, and with further validation and confirmation, the blastocoele fluid therefore has the potential to provide an alternative strategy for embryo sex determination for preventing the inheritance of X–linked disorders. However, the current requirement to target multi-copy genes due to reduced sensitivity limits this type of approach for use with most other inherited disorders that are caused by mutations in single copy genes [Gutierrez–Mateo et al., 2009].

For increased sensitivity, the nuclear DNA isolated from blastocoele fluid can undergo a targeted amplification or whole genome amplification before downstream genetic analysis is performed. The proportion of blastocoele fluid samples that successfully undergo whole genome amplification and produce detectable levels of DNA ranges between 40 and 98% [Palini et al., 2013; Gianaroli et al., 2014; Tobler et al., 2015; Magli et al., 2016].
For trophectoderm biopsy, there is a generally accepted low failure rate of whole genome amplification, but it may occur due to the presence of lysed cells, absence of cells, or sample processing errors [Tobler et al., 2015]. Therefore, this procedure may need further optimization when applied to the DNA in blastocoele fluid. The lower levels of positive amplification compared with trophectoderm cells suggest that there may be an absence of DNA within some blastocoele fluid samples [Elizabeth R. Hammond et al., 2016]. For those samples that do contain DNA, it may be of lower quantity and quality, which may result from DNA fragmentation occurring within the blastocoele cavity [Gianaroli et al., 2014; Tobler et al., 2015; Magli et al., 2016]. It should be noted however that the use of whole genome amplification for the very low level of DNA within the blastocoele fluid may be particularly susceptible to false positive results. As with single cells, this low level of DNA may be prone to uneven amplification and allele dropout [Huang et al., 2015].

If DNA amplification methodologies can be optimized to overcome the limitations posed by the low quantity and quality of the genetic material in the blastocoele fluid, the important question regarding the origin of the DNA remains. Does the genetic material found to be present in the blastocoele fluid reflect the genome of the embryo? As an alternative strategy for Preimplantation Genetic Screening (PGS), studies have aimed to test whether the amplifiable DNA present in the blastocoele fluid produces array Comparative Genomic Hybridization (aCGH) results concordant with DNA extracted from Polar Body, blastomere or trophectoderm biopsy [Elizabeth R. Hammond et al., 2016]. The earliest attempts proved to be largely unsuccessful, with only 25–33% of the blastocoele fluid DNA producing concordant results with trophectoderm biopsy or the remaining embryo, if it was available for analysis, although fewer than 10 embryos were analyzed in each study [Perloe et al., 2013; Poli et al., 2013]. A more recent study compared the blastocoele fluid DNA with previous results obtained from either Polar Body or blastomere biopsy, in addition to a separate chromosomal analysis from trophectoderm biopsy for 51 embryos. Of those blastocoele fluid samples that underwent successful whole genome amplification (77%), the ploidy condition was concordant with either the Polar Body or blastomere biopsy in 95% of samples. Compared with trophectoderm biopsy, the blastocoele fluid DNA showed 97% concordance [Gianaroli et al., 2014]. It should be noted however that such a high concordance, especially with respect to Polar Body biopsy, may not be expected due to the presence of mitotic errors, paternal aneuploidy, resolution of aneuploidy or mosaicism confined to either the trophectoderm or inner cell mass by the blastocyst stage of development [Taylor et al., 2014]. In a later study by the same group, a larger cohort of 116 blastocysts underwent both blastocentesis and trophectoderm biopsy, having also had a previous analysis from either Polar Body or blastomere biopsy [Magli et al., 2016]. The rate of DNA detection and whole genome amplification was improved (82%) and the concordance with trophectoderm biopsy remained constant at 97% [Magli et al., 2016]. Given the high level of concordance, the results from these later studies are promising and support the potential use of the blastocoele fluid DNA for Preimplantation Genetic Screening (PGS).

The conflicting results of these aforementioned studies occurred despite both laboratories using similar methodologies, including the same method for performing blastocentesis, whole genome amplification and array Comparative Genomic Hybridization (aCGH) [Gianaroli et al., 2014; Tobler et al., 2015; Magli et al., 2016]. Inconsistent concordance levels could suggest that there are other differences in the two laboratories, such as subtle variations in the way that the methodologies were implemented, modifications in data analysis and interpretation, or simply dissimilarities in the cohorts of embryos used. Ultimately, due to the lower levels of whole genome amplification success (63–82%) compared with trophectoderm biopsy, and difficulties isolating the blastocoele fluid, these studies had a limited sample size which reduces the power of the analysis. Therefore, additional studies, with larger sample sizes, are needed to assess the potential clinical use of blastocoele fluid DNA for Preimplantation Genetic Screening (PGS) and Preimplantation Genetic Diagnosis (PGD). Another important consideration is whether any contaminating genetic material arises during blastocoele isolation and analysis. Ultimately DNA fingerprinting technology with the ability to detect low levels of complex mixtures can be utilized to address this issue [Elizabeth R. Hammond et al., 2016].

According to Elizabeth R. Hammond et al. (2016) scientific investigation, there are two proposed mechanisms of DNA release and potential sources of DNA contamination in embryo culture medium at the cleavage stage and in blastocoele fluid and culture medium during blastocyst development. (I) DNA may be released from the cleavage–stage embryo by cellular fragmentation, apoptosis and necrosis in fragmentation bodies and during cell cleavage. Apoptotic and necrotic mechanisms may result in the release of fragmented DNA. The DNA must pass through the zona pellucida, and may be in the form of extracellular vesicles for cell communication. Maternal contamination may arise from cumulus cells that remain adhered to the zona pellucida following denudation prior to ICSI and following IVF insemination. It is also possible that maternal contamination may arise from the Polar Bodies following their extrusion from the oocyte. Paternal contamination may arise from sperm that adhere to the zona pellucida following IVF insemination. The possibility of paternal contamination is thus removed when analyzing culture medium from ICSI inseminated embryos. Low levels of genetic material may contaminate the culture medium following manufacture, or alternatively, contamination may arise during the culture period from the surrounding environment. (B) During blastocyst development, DNA may be released into the blastocoele fluid and culture medium from cells of the inner cell mass and trophectoderm by cell lysis, apoptosis or shedding of cellular debris. Nuclei from lysed cells may undergo deterioration and release whole chromosomes into the blastocoele fluid and the culture medium. Cells undergoing apoptosis as part of a controlled elimination process may result in the release of fragmented DNA. DNA may be packaged in extracellular vesicles for communication between cells of the trophectoderm and inner cell mass. Potential sources of contamination may arise from cumulus cells, sperm and Polar Bodies that persist to the blastocyst stage. The culture medium may also become contaminated following manufacture or during the culture period [Elizabeth R. Hammond et al., 2016].

While the presence of nuclear DNA has been investigated, to date no study has assessed the mitochondrial DNA (mtDNA) in the blastocoele fluid [Elizabeth R. Hammond et al., 2016]. The mitochondrial DNA (mtDNA) is a double stranded 16.6 kb genome that encodes for several subunits of the electron transport chain, making it directly involved in cellular oxidative phosphorylation. The mitochondrial DNA (mtDNA) of the embryo is maternally inherited, with the oocyte harbouring around 500 000 copies [Barritt et al., 2002; Van Blerkom, 2011]. The amount of mitochondrial DNA (mtDNA) remains stable throughout the cleavage stages with mitochondrial DNA (mtDNA) replication beginning at the blastocyst stage or post–implantation [Cree et al., 2008; St John et al., 2010]. Recently, it has been reported that elevated levels of mitochondrial DNA (mtDNA), measured in trophectoderm biopsies, is associated with aneuploidy and decreased implantation potential for euploid embryos [Diez–Juan et al., 2015; Fragouli et al., 2015]. From a biological standpoint, the distribution of the mitochondrial DNA (mtDNA) between different cells of the trophectoderm and inner cell mass during progression to the blastocyst stage is unknown. An investigation into the levels of mitochondrial DNA (mtDNA) in the blastocoele fluid in relation to aneuploidy and implantation potential may be warranted to assess whether a similar relationship exists for blastocoele fluid compared with trophectoderm cells. Due to the high number of copies in the preimplantation embryo, mitochondrial DNA (mtDNA) analysis from the blastocoele fluid may prove less challenging compared with that of nuclear DNA. It is possible that quality control mechanisms exist whereby the blastocyst may eliminate mutated mitochondrial DNA (mtDNA) into the blastocoele fluid, or alternatively levels of both wild–type or mutated mitochondrial DNA (mtDNA) in the blastocoele fluid may be altered when the embryo is aneuploid. These aspects of mitochondrial segregation should be studied to further understand the biology of the preimplantation embryo [Elizabeth R. Hammond et al., 2016].

2.3. Embryo culture medium

Recent evidence suggests that DNA can be detected in spent embryo culture medium [Stigliani et al., 2013, 2014; Assou et al., 2014; Wu et al., 2015]. In addition, miRNAs have been detected in spent blastocyst medium. Two miRNAs, thought to be of trophectoderm origin, have been identified as potential candidate biomarkers of implantation in euploid embryos [Capalbo et al., 2016]. The origin of the DNA within spent embryo culture medium however has not been fully characterized, and it is unclear whether the DNA originates from the embryo. It is possible that the embryo may release DNA into the surrounding culture medium, under a similar mechanism that occurs for blastocoele fluid. However, compared with blastocoele fluid, there is a higher risk that contaminating DNA may be present within the spent culture medium. This could occur for a number of reasons, namely nucleic acid contamination from the human serum albumin component of the culture medium, maternal or paternal contamination from cumulus cells, Polar Bodies or sperm, or contaminating DNA arising from the surrounding environment during the culture period mitochondrial DNA (mtDNA) [Elizabeth R. Hammond et al., 2016].

Both nuclear DNA and mitochondrial DNA (mtDNA) have been detected in the spent culture medium for embryos during preimplantation development [Stigliani et al., 2013, 2014; Assou et al., 2014; Wu et al., 2015]. In a preliminary analysis, spent culture medium was analyzed for the presence of DNA in a cohort of Day 2 (166 embryos) and Day 3 (634 embryos) cleavage stage embryos. It was found that DNA could be detected in 99% of the spent embryo culture medium, indicating that DNA within the culture medium was a common phenomenon [Stigliani et al., 2013]. In a subset of 326 samples, it was determined that nuclear DNA and mtDNA were present in 63 and 99% of the samples, respectively. These results show that there is higher mtDNA content compared with the nuclear DNA, which may reflect the high mtDNA copy number in the preimplantation embryo. This could also be the case for contaminating tissue such as cumulus cells, although it should be noted that the mtDNA copy number of cumulus cells is significantly lower (10 copies) compared with cells of the preimplantation embryo [Cree et al., 2015]. The study also found that a higher mtDNA copy number was associated with increased levels of embryo fragmentation, which may reflect a loss of cytoplasm from the embryo following fragmentation. Additionally, this relationship was particularly apparent for patients aged 35 years and older, showing that female age appeared to affect the level of mtDNA detected [Stigliani et al., 2013].

A later study by the same group analyzed the mtDNA/nuclear DNA ratio in 605 spent Day 3 culture medium samples in relation to blastocyst quality. Converse to the first study, a higher mtDNA/nuclear DNA ratio was predictive of blastulation for good quality cleavage stage embryos with mild levels of fragmentation, showing that the relationship between embryo quality and the level of mitochondrial DNA (mtDNA) detected in spent culture medium remains unclear [Stigliani et al., 2014]. Additionally, it was found that a higher mtDNA/nuclear DNA ratio was associated with implantation following Day 3 transfer in a cohort of 94 embryos [Stigliani et al., 2014]. While the relationship to embryo quality does indicate that the embryo itself has a role to play in the quantity of DNA present in the culture medium, it does not definitively prove that the genetic material observed is in part, or exclusively, released from the embryo. Taken together, the results of these two studies suggest that the presence of nuclear DNA and mitochondrial DNA (mtDNA) in the embryo culture medium is a common phenomenon occurring for cleavage stage embryos, and that the mitochondrial DNA (mtDNA) content may be related to fragmentation levels, blastulation and implantation. However, it is necessary for this to be analyzed in relation to control droplets of medium that have not been exposed to embryos, to assess the presence of background nucleic acid contamination in the culture medium system. Further, no study has assessed the levels of nuclear DNA and mitochondrial DNA (mtDNA) in Day 5 or 6 culture medium and compared this to Day 3 culture medium in a sequential medium system. An experiment like this should be performed in order to characterize the DNA profile occurring throughout preimplantation development. A critical study should also investigate the effect of different commercial culture systems, as continuous culture to the blastocyst stage may prove to be more applicable for the assessment of nucleic acids in spent embryo culture medium, due to DNA accumulation throughout preimplantation development [Elizabeth R. Hammond, 2016].

2.4. Possible mechanisms of DNA release and potential sources of contamination

Determining the origin of the DNA within blastocoele fluid and spent embryo culture medium is of primary importance. This is necessary to assess its potential for use in a clinical setting, because the extremely low levels
of DNA present are prone to false positive amplification. Future studies should employ rigorous controls to further validate these methods of assessment. This should include culture media controls that are cultured alongside embryos in an identical manner, but do not come into contact with embryos, and no template negative controls following all amplification procedures. If the genetic material present within the blastocoele fluid does originate from cells of the blastocyst, then the mechanisms of release need to be determined [Elizabeth R. Hammond, 2016]. The observation that full karyotypes can be obtained from the blastocoele fluid DNA may indicate that whole cell nuclei may be present in the cavity. It is thought however that whole cells are not the source of the DNA isolated by blastocentesis, as the use of a 5.5 µM ICSI pipette would not allow cells to enter the pipette during isolation [Tobler et al., 2015]. Partially lysed cells may still be isolated, although this has not been visually reported in any of the blastocentesis studies. It is unlikely that blastocentesis itself promotes the lysis of embryonic cells, due to the procedure being performed gently, although damaged or partially lysed cells may become dislodged and isolated when the fluid is withdrawn by the pipette. Conversely, any genetic material that is released into the culture medium from the embryo must pass through the zona pellucida, with the only exception being for blastocysts that have begun the process of hatching where cells of the trophectoderm begin to herniate from the zona pellucida in preparation for implantation. It is thought that the zona pellucida glycoprotein membrane permits DNA and other molecules such as metabolites through relatively unhindered [Stigliani et al., 2013]. Supporting this, studies have shown that metabolic profiles for embryos can be obtained from spent culture medium [Kirkegaard et al., 2014; Bellver et al., 2015]. Given that the DNA studies performed to date have only used cohorts of Intracytoplasmic Sperm Injection (ICSI) inseminated embryos, it is possible that the Intracytoplasmic Sperm Injection (ICSI) Injection site may produce an outlet for minute amounts of embryo cytoplasm prior to the first cleavage, and for cellular fragments and debris during subsequent cleavages. To investigate this possibility, In Vitro Fertilization (IVF) embryos should also be analyzed for levels of DNA in the embryo culture medium [Elizabeth R. Hammond, 2016].

In the majority of studies, Intracytoplasmic Sperm Injection (ICSI) inseminated embryos have been investigated thus far in order to remove the possibility of paternal sperm contamination [Stigliani et al., 2013, 2014; Wu et al., 2015]. Prior to Intracytoplasmic Sperm Injection (ICSI), the cumulus cells are stripped using hyaluronidase, however some tightly associated cumulus cells may remain bound to the zona pellucida during preimplantation development. The presence of cumulus cells throughout preimplantation development allows for the possibility of maternal contamination within the culture medium. Indeed, the incidence of maternal contamination may be reduced by thoroughly denuding the oocytes prior to Intracytoplasmic Sperm Injection (ICSI) in order to remove all the adhering cumulus cells. However, prolonged exposure to hyaluronidase and over manipulation may be detrimental to the developmental competence of the oocyte. Alternatively, cumulus cells could undergo an additional denudation at Day 3 of culture, prior to medium sampling at the blastocyst stage in a sequential medium system. Another potential source of maternal contamination is the Polar Bodies, as they may release their genetic material into the surrounding culture medium following their extrusion from the oocyte, although the amount of nuclear genetic material that they would contribute may be negligible [Ottolini et al., 2015].

In order to validate the embryonic origin of the DNA in the culture medium, it will be necessary to perform DNA fingerprinting analysis. This would involve comparing maternal tissue, such as cumulus cells or blood, to the culture medium of individual embryos [Elizabeth R. Hammond, 2016]. For blastocentesis, one study aimed to compare the DNA fingerprints of the blastocoele fluid with that of the embryo, but found that the number of loci successfully amplified for blastocoele fluid was insufficient to allow for an accurate analysis [Poli et al., 2013]. The low yield and compromised integrity of the blastocoele fluid DNA and embryo culture medium DNA will make it challenging to discriminate mixed profiles of DNA; however, these investigations are necessary in order to validate the origin of the DNA for downstream genetic testing and interpretation [Elizabeth R. Hammond, 2016].

The DNA isolated from blastocoele fluid and embryo culture medium may be fragmented and degraded, possibly due to cell death processes occurring throughout development [Elizabeth R. Hammond, 2016]. The arresting early cleavage stage embryo undergoes cell cycle arrest rather than cell death; however, fragmentation is associated with both apoptosis and necrosis [Betts and Madan, 2008; Chi et al., 2011]. At the blastocyst stage, apoptosis is thought to be a normal developmental event and may be involved in the regulation of cell number [Hardy et al., 1989; Hardy et al., 2003]. Fragmenting nuclei and DNA appear following compaction, and apoptotic cells are detected in both the inner cell mass and trophectoderm, indicating a controlled elimination of cells as part of normal preimplantation development [Hardy et al., 1989, 2003; Spanos et al., 2002]. Therefore, it is likely that some of the DNA present in the blastocoele fluid may arise through these apoptotic processes and the DNA may be fragmented as a consequence [Elizabeth R. Hammond, 2016]. There is also evidence that aneuploid cells may be excluded from the embryo and end up in the blastocoele fluid as a process of aneuploidy resolution, although further studies are needed to confirm this [Tobler et al., 2015]. The high rate of whole genome amplification failure for the blastocoele fluid DNA may be due to highly fragmented DNA within the blastocoele cavity, in conjunction with the low quantity of DNA isolated, but this requires further characterization [Tobler et al., 2015]. The nuclear DNA isolated from the embryo culture medium also has a low–molecular weight, where cellular fragmentation may also play a role [Stigliani et al., 2013].

Conclusion

In order to reduce the number of embryos transferred, it is vital to have very efficient selection criteria to identify those embryos that are more likely to implant and develop into pregnancy. Moreover, it is also important to select spare embryos with the ability to implant for Cryopreservation and future transfer. Traditionally morphological criteria have been mostly used to determine which embryos are more suitable to implant and develop. The morphological criteria are based on the number of blastomeres (cleavage rate), size and shape of the blastomeres and the amount of anuclear fragments.

The development of Preimplantation Genetic Diagnosis (PGD) techniques has given many couples with a high risk of transmitting genetic pathology the chance to have children without the disease. Allowing biopsied blastomeres to multiply in vitro will increase the number of cells available for analysis and thus improve the results of the genetic study [Geber et al., 1995b]; moreover, Preimplantation Genetic Diagnosis (PGD) might be offered to a greater number of patients, increasing the range of indications. Also important is the fact that creating a hole in the zona pellucida (Assisted Hatching) might improve the implantation rates as previously demonstrated [Cohen et al., 1990]. One criticism of this technique is the need to micromanipulate all embryos. This point, however, is not very important nowadays as many IVF centres worldwide are performing Intracytoplasmic Sperm Injection (ICSI), Preimplantation Genetic Diagnosis (PGD) or Assisted Hatching, methods that are based on micromanipulation techniques.

To our knowledge, blastocoele fluid and embryo culture medium could be an alternative source of DNA for genetic analysis provided that there is some assurance that the DNA is of adequate quality, can undergo reliable amplification, and is largely of embryonic origin. Currently, there is a lot of preliminary evidence suggesting that blastocoele fluid and embryo culture medium assessment may have the potential to be utilized as a method of genetic analysis, for either direct genetic assessment of the embryo or as a biomarker of viability. Clinically, blastocentesis may represent an alternative approach to trophectoderm biopsy. Given that blastocentesis can occur as part of the Vitrification process, the embryo would undergo fewer manipulations, and cells would not need to be removed from the embryo itself. The genetic material present in the embryo culture medium may also have the potential to be used for genetic testing in a completely non–invasive manner. Further optimization of the methodologies will need to be performed however, along with larger study cohorts and a full assessment of DNA origin before the use of blastocoele fluid or spent embryo medium could be advised [Elizabeth R. Hammond, 2016].

Minimally invasive methodologies for the genetic assessment of embryos may represent an exciting development for the future of Preimplantation Genetic Screening (PGS) and Preimplantation Genetic Diagnosis (PGD). In recent years, there have been significant advancements in the molecular platforms that are used to screen embryos for aneuploidy and inherited disorders. These advancements have resulted in the ability to obtain a highly reliable genetic diagnosis from the limited amount of biological material available following embryo biopsy. With further research, it is possible that the genetic material present in blastocoele fluid or embryo culture medium may be utilized for the genetic assessment of embryos. If these methodologies accurately represent the genetics of the embryo and can be optimized to overcome the very limited amount of genetic material available for analysis, then they may offer the additional benefits of being minimally invasive to the embryo and less technical to perform.

The ability to screen embryos for aneuploidy or inherited disorders in a minimally invasive manner may represent a major advancement for the future of embryo viability assessment. Recent studies have demonstrated that both blastocoele fluid and embryo culture medium contain genetic material, which can be isolated and subjected to downstream genetic analysis. The blastocoele fluid may represent an alternative source of nuclear DNA for aneuploidy testing, although the degree to which the isolated genetic material is solely representative of the developing embryo is currently unclear. In addition to nuclear DNA, mitochondrial DNA (mtDNA) can be detected in the embryo culture medium. Currently, the origin of this nuclear and mtDNA has not been fully evaluated and there are several potential sources of contamination that may contribute to the genetic material detected in the culture medium. There is however evidence that the mtDNA content of the culture medium is related to embryo fragmentation levels and its presence is predictive of blastulation, indicating that embryo development may influence the levels of genetic material detected. If the levels of genetic material are strongly related to aspects of embryo quality, then this may be a novel biomarker of embryo viability. If the genetic material does have an embryo origin, the mechanisms by which DNA may be released into the blastocoele fluid and embryo culture medium are unknown, although apoptosis may play a role. While the presence of this genetic material is an exciting discovery, the DNA in the blastocoele fluid and embryo culture medium appears to be of low yield and integrity, which makes it challenging to study. Further research aimed at assessing the methodologies used for both isolating and analyzing this genetic material, as well as tracing its origin, are needed in order to evaluate its potential for clinical use. Should such methodologies prove to be routinely successful and the DNA recovered demonstrated to be embryonic in origin, then they may be used in a minimally invasive and less technical methodology for genetic analysis and embryo viability assessment than those currently available [Elizabeth R. Hammond, 2016].

It is unknown whether the genetic material released by the preimplantation embryo is involved in cellular communication. It has been recently reported that miRNAs are secreted into the blastocoele fluid, suggesting that donor cells exert a miRNA gene regulatory effect on recipient cells through the blastocoele cavity [Cimadomo et al., 2015]. It is possible that the DNA contained in the blastocoele fluid and embryo culture medium may be associated with extra–cellular vesicles as a mode of communication with other cells of the embryo or endometrial cells, and this should be investigated. A summary of the potential mechanisms by which genetic material may be released into the blastocoele fluid and embryo culture medium, and the potential sources of contamination are outlined for the cleavage stage embryo [Elizabeth R. Hammond, 2016].

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Embryo's Noninvasive Chromosome Screening: Main Issues. Perspectives

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