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Mitochondrial DNA Assessment to Determine Oocyte and Embryo Viability Elpida Fragouli, PhD1

Dagan Wells, PhD1,2

1 Reprogenetics UK, Oxford, United Kingdom 2 Nuffield Department of Obstetrics and Gynaecology, University of

Oxford, Oxford, United Kingdom

Address for correspondence Elpida Fragouli, PhD, Reprogenetics UK, Oxford Business Park North, Oxford OX4 2HW, United Kingdom (e-mail: [email protected]).

Abstract

Keywords

► ► ► ► ►

mitochondria mitochondrial DNA oocyte embryo biomarker

Mitochondria are the key regulators of multiple vital cellular processes, including apoptosis, calcium homeostasis, and the generation of ATP via the metabolic pathway known as oxidative phosphorylation. Unlike other cellular organelles, mitochondria contain one or more copies of their own genome (mtDNA). The mtDNA encodes a total of 13 genes with critical functions in cellular metabolism. The energy required to support the normal progress of preimplantation embryo development is provided in the form of ATP generated by the mitochondria. It has been suggested that cellular bioenergetic capacity and suboptimal levels of mitochondria-generated ATP could contribute to a variety of embryo developmental defects, and therefore adversely affect in vitro fertilization success rates. During this review, we discuss the role of mitochondria and their genome during oogenesis and early embryo development. We also assess whether analysis of mitochondria and their genome could be used as biomarkers to determine oocyte quality and embryo viability.

Mitochondria are organelles that serve as key regulators of multiple vital cellular processes, including apoptosis, calcium homeostasis, and the generation of ATP via the metabolic pathway known as oxidative phosphorylation (OXPHOS).1,2 A unique feature of mitochondria, compared with the other organelles of animal cells, is that they contain one or more copies of their own genome. For this reason, mitochondria could be considered as endosymbionts in the eukaryotic cell.3 The mitochondrial genome (mtDNA) is a circular doublestranded molecule, of 16.6 kb in size, located in the inner mitochondrial membrane. The human mtDNA encodes a total of 13 genes with important roles in cellular metabolism. Specifically, the genes are responsible for the construction of the subunits of Complexes I, III, IV, and V of the electron transport chain (ETC). All remaining genes for the abovementioned Complexes along with those of Complex II are encoded by the nuclear DNA.4 In addition to these 13 genes, the human mtDNA encodes for 22 tRNAs and 2 rRNAs, with the rest of its transcriptional and translational genes also being encoded by the nuclear genome.4

Issue Theme Ovarian Aging, from Bench to Bedside; Guest Editor, Emre Seli, MD

Another unusual feature of the mitochondria and their genome is that they are exclusively inherited from the mother to her children, a process that does not follow classic Mendelian laws.5 Any mitochondria derived from sperm that enter the cytoplasm of the oocyte do not persist.6 In most cases, the inherited mtDNA molecules are considered homoplasmic (i.e., genetically homogeneous). Clinically significant heteroplasmy, in which a proportion of the mtDNA molecules within the cell carry a defective gene affecting their function, is infrequent. The likelihood of heteroplasmy persisting through the generations is reduced by a mechanism regulating mitochondrial segregation during oogenesis known as the “genetic bottleneck.”7 This involves the elimination of the vast majority of the oocyte mtDNA molecules, with only a small population ultimately passed to the next generation.8 In most cases, this genetic bottleneck results in either the removal of mtDNA variants harboring mutations or their increase past a critical level of heteroplasmy, causing mitochondrial disease in the child.

Copyright © 2015 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel: +1(212) 584-4662.

DOI http://dx.doi.org/ 10.1055/s-0035-1567821. ISSN 1526-8004.

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Semin Reprod Med 2015;33:401–409

Mitochondrial DNA Assessment to Determine Oocyte and Embryo Viability

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It should be noted that the location of the mtDNA in the mitochondrion as well as its lack of histones leave it vulnerable to the deleterious effects of reactive oxygen species (ROS), and make it particularly prone to mutations.9 Findings obtained from the assessment of oocytes and embryos carrying mtDNA mutations suggest that the genetic bottleneck takes place toward the end of oogenesis and may be influenced by the presence of specific mutations.10 Preimplantation embryo development is a dynamic process, consisting of continuous mitotic divisions and other cellular events requiring an adequate supply of energy. This is mainly provided in the form of ATP generated by the mitochondria.11 Experiments in animal models (mouse) have demonstrated that after embryonic genome activation and blastocyst formation, the generation of ATP is upregulated so as to satisfy the energetic requirements of further differentiation and development, and to support processes required for implantation.12 Evidence for a direct relationship between the ATP content of human oocytes, the developmental potential of the resulting embryos, and the outcome of in vitro fertilization (IVF) cycles has also been presented.13 Despite the great progress made in the field of IVF and associated technologies in recent years, the success rates of infertility treatments remain relatively low, especially in women of advanced reproductive age. The presence of abnormal numbers of chromosomes (aneuploidy) in gametes and embryos is thought to be responsible for a significant number of the unsuccessful IVF cycles.14,15 The use of preimplantation genetic screening (PGS) enables the detection of lethal aneuploidy in embryos, allowing those with the correct number of chromosomes to be identified and transferred to the uterus. This strategy has been shown to improve clinical outcomes in recent randomized clinical trials.16,17 However, even the transfer of a chromosomally normal embryo of good morphology cannot guarantee that a viable pregnancy will result and thus it is clear that other factors, invisible to current cytogenetic and microscopic evaluations, play an important role in determining the potential of an embryo. It has been suggested that cellular bioenergetic capacity and suboptimal levels of mitochondria-generated ATP could contribute to a variety of embryo developmental defects, and therefore adversely affect IVF success rates.11 Hence, the concept of assessing oocyte quality and embryo viability by examining the mitochondria and their genome has been the subject of multiple recent investigations. Some of these studies and their findings will be discussed in the following sections of this review.

a comparatively small number of truncated cristae surrounding a high-electron density matrix.11,19 Nonetheless, oocyte mitochondria are able to generate ATP via the OXPHOS reaction, providing energy required from fertilization through to the blastocyst stage.13,20 As preimplantation development progresses, the mitochondria begin to mature, elongating, increasing the number of cristae, and with a matrix of progressively decreasing electron density.11 Studies have suggested that mitochondria complete this maturation process after the embryo has undergone the first cellular differentiation into trophectoderm (TE) and inner cell mass (ICM) and has become a blastocyst.21,22 The timing of the mitochondrial genome replication is different from that of the actual organelle. Specifically, during the initial embryonic cleavage divisions, the amounts of mtDNA remain stable, as the oocyte distributes mitochondria and mtDNA to the resulting blastomeres.1,23–25 A study quantifying the mtDNA of individual blastomeres originating from the same cleavage stage embryo concluded that there was a direct relationship between blastomere volume and mtDNA copy number.26 Unlike mitochondrial replication, which begins post–embryo implantation, that of the mtDNA starts after blastocyst differentiation, and is first observed in the TE of the embryo.1,2,27–29 It is thought that the TE part of blastocysts starts replicating mtDNA first due to its advanced state of differentiation, being committed to form the placenta of the fetus (i.e., loss of pluripotency). The ICM, on the other hand, retains pluripotency until later stages of development, and consequently mtDNA replication begins later in the cells comprising this part of the embryo.2 The regulation of mtDNA replication involves several nuclear-encoded genes, including the transcription factor TFAM which stabilizes mtDNA, and Peo1/SSbp1 which unwinds it. Additionally, the polymerase POLGA/B plays a very important role in maintaining the efficiency and fidelity of mtDNA replication.30 Other factors include the mitochondrial helicase Twinkle and the mitochondrial single-stranded binding protein.31,32 These proteins along with the transcription factors TFB1M, TFB2M, the mtRNA polymerase, and mTERF1 constitute the core mtDNA nucleoid and ensure the correct transcription, replication, and packaging of the mitochondrial genome.33 The nuclear genome also controls mitochondrial biogenesis via the AMP-activated kinase, and the transcription factors PPARg coactivator-1 a/b (PGC-1) and Nrf-1/2.34 It should be noted that each mitochondrion contains between 1 and 15 copies of the mtDNA,35 whereas each nucleoid is thought to consist from 1 to 3 mtDNA molecules.36

The Replication of Mitochondria and mtDNA

Assessment of Mitochondria and mtDNA to Predict Oocyte Quality

It is thought that appreciable levels of mitochondrial organelle replication do not occur until around the time of implantation.18 This means that the energy requirements of the early embryo, up until the blastocyst stage, must be supported by the activity of mitochondria that are primarily derived from the oocyte. The mitochondria found in the mature metaphase II oocyte are structurally distinct from those found in most somatic cells, with spherical shape (a diameter of
1 μM) and Seminars in Reproductive Medicine

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Oocyte genetics and physiology are the most important elements determining the capacity of an embryo to progress through preimplantation development, implant, and ultimately result in a healthy live birth. It is well established that the vast majority of aneuploidies seen in pregnancies and miscarriages are due to chromosome segregation errors taking place during oogenesis. Results obtained during the

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cytogenetic analysis of a large number of human oocytes have demonstrated a relationship between advancing female age and increasing aneuploidy rates.14,37–42 It is also widely accepted that mRNA transcripts and proteins stored in the oocyte are responsible for supporting the initial mitotic divisions following fertilization, until activation of the embryonic genome occurs around the 4- to 8-cell stages. The energy used by the embryo during the first few stages of preimplantation development is also derived from the oocyte, either in the form of intracellular ATP or produced after fertilization by mitochondria inherited from the oocyte.13,20 The number of mitochondria in mammalian cells has a wide range, varying from a few hundred to many thousands. This is determined by the volume of the cell and its energy requirements. The human mature oocyte is the largest cell in the body and therefore, unsurprisingly, contains a very large number of mitochondria and a considerable amount of mtDNA.1 Oocyte mitochondria begin replicating during fetal development, with the oogonial cells containing 200 individual organelles.43 As the developing oocyte progresses through maturation, its mitochondria continue replicating. Data obtained from different investigations vary considerably, but suggest that a mature metaphase II oocyte contains 100,000 mitochondria and between 50,000 and 550,000 copies of the mitochondrial genome.1,19,23,24,44,45 The critical role of the oocyte in supporting the developmental and energy needs of the embryo has led many researchers to investigate mitochondria and mtDNA within this type of cell. Some of these studies were interested in assessing the effects that different factors, such as advancing female age, have on oocyte mitochondria and the mitochondrial genome, whereas others have asked whether certain measurable features of mitochondria and mtDNA could serve as biomarkers of oocyte quality. A recent investigation examined mitochondrial function and mtDNA quantity in cumulus cells (CCs) removed from oocytes collected from 11 women undergoing IVF due to endometriosis (test group), and 39 women undergoing IVF due to other indications (control group).46 The aim was to establish whether the presence of endometriosis would adversely affect the follicular environment, and hence lead to oocytes of suboptimal quality. Their findings confirmed this hypothesis. Specifically, the CCs surrounding oocytes collected from women with endometriosis showed significantly less ATP production (65%, p ¼ 0.0147), compared with the amounts observed in the control group. They did not, however, observe any obvious changes in mtDNA quantities between the two sets of samples. It was concluded that mitochondrial dysfunction in the CCs of women with endometriosis may contribute to events such as defective apoptosis, and increased oxidative stress. These could in turn prevent the CCs from adequately supporting the developing oocyte they surround, affecting its function and the fertility of the female patient.46 Boucret and colleagues47 measured the mtDNA quantity in a total of 74 immature oocytes and their corresponding CCs. These were donated by 26 women with diminished ovarian reserve (DOR) and 21 women with normal ovarian reserve

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(NOR). The researchers aimed to determine whether mitochondria were associated with the genesis of DOR pathology. In addition to mtDNA quantification, the authors examined the expression of 13 genes involved in key mitochondrial functions such as apoptosis and antioxidant activity, or in mitochondrial biogenesis. Of these 13 genes, the PPARGC1A (peroxisome proliferator-activated receptor-gamma coactivator 1-α) was downregulated in the DOR CC samples, compared with the NOR group. This particular gene is the main regulator of mitochondrial biogenesis and respiration.48,49 It was also evident that oocytes and CCs of DOR patients contained lower levels of mtDNA, compared with NOR patients.47 There was, however, a difference in the average female ages of the two groups, with the NOR patients being significantly younger (30.6 years) than the DOR patients(33.7 years). So the possibility that the reduced mtDNA amounts observed in the DOR-derived oocytes could be related to a combination of ovarian pathology and female age cannot be excluded. Aging is associated with a general cellular and mitochondrial impairment affecting the physiologic function of the associated tissue.50 Tissues with slow turnover of mitochondria, such as the ovary, are led to a physiologic deficit via mitochondria-regulated apoptosis.51 The mtDNA also becomes compromised as a tissue ages due to the acquisition of mutations. An example is the presence of a 4,977 base pair (bp) deletion detected in some of the oocytes derived from reproductively older women.52 The possible impact of mitochondrial aging on female reproductive capacity was investigated by Duran and colleagues.50 The study involved examination of the ATP content, the mitochondria number, and the presence of the 4,977 bp deletion in individual human oocytes at different stages of maturation (i.e., germinal vesicle [GV]; meiosis I [MI]; meiosis II [MII]) donated by women of an average age of 35.2 years. The results obtained revealed that mitochondrial numbers, predicted via mtDNA quantification, were more closely associated with reproductive age (as determined by measurement of FSH [follicle stimulating hormone] levels) rather than chronological age. Additionally, ATP content was shown to increase as the oocyte matured, but its amount was also not related to chronological age. As far as the 4,977bp deletion was concerned, this was identified only in arrested or degenerate oocytes.50 Findings obtained in another study performed by our group, which also assessed mtDNA levels in relation to female age, were contradictory to those of Duran et al.50 We employed a novel microarray capable of simultaneously detecting chromosome abnormalities and quantifying mtDNA levels in a sample, to examine a total of 27 first polar bodies (PBs) biopsied from oocytes generated by women of various ages.53 These samples were divided into two groups according to female age (30–37 years and 38–45 years). This analysis showed that PB samples from older women tended to contain lower mtDNA quantities (p ¼ 0.04) compared with those obtained from younger women.53 The discrepancy between the two studies could be due to the fact that our research employed polar body analysis as a Seminars in Reproductive Medicine

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Mitochondrial DNA Assessment to Determine Oocyte and Embryo Viability proxy for direct oocyte testing. While it seems reasonable to suppose that the quantity of mitochondria in the polar body is in direct proportion to the number present in the corresponding oocyte, the fact that there is some variation in the cytoplasmic volume of polar bodies could introduce a source of error. Nonetheless, results obtained by other researchers using different methods of quantification and employing direct oocyte testing support the notion that the quantity of mtDNA in human oocytes declines with advancing age.25,54 Taken together, the data from these investigations provide strong evidence for a role of mitochondria and/or mtDNA dysfunction in the loss of oocyte competence in older women. Aneuploidy is the main genetic factor responsible for embryonic demise and can have a meiotic or post-zygotic origin. Female meiosis is much more vulnerable to chromosome malsegregation than male meiosis, perhaps related to the prolonged cell cycle arrest experienced by the female gamete. A recent investigation assessed the presence of a possible relationship between mitochondrial haplogroup and aneuploidy of female origin.55 There are several distinct mtDNA haplogroups characterized by polymorphic variation in the sequence of the mtDNA, some of which may be associated with altered mitochondrial function, potentially affecting the efficiency of energy production, signaling pathways, and possibly influencing the fidelity of chromosome segregation.56 Gianaroli and colleagues examined the sequence of the polymorphic HVRI D-loop region of the mtDNA and also the chromosomal status of 66 first and 66 second PB pairs. For 51 of these, the corresponding oocyte was also available for analysis. The samples were donated by 16 patients of a similar age (40 years). The majority (76%) of the investigated PBs and their corresponding oocytes were characterized as being aneuploid, but the frequency of chromosome abnormality was found to vary depending on the specific mitochondrial haplogroup. Hence, the sister haplogroups J and T were associated with significantly (p ¼ 0.001) higher aneuploidy rates in the PBs and/or corresponding oocytes, compared with haplogroup H. The authors hypothesized that this could be due to reduced ETC efficiency and diminished ATP production in mitochondria of the J haplogroup.57 Such events may in turn affect various aspects of meiosis and oogenesis, including chromosome segregation.55 The data from the studies discussed in this section clearly indicate that the integrity of the mitochondria and their genome plays an important function in determining oocyte competence and quality. It was evident from most of the data that lower quantities of mitochondria and mtDNA, leading to reduced generation of ATP, were associated with a general oocyte dysfunction and possibly an increased risk of aneuploidy. It has therefore been postulated that providing oocytes of suboptimal quality with additional mitochondria could improve their chances of energetically supporting the developing embryo.58 Supplementation of mitochondria in an oocyte initially took place in the form of cytoplasmic transfer.58 During this procedure, 5 to 15% of the ooplasm from “good quality” oocytes derived from young donors was micro-injected into oocytes obtained from reproductively older women, or Seminars in Reproductive Medicine

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patients experiencing repetitive implantation failure. Inevitably the cytoplasm transferred included mitochondria from the donor (as well as other cytoplasmic components). Clinical application of this procedure led to the birth of several children to women who had previously been unable to achieve pregnancies.58,59 The problem with this methodology, however, was that some of the children born following treatment displayed mtDNA heteroplasmy. In other words, their cells contained two distinct mtDNA types, one from the donor and another originating from the recipient oocyte.60 For some, the concept of assisted reproduction involving the introduction of genetic material from a third party (i.e., mtDNA from the ooplasm donor) raises ethical questions. Additionally, the relative paucity of information concerning the biological and clinical effects of cytoplasm donation led to fears that there could be unpredicted adverse impacts. Although there is no clear evidence that cytoplasm transfer, in general, and mitochondrial heteroplasmy, in particular, have any negative clinical consequences in humans, concerns over safety ultimately led to ooplasm transfer being banned in the United States and Canada. An alternative approach of mitochondrial supplementation, with the aim of “rescuing” defective oocytes, is the use of autologous mitochondria, that is, ones coming from the same patient. These mitochondria could be removed from one group of the patient’s own oocytes, and be injected into another group.61 Alternatively, the mitochondria could be sourced from the patient’s somatic cells (e.g., from their cumulus cells). The concept of autologous mitochondrial transfer overcomes ethical objections related to the use of genetically distinct mitochondria from an unrelated donor and also circumvents theoretical clinical/biological issues potentially associated with heteroplasmy. Some researchers have proposed the injection of mitochondria obtained from oocyte “precursor” cells.62 This intriguing strategy is just beginning to find clinical use, but its general effectiveness is yet to be proven in the context of a well-controlled trial and further data will be required before wide application can be contemplated.

Assessment of Mitochondria and mtDNA to Predict Embryo Viability After fertilization, the zygote commits itself to a series of mitotic divisions which divide it into progressively smaller cells, known as blastomeres, without changing its overall size. The first few mitotic divisions take place under the control of the maternal mRNAs and proteins, but they transition to the control of the embryonic genome, when this becomes activated at the 4- to 8-cell stages.63 Following embryonic genome activation, the cleavage stage embryo changes its morphology to become a morula (individual blastomeres are no longer visualized), and then goes through the first cellular differentiation into TE and ICM and becomes a blastocyst. The blastocyst stage of preimplantation development is reached 5 to 6 days post-fertilization. During the cleavage stage, the oocyte mitochondria and the DNA they contain are dispersed into the blastomeres,

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although the organelles are not necessarily evenly distributed within the cells. It is thought that little, if any, replication of mitochondria or mtDNA occurs during this phase. Data obtained from animal models (mouse and pig) suggest a minimum threshold of 50,000 to 100,000 oocyte mtDNA copies is required to support normal embryo development.8,27,64,65 As already discussed, significant level of mtDNA replication, leading to a net increase in the mtDNA content of the embryo, begins around the time of blastocyst formation, and is first observed in TE cells of the embryo.2,61 Blastocyst mitochondria were visualized in great detail by Van Blerkom66 with the use of fluorescent probes, such as JC-1 and MitoTracker Orange. These experiments indicated that each TE cell of human-expanded blastocysts contains 150 mitochondria, whereas the number of these organelles in the ICM cells was lower, as replication in this embryonic part begins later.2,66 The appropriate function of mitochondria and their genome during these early stages of life is critical, as vital processes related to metabolism, synthesis, cell division, and differentiation require significant quantities of energy. It has been shown that adverse or suboptimal in vitro culture conditions, including the presence of glucose early in development, or calcium and/or pH fluctuations have a direct effect both on mitochondrial function and embryo metabolism.67–70 The effects of in vitro culture conditions on embryo morphology and the presence of cellular fragmentation were assessed by Stigliani and colleagues.71 Specifically, their study examined whether cell-free genomic and mtDNA of embryonic origin were present in the surrounding culture medium, and whether this was correlated with morphological features of the corresponding cleavage stage embryos. A total of 800 culture medium samples were examined via realtime PCR for this purpose. Interestingly, the levels of mtDNA were significantly higher in samples derived from fragmented embryos, and those generated by women of advanced reproductive age (35 years or over) (p ¼ 0.0247 and p ¼ 0.0149, respectively).71 In a separate investigation performed by the same group, the relationship between the mtDNA/gDNA ratio in spent cleavage stage culture medium and embryo development to the blastocyst stage, as well as the ability to establish an ongoing pregnancy, was examined.72 The analysis of 699 spent culture medium samples demonstrated that a high mtDNA/gDNA ratio in samples collected on day 3 of preimplantation development was associated with superior blastocyst morphology and higher implantation potential. The authors therefore concluded that examination of the mtDNA/gDNA ratio could serve as a noninvasive biomarker of embryo quality and viability.72 Our research group recently performed a thorough examination of the quantity of mtDNA in human embryos during the cleavage and blastocyst stages.73 Our study considered the relationship between embryo mtDNA content, female age, chromosomal status, and implantation potential. Additionally, we investigated the mtDNA sequence of blastocyst stage embryos, searching for mutations, deletions, and polymorphisms. Cleavage stage analysis involved the evaluation of

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blastomeres removed from 39 embryos previously characterized as euploid after cytogenetic analysis. Assessment of blastocysts focused on 340 TE biopsy specimens, 123 of which had been shown to be aneuploid after chromosome screening while the remaining 217 were euploid. Cytogenetic assessment took place either via array comparative genomic hybridization (aCGH) or next-generation sequencing (NGS). NGS was also employed for the analysis of the mtDNA sequence and provided information on the amount of mtDNA in the samples tested. However, in most cases, quantification of mtDNA levels was achieved using a real-time PCR methodology. Quantification of mtDNA demonstrated that cleavage stage embryos generated by younger women (average age: 33.7 years) contained significantly higher levels (p ¼ 0.01) compared with embryos produced by older women (average age: 39.2 years). This mirrors the age-related decline in mtDNA quantity seen in oocytes.25,50,53,54 The results are consistent with the notion that there is no significant mtDNA replication between fertilization and the blastocyst stage of development, and suggest that the great majority of the mtDNA within cleavage stage embryos is derived from the oocyte. Interestingly, the analysis TE samples showed a trend in the opposite direction, with mtDNA levels increasing significantly (p ¼ 0.003) with advancing female age (younger age group: average age 34.8 years, older age group: average age 39.8). This association was apparent for both chromosomally normal and abnormal blastocysts. These data suggest that an appreciable quantity of mtDNA replication has already occurred by the time blastocyst formation is complete. The relative increase in the quantity of mtDNA seen in blastocysts from reproductively older women may reflect higher energy requirements in comparison with embryos generated by younger patients (i.e., a larger number of mitochondria needed to provide adequate levels of ATP). Real-time PCR assessment of TE samples also revealed a significant increase (p ¼ 0.025) in mtDNA levels of aneuploid blastocysts, compared with chromosomally normal embryos. This was confirmed by NGS analysis of an independent population of blastocysts (p ¼ 0.006). It is possible that high levels of mtDNA are indicative of some form of mitochondrial dysfunction. Thus, blastocysts with unusually high levels of mtDNA may experience suboptimal ATP production and be predisposed to the failure of energetic cellular processes, including chromosome segregation. However, an alternative possibility is that the increase in mtDNA levels may represent a cellular “stress” response. Stress factors, including aneuploidy, could induce replication of mitochondria, providing additional energy capacity for cells as they attempt to normalize intracellular conditions. When we examined mtDNA quantity in relation to embryo viability, we observed that euploid blastocysts which successfully implanted after transfer tended to contain lower mtDNA levels than those failing to produce an ongoing pregnancy (p ¼ 0.007). Detailed analysis of the data produced enabled the establishment of an mtDNA quantity threshold for blastocyst stage embryos, above which implantation was never observed. The predictive value of this threshold was confirmed in a blinded prospective study. Seminars in Reproductive Medicine

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Mitochondrial DNA Assessment to Determine Oocyte and Embryo Viability From this research, it was evident that 30% of the chromosomally normal blastocysts that did not implant had unusually high (above the threshold) mtDNA levels. It should be noted that there was no correlation between mtDNA level and blastocyst morphology and that the threshold remained the same regardless of the clinic in which IVF cycles were undertaken. These results are illustrated in ►Fig. 1. Interestingly, the clear correlation between mtDNA quantity and embryo viability seen at the blastocyst stage was not seen at the cleavage stage in our study. This suggests that problems associated with increased mtDNA are only revealed after replication of the mitochondrial genome is initiated, around the time of blastocyst formation. It is also noteworthy that NGS analysis did not reveal any increase in DNA sequence mutation in blastocysts with elevated mtDNA levels. Although not conclusive, this argues against the possibility that increased quantities of mtDNA could be indicative of a compensatory mechanism, by which cells increase the quantities of mitochondria to offset inefficient ATP production associated with defective organelles carrying mutations in key genes. Another possible explanation for the increase in mtDNA in nonviable blastocysts is that embryos that are compromised in some way may have greater metabolic activity, requiring more ATP production. In other words, the increase in mtDNA

Fig. 1 Quantification of mtDNA from TE samples demonstrated that euploid blastocysts capable of leading to ongoing pregnancies consisted of significantly lower mtDNA levels (p ¼ 0.007), compared with those which, after transfer, did not implant. An mtDNA quantity threshold was also established. Blastocysts generating viable pregnancies contained mtDNA quantities below this threshold, whereas mtDNA quantities above it were associated with failure to achieve an ongoing clinical pregnancy. Seminars in Reproductive Medicine

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might not reflect a means of offsetting the inefficiencies of defective organelles, but may represent a way of supporting elevated energetic demands. These observations are consistent with the “quiet embryo” hypothesis proposed by Leese.70 The clear and direct association between blastocyst mtDNA quantity and implantation potential means that quantification of this feature could serve as a valuable biomarker for the prediction of embryo viability.73 Similar results to ours regarding mtDNA quantity and blastocyst chromosome status were obtained in a study performed with the use of NGS.74 A total of 454 blastocysts underwent a TE biopsy, followed by cytogenetic analysis via NGS, for the purposes of PGS. The authors evaluated the relative mtDNA copy number for blastocysts characterized as being euploid after TE analysis, and also those characterized as being chromosomally abnormal. Data comparison demonstrated that aneuploid blastocysts contained significantly (p ¼ 0.017) higher levels of mtDNA than those seen in chromosomally normal embryos.74 These findings further confirm the presence of a relationship between chromosome segregation and mitochondrial function. A real-time PCR approach was employed by Murakoshi and colleagues26 to quantify the mtDNA of human oocytes and cleavage stage embryos. Their study assessed the number of mtDNA copies in relation to female age, as well as cellular volume. To address the second aim, the authors disaggregated cleavage stage embryos to their individual blastomeres and analyzed mtDNA content of each cell separately. The results obtained demonstrated that oocytes and embryos generated by women older than 40 years contain, on average, fewer mtDNA copies (p < 0.05) compared with those obtained from younger patients. This is in agreement with our cleavage stage data73 and is also concordant with results obtained from oocyte investigations.53 Not surprisingly, a positive correlation was identified between blastomere volume and mtDNA copy number, larger blastomeres containing more copies of mtDNA (and more mitochondria) (p < 0.01).26 The relationship between mtDNA quantity and embryo viability was also assessed in a recent investigation performed by Diez-Juan and colleagues.75 These authors attempted to quantify the mtDNA copy number of blastomeres biopsied from 225 cleavage stage embryos and TE samples removed from 65 blastocysts. The samples were first subjected to whole genome amplification (WGA) and aCGH for the purpose of aneuploidy screening, and had all been identified to be chromosomally normal prior to investigation of mtDNA content. Similar to our investigation, real-time PCR was employed to assess embryonic mtDNA quantities. However, a technical difference was that the strategy employed involved the use of a single copy nuclear sequence (β-actin) as a reference for the normalization of input DNA. The purpose of normalization is to ensure that any variation related to technical issues (e.g., differences in the efficiency of WGA, or the number of cells within the biopsy specimen) are adjusted for. The use of single copy sequences, such as β-actin, for normalization of mtDNA can be problematic, especially at the single cell level (i.e., for blastomeres). The difficulty arises due to allele dropout (ADO), a phenomenon which typically

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affects 5 to 10% of single cell PCR analyses, and has the effect of making it seem that there is more mtDNA than there truly is. Since ADO occurs during the initial WGA, performing replicate real-time PCR amplifications cannot circumvent this problem. A further limitation of the study is that embryo assessments were retrospective and analyses do not appear to have taken place in a blinded fashion. In agreement with our data,73 the Diez-Juan et al75 study demonstrated that TE samples removed from blastocysts capable of producing ongoing pregnancies contain significantly lower amounts of mtDNA, compared with biopsy specimens from embryos that fail to implant (p ¼ 0.027). However, the blastomere results obtained during this study disagreed with our findings, suggesting that the association of mtDNA content and viability seen in blastocysts can also be observed at the cleavage stage. There are several possible explanations for this discrepancy. Considering that ADO is particularly common following single cell WGA, it is likely that an appreciable number of the blastomeres analyzed by Diez-Juan and colleagues did not yield reliable results. Additionally, as observed by Murakoshi et al,26 the mtDNA copy number is closely related to blastomere volume. Variability in cell size was not taken into account in the investigation of Diez-Juan et al and thus there is a possibility that the reported link between reduced embryo viability and high mtDNA levels at the cleavage stage may actually be a reflection of the volume of the cell biopsied. The removal of a large blastomere (containing more mtDNA) deprives the embryo of a significant proportion of its mass and reduces the total number of mitochondria available for energy production to a greater extent than removal of a smaller cell. This could have an impact on implantation potential. Similarly, the biopsy of a cell containing a disproportionately large amount of the mitochondria of the embryo (a consequence of uneven distribution of the organelles to different cells during mitosis) could lead to reduced embryo viability due to impaired ATP production following cell removal. Thus, the reported association between cleavage stage viability and mtDNA content may not be related to an inherent embryo deficiency, but might actually be an unintended consequence of the testing process itself. Clearly, further work is needed to clarify whether mtDNA analysis performed on single blastomeres is of clinical value.

factors related to reproductive senescence. While much currently remains unknown, what is already evident is that mtDNA is of great relevance to human reproduction and that diagnostics or interventions focused on mitochondria are likely to have an important role in the treatment of infertility in the future.

Conclusion

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To summarize, the role of the mitochondria and their genome is critical both during oogenesis and early preimplantation development. Oocytes with reduced amounts of mtDNA and/ or defective mitochondria are likely to be compromised and unable to adequately support the energy requirements of the developing embryo. At the blastocyst stage, unusually large quantities of mtDNA are seen in 30% of euploid embryos that fail to form a viable pregnancy. This suggests that mtDNA quantification could represent a new biomarker, second only to aneuploidy in terms of its importance for embryo viability. The studies discussed in this review implicate mitochondria and their genome in the genesis of aneuploidy and other

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Acknowledgments D. W. is supported by the NIHR Biomedical Research Centre Oxford.

References

2

3

4

5

6

7

8

9

10

11

13

14 15

16

Reynier P. Low oocyte mitochondrial DNA content in ovarian insufficiency. Hum Reprod 2005;20(3):593–597 St John JC, Facucho-Oliveira J, Jiang Y, Kelly R, Salah R. Mitochondrial DNA transmission, replication and inheritance: a journey from the gamete through the embryo and into offspring and embryonic stem cells. Hum Reprod Update 2010;16(5):488–509 Dumollard R, Carroll J, Duchen MR, Campbell K, Swann K. Mitochondrial function and redox state in mammalian embryos. Semin Cell Dev Biol 2009;20(3):346–353 Anderson S, Bankier AT, Barrell BG, et al. Sequence and organization of the human mitochondrial genome. Nature 1981; 290(5806):457–465 Ashley MV, Laipis PJ, Hauswirth WW. Rapid segregation of heteroplasmic bovine mitochondria. Nucleic Acids Res 1989;17(18): 7325–7331 Bartmann AK, Salata Romao G, da Silveira Ramos E, et al. Why do older women have poor implantation rates? A possible role of the mitochondria. JARG 2004;21:79–83 Marchington DR, Macaulay V, Hartshorne GM, Barlow D, Poulton J. Evidence from human oocytes for a genetic bottleneck in an mtDNA disease. Am J Hum Genet 1998;63(3):769–775 Wai T, Ao A, Zhang X, Cyr D, Dufort D, Shoubridge EA. The role of mitochondrial DNA copy number in mammalian fertility. Biol Reprod 2010;83(1):52–62 Jansen RP. Germline passage of mitochondria: quantitative considerations and possible embryological sequelae. Hum Reprod 2000;15(Suppl 2):112–128 Monnot S, Gigarel N, Samuels DC, et al. Segregation of mtDNA throughout human embryofetal development: m.3243A>G as a model system. Hum Mutat 2011;32(1):116–125 Van Blerkom J. Mitochondrial function in the human oocyte and embryo and their role in developmental competence. Mitochondrion 2011;11(5):797–813 Houghton FD. Energy metabolism of the inner cell mass and trophectoderm of the mouse blastocyst. Differentiation 2006; 74(1):11–18 Van Blerkom J, Davis PW, Lee J. ATP content of human oocytes and developmental potential and outcome after in-vitro fertilization and embryo transfer. Hum Reprod 1995;10(2):415–424 Fragouli E, Alfarawati S, Spath K, et al. The origin and impact of embryonic aneuploidy. Hum Genet 2013;132(9):1001–1013 Wells D, Kaur K, Grifo J, et al. Clinical utilisation of a rapid low-pass whole genome sequencing technique for the diagnosis of aneuploidy in human embryos prior to implantation. J Med Genet 2014;51(8):553–562 Yang Z, Liu J, Collins GS, et al. Selection of single blastocysts for fresh transfer via standard morphology assessment alone and with array CGH for good prognosis IVF patients: results from a randomized pilot study. Mol Cytogenet 2012;5(1):24

Seminars in Reproductive Medicine

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Downloaded by: University of British Columbia. Copyrighted material.

1 May-Panloup P, Chrétien MF, Jacques C, Vasseur C, Malthièry Y,

Mitochondrial DNA Assessment to Determine Oocyte and Embryo Viability

Fragouli, Wells

17 Forman EJ, Upham KM, Cheng M, et al. Comprehensive chromo-

38 Kuliev A, Cieslak J, Ilkevitch Y, Verlinsky Y. Chromosomal abnor-

some screening alters traditional morphology-based embryo selection: a prospective study of 100 consecutive cycles of planned fresh euploid blastocyst transfer. Fertil Steril 2013;100(3): 718–724 Larsson NG, Wang J, Wilhelmsson H, et al. Mitochondrial transcription factor A is necessary for mtDNA maintenance and embryogenesis in mice. Nat Genet 1998;18(3):231–236 Motta PM, Nottola SA, Makabe S, Heyn R. Mitochondrial morphology in human fetal and adult female germ cells. Hum Reprod 2000; 15(Suppl 2):129–147 Dumollard R, Duchen M, Carroll J. The role of mitochondrial function in the oocyte and embryo. Curr Top Dev Biol 2007; 77:21–49 Van Blerkom J. Development of human embryos to the hatched blastocyst stage in the presence or absence of a monolayer of Vero cells. Hum Reprod 1993;8(9):1525–1539 Sathananthan AH, Trounson AO. Mitochondrial morphology during preimplantational human embryogenesis. Hum Reprod 2000; 15(Suppl 2):148–159 Steuerwald N, Barritt JA, Adler R, et al. Quantification of mtDNA in single oocytes, polar bodies and subcellular components by real-time rapid cycle fluorescence monitored PCR. Zygote 2000;8(3):209–215 Reynier P, May-Panloup P, Chrétien MF, et al. Mitochondrial DNA content affects the fertilizability of human oocytes. Mol Hum Reprod 2001;7(5):425–429 Chan CC, Liu VW, Lau EY, Yeung WS, Ng EH, Ho PC. Mitochondrial DNA content and 4977 bp deletion in unfertilized oocytes. Mol Hum Reprod 2005;11(12):843–846 Murakoshi Y, Sueoka K, Takahashi K, et al. Embryo developmental capability and pregnancy outcome are related to the mitochondrial DNA copy number and ooplasmic volume. J Assist Reprod Genet 2013;30(10):1367–1375 Pikó L, Taylor KD. Amounts of mitochondrial DNA and abundance of some mitochondrial gene transcripts in early mouse embryos. Dev Biol 1987;123(2):364–374 Thundathil J, Filion F, Smith LC. Molecular control of mitochondrial function in preimplantation mouse embryos. Mol Reprod Dev 2005;71(4):405–413 Spikings EC, Alderson J, St John JC. Regulated mitochondrial DNA replication during oocyte maturation is essential for successful porcine embryonic development. Biol Reprod 2007;76(2): 327–335 Tyynismaa H, Suomalainen A. Mouse models of mtDNA replication diseases. Methods 2010;51(4):405–410 Spelbrink JN, Li FY, Tiranti V, et al. Human mitochondrial DNA deletions associated with mutations in the gene encoding Twinkle, a phage T7 gene 4-like protein localized in mitochondria. Nat Genet 2001;28(3):223–231 Korhonen JA, Gaspari M, Falkenberg M. TWINKLE Has 5′ -> 3′ DNA helicase activity and is specifically stimulated by mitochondrial single-stranded DNA-binding protein. J Biol Chem 2003;278(49): 48627–48632 Bogenhagen DF, Rousseau D, Burke S. The layered structure of human mitochondrial DNA nucleoids. J Biol Chem 2008;283(6): 3665–3675 Seo AY, Joseph AM, Dutta D, Hwang JC, Aris JP, Leeuwenburgh C. New insights into the role of mitochondria in aging: mitochondrial dynamics and more. J Cell Sci 2010;123(Pt 15):2533–2542 Satoh M, Kuroiwa T. Organization of multiple nucleoids and DNA molecules in mitochondria of a human cell. Exp Cell Res 1991; 196(1):137–140 Brown TA, Tkachuk AN, Shtengel G, et al. Superresolution fluorescence imaging of mitochondrial nucleoids reveals their spatial range, limits, and membrane interaction. Mol Cell Biol 2011; 31(24):4994–5010 Sandalinas M, Márquez C, Munné S. Spectral karyotyping of fresh, non-inseminated oocytes. Mol Hum Reprod 2002;8(6):580–585

malities in a series of 6,733 human oocytes in preimplantation diagnosis for age-related aneuploidies. Reprod Biomed Online 2003;6(1):54–59 Pellestor F, Andréo B, Arnal F, Humeau C, Demaille J. Maternal aging and chromosomal abnormalities: new data drawn from in vitro unfertilized human oocytes. Hum Genet 2003;112(2): 195–203 Fragouli E, Wells D, Thornhill A, et al. Comparative genomic hybridization analysis of human oocytes and polar bodies. Hum Reprod 2006;21(9):2319–2328 Fragouli E, Escalona A, Gutiérrez-Mateo C, et al. Comparative genomic hybridization of oocytes and first polar bodies from young donors. Reprod Biomed Online 2009;19(2):228–237 Fragouli E, Katz-Jaffe M, Alfarawati S, et al. Comprehensive chromosome screening of polar bodies and blastocysts from couples experiencing repeated implantation failure. Fertil Steril 2010; 94(3):875–887 Eichenlaub-Ritter U, Wieczorek M, Lüke S, Seidel T. Age related changes in mitochondrial function and new approaches to study redox regulation in mammalian oocytes in response to age or maturation conditions. Mitochondrion 2011;11(5):783–796 Chen X, Prosser R, Simonetti S, Sadlock J, Jagiello G, Schon EA. Rearranged mitochondrial genomes are present in human oocytes. Am J Hum Genet 1995;57(2):239–247 Cummins JM. Fertilization and elimination of the paternal mitochondrial genome. Hum Reprod 2000;15(Suppl 2):92–101 Hsu AL, Townsend PM, Oehninger S, Castora FJ. Endometriosis may be associated with mitochondrial dysfunction in cumulus cells from subjects undergoing in vitro fertilizationintracytoplasmic sperm injection, as reflected by decreased adenosine triphosphate production. Fertil Steril 2015;103(2): 347–352.e1 Boucret L, Chao de la Barca JM, Morinière C, et al. Relationship between diminished ovarian reserve and mitochondrial biogenesis in cumulus cells. Hum Reprod 2015;30(7):1653–1664 Puigserver P, Wu Z, Park CW, Graves R, Wright M, Spiegelman BM. A cold-inducible coactivator of nuclear receptors linked to adaptive thermogenesis. Cell 1998;92(6):829–839 Wu Z, Puigserver P, Andersson U, et al. Mechanisms controlling mitochondrial biogenesis and respiration through the thermogenic coactivator PGC-1. Cell 1999;98(1):115–124 Duran HE, Simsek-Duran F, Oehninger SC, Jones HW Jr, Castora FJ. The association of reproductive senescence with mitochondrial quantity, function, and DNA integrity in human oocytes at different stages of maturation. Fertil Steril 2011;96(2): 384–388 Kitagawa T, Suganuma N, Nawa A, et al. Rapid accumulation of deleted mitochondrial deoxyribonucleic acid in postmenopausal ovaries. Biol Reprod 1993;49(4):730–736 Keefe DL, Niven-Fairchild T, Powell S, Buradagunta S. Mitochondrial deoxyribonucleic acid deletions in oocytes and reproductive aging in women. Fertil Steril 1995;64(3):577–583 Konstantinidis M, Alfarawati S, Hurd D, et al. Simultaneous assessment of aneuploidy, polymorphisms, and mitochondrial DNA content in human polar bodies and embryos with the use of a novel microarray platform. Fertil Steril 2014;102(5): 1385–1392 de Boer KA, Jansen RP, Leigh DA, et al. O-165 quantification of mtDNA copy number in the human secondary oocyte. Hum Reprod 1999;14(9):2419 Gianaroli L, Luiselli D, Crivello AM, et al. Mitochondrial DNA analysis and numerical chromosome condition in human oocytes and polar bodies. Mol Hum Reprod 2015;21(1):46–57 Kenney MC, Chwa M, Atilano SR, et al. Mitochondrial DNA variants mediate energy production and expression levels for CFH, C3 and EFEMP1 genes: implications for age-related macular degeneration. PLoS ONE 2013;8(1):e54339

18

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25

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27

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29

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32

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45 46

47

48

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50

51

52

53

54

55

56

Downloaded by: University of British Columbia. Copyrighted material.

408

Fragouli, Wells

57 Marcuello A, Martínez-Redondo D, Dahmani Y, et al. Human

67 Barnett DK, Clayton MK, Kimura J, Bavister BD. Glucose and

mitochondrial variants influence on oxygen consumption. Mitochondrion 2009;9(1):27–30 Cohen J, Scott R, Alikani M, et al. Ooplasmic transfer in mature human oocytes. Mol Hum Reprod 1998;4(3):269–280 Barritt J, Willadsen S, Brenner C, Cohen J. Cytoplasmic transfer in assisted reproduction. Hum Reprod Update 2001;7(4): 428–435 Harvey AJ, Gibson TC, Quebedeaux TM, Brenner CA. Impact of assisted reproductive technologies: a mitochondrial perspective of cytoplasmic transplantation. Curr Top Dev Biol 2007; 77:229–249 St John J. The control of mtDNA replication during differentiation and development. Biochim Biophys Acta 2014;1840(4): 1345–1354 Chappel S. The role of mitochondria from mature oocyte to viable blastocyst. Obstet Gynecol Int 2013;2013:183024 Braude P, Bolton V, Moore S. Human gene expression first occurs between the four- and eight-cell stages of preimplantation development. Nature 1988;332(6163):459–461 El Shourbagy SH, Spikings EC, Freitas M, St John JC. Mitochondria directly influence fertilisation outcome in the pig. Reproduction 2006;131(2):233–245 Harvey A, Gibson T, Lonergan T, Brenner C. Dynamic regulation of mitochondrial function in preimplantation embryos and embryonic stem cells. Mitochondrion 2011;11(5):829–838 Van Blerkom J. Mitochondria as regulatory forces in oocytes, preimplantation embryos and stem cells. Reprod Biomed Online 2008;16(4):553–569

phosphate toxicity in hamster preimplantation embryos involves disruption of cellular organization, including distribution of active mitochondria. Mol Reprod Dev 1997;48(2):227–237 Lane M, Bavister BD. Calcium homeostasis in early hamster preimplantation embryos. Biol Reprod 1998;59(4):1000–1007 Squirrell JM, Lane M, Bavister BD. Altering intracellular pH disrupts development and cellular organization in preimplantation hamster embryos. Biol Reprod 2001;64(6):1845–1854 Leese HJ. Quiet please, do not disturb: a hypothesis of embryo metabolism and viability. BioEssays 2002;24(9):845–849 Stigliani S, Anserini P, Venturini PL, Scaruffi P. Mitochondrial DNA content in embryo culture medium is significantly associated with human embryo fragmentation. Hum Reprod 2013;28(10): 2652–2660 Stigliani S, Persico L, Lagazio C, Anserini P, Venturini PL, Scaruffi P. Mitochondrial DNA in Day 3 embryo culture medium is a novel, non-invasive biomarker of blastocyst potential and implantation outcome. Mol Hum Reprod 2014;20(12):1238–1246 Fragouli E, Spath K, Alfarawati S, et al. Altered levels of mitochondrial DNA are associated with female age, aneuploidy, and provide an independent measure of embryonic implantation potential. PLoS Genet 2015;11(6):e1005241 Tan Y, Yin X, Zhang S, et al. Clinical outcome of preimplantation genetic diagnosis and screening using next generation sequencing. Gigascience 2014;3(1):30 Diez-Juan A, Rubio C, Marin C, et al. Mitochondrial DNA content as a viability score in human euploid embryos: less is better. Fertil Steril 2015;104(3):534–541.e1

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