Mitochondria play a vital role in embryo development. They are the principal site of energy production and have various other critical cellular functions. Despite the importance of this organelle, little is known about the extent of variation in mitochondrial DNA (mtDNA) between individual human embryos prior to implantation. This study investigated the biological and clinical relevance of the quantity of mtDNA in 379 embryos. These were examined via a combination of microarray comparative genomic hybridisation (aCGH), quantitative PCR and next generation sequencing (NGS), providing information on chromosomal status, amount of mtDNA, and presence of mutations in the mitochondrial genome. The quantity of mtDNA was significantly higher in embryos from older women (P=0.003). Additionally, mtDNA levels were elevated in aneuploid embryos, independent of age (P=0.025). Assessment of clinical outcomes after transfer of euploid embryos to the uterus revealed that blastocysts that successfully implanted tended to contain lower mtDNA quantities than those failing to implant (P=0.007). Importantly, an mtDNA quantity threshold was established, above which implantation was never observed. Subsequently, the predictive value of this threshold was confirmed in an independent blinded prospective study, indicating that abnormal mtDNA levels are present in 30% of non-implanting euploid embryos, but are not seen in embryos forming a viable pregnancy. NGS did not reveal any increase in mutation in blastocysts with elevated mtDNA levels. The results of this study suggest that increased mtDNA may be related to elevated metabolism and are associated with reduced viability, a possibility consistent with the ‘quiet embryo’ hypothesis. Importantly, the findings suggest a potential role for mitochondria in female reproductive aging and the genesis of aneuploidy. Of clinical significance, we propose that mtDNA content represents a novel biomarker with potential value for in vitro fertilisation (IVF) treatment, revealing chromosomally normal blastocysts incapable of producing a viable pregnancy.
References
[1]
May-Panloup P, Chrétien MF, Jacques C, Vasseur C, Malthièry Y, et al. (2005) Low oocyte mitochondrial DNA content in ovarian insufficiency. Hum Reprod 20: 593–597. pmid:15608038 doi: 10.1093/humrep/deh667
[2]
Dumollard R, Carroll J, Duchen MR, Campbell K, Swann K (2009) Mitochondrial function and redox state in mammalian embryos. Semin Cell Dev Biol 20: 346–353. pmid:19530278 doi: 10.1016/j.semcdb.2008.12.013
[3]
St John JC, Facucho-Oliveira J, Jiang Y, Kelly R, Salah R (2010) Mitochondrial DNA transmission, replication and inheritance: a journey from the gamete through the embryo and into offspring and embryonic stem cells. Hum Reprod Update 16: 488–509. doi: 10.1093/humupd/dmq002. pmid:20231166
[4]
Bentov Y, Casper RF (2013) The aging oocyte—can mitochondrial function be improved? Fertil Steril 99: 18–22. doi: 10.1016/j.fertnstert.2012.11.031. pmid:23273985
[5]
Tilly JL, Sinclair DA (2013) Germline energetics, aging, and female infertility. Cell Metab 17: 838–850. doi: 10.1016/j.cmet.2013.05.007. pmid:23747243
[6]
Anderson S, Bankier AT, Barrell BG, de Bruijn MH, Coulson AR, et al. (1981) Sequence and organization of the human mitochondrial genome. Nature 290: 457–465. pmid:7219534 doi: 10.1038/290457a0
[7]
Palmer CS, Osellame LD, Stojanovski D, Ryan MT (2011) The regulation of mitochondrial morphology: intricate mechanisms and dynamic machinery. Cell Signal 23: 1534–1545. doi: 10.1016/j.cellsig.2011.05.021. pmid:21683788
[8]
Eichenlaub-Ritter U, Wieczorek M, Lüke S, Seidel T (2011) Age related changes in mitochondrial function and new approaches to study redox regulation in mammalian oocytes in response to age or maturation conditions. Mitochondrion 11: 783–796. doi: 10.1016/j.mito.2010.08.011. pmid:20817047
[9]
Chen X, Prosser R, Simonetti S, Sadlock J, Jagiello G, et al. (1995) Rearranged mitochondrial genomes are present in human oocytes. Am J Hum Genet 57: 239–247. pmid:7668249
[10]
Motta PM, Nottola SA, Makabe S, Heyn R (2000). Mitochondrial morphology in human fetal and adult germ cells. Hum Reprod 15: 129–147. pmid:11041520 doi: 10.1093/humrep/15.suppl_2.129
[11]
Cummins JM (2000) Fertilization and elimination of the paternal mitochondrial genome. Hum. Reprod 15: 92–101. pmid:11041517 doi: 10.1093/humrep/15.suppl_2.92
[12]
Steuerwald N, Barritt JA, Adler R, Malter H, Schimmel T, et al. (2000) Quantification of mtDNA in single oocytes, polar bodies and subcellular components by real-time rapid cycle fluorescence monitored PCR. Zygote 8: 209–215. pmid:11014500 doi: 10.1017/s0967199400001003
[13]
Reynier P, May-Panloup P, Chrétien MF, Morgan CJ, Jean M, et al. (2001) Mitochondrial DNA content affects the fertilizability of human oocytes. Mol Hum Reprod 7: 425–429. pmid:11331664 doi: 10.1093/molehr/7.5.425
[14]
Barritt JA, Kokot M, Cohen J, Steuerwald N, Brenner CA (2002) Quantification of human ooplasmic mitochondria. Reprod Biomed Online 4: 243–247. pmid:12709274 doi: 10.1016/s1472-6483(10)61813-5
[15]
Lin DP, Huang CC, Wu HM, Cheng TC, Chen CI, et al. (2004) Comparison of mitochondrial DNA contents in human embryos with good or poor morphology at the 8-cell stage. Fertil Steril 81: 73–79. pmid:14711547 doi: 10.1016/j.fertnstert.2003.05.005
[16]
Chan CC, Liu VW, Lau EY, Yeung WS, Ng EH, et al. (2005) Mitochondrial DNA content and 4977 bp deletion in unfertilized oocytes. Mol Hum Reprod 11: 843–846. pmid:16421213 doi: 10.1093/molehr/gah243
[17]
Van Blerkom J, Davis PW, Lee J (1995) ATP content of human oocytes and developmental potential and outcome after in-vitro fertilization and embryo transfer. Hum Reprod 10: 415–424. pmid:7769073
[18]
Fragouli E, Alfarawati S, Spath K, Jaroudi S, Sarasa J, et al. (2013). The origin and impact of embryonic aneuploidy. Hum Genet 132: 1001–1013. doi: 10.1007/s00439-013-1309-0. pmid:23620267
[19]
Van Blerkom J (2011) Mitochondrial function in the human oocyte and embryo and their role in developmental competence. Mitochondrion 11: 797–813. doi: 10.1016/j.mito.2010.09.012. pmid:20933103
[20]
Houghton F (2006) Energy metabolism of the inner cell mass and trophectoderm of the mouse blastocyst. Differentiation 74: 11–18. pmid:16466396 doi: 10.1111/j.1432-0436.2006.00052.x
[21]
Barritt JA, Cohen J, Brenner CA (2000) Mitochondrial DNA point mutation in human oocytes is associated with maternal age. Reprod Biomed Online 1: 96–100. pmid:12804188 doi: 10.1016/s1472-6483(10)61946-3
[22]
Konstantinidis M, Alfarawati S, Hurd D, Paolucci M, Shovelton J, et al. (2014) 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: in press. doi: 10.1016/j.fertnstert.2014.07.1233
[23]
Narita A (1995) Endogenous factors affecting sterility in oocytes of aged animals. Jap J Fertil Steril 40: 57–65.
[24]
Duran HE, Simsek-Duran F, Oehninger SC, Jones HW Jr, Castora (2011) The association of reproductive senescence with mitochondrial quantity, function, and DNA integrity in human oocytes at different stages of maturation. Fertil Steril 96: 384–388. doi: 10.1016/j.fertnstert.2011.05.056. pmid:21683351
[25]
Piko L, Taylor KD (1987) Amounts of mitochondrial DNA and abundance of some mitochondrial gene transcripts in early mouse embryos. Dev Biol 123: 364–374. pmid:2443405 doi: 10.1016/0012-1606(87)90395-2
[26]
Simsek-Duran F, Li F, Ford W, Swanson RJ, Jones HW Jr, et al. (2009) The effect of aging on the metabolic function and structure of mitochondria in hamster oocytes. FASEB J 855: 10.
[27]
Monnot S, Samuels DC, Hesters L, Frydman N, Gigarel N, et al. (2013) Mutation dependance of the mitochondrial DNA copy number in the first stages of human embryogenesis. Hum Mol Genet 22: 1867–1872. doi: 10.1093/hmg/ddt040. pmid:23390135
[28]
Hsieh RH, Au HK, Yeh TS, Chang SJ, Cheng YF, et al. (2004) Decreased expression of mitochondrial genes in human unfertilized oocytes and arrested embryos. Fertil Steril 81: 912–918. pmid:15019829 doi: 10.1016/j.fertnstert.2003.11.013
[29]
Hsieh RH, Tsai NM, Au HK, Chang SJ, Wei YH, et al. (2002) Multiple rearrangements of mitochondrial DNA in unfertilized human oocytes. Fertil Steril 77: 1012–1017. pmid:12009360 doi: 10.1016/s0015-0282(02)02994-1
[30]
Fragouli E, Alfarawati S, Daphnis DD, Goodall NN, Mania A, et al. (2011) Cytogenetic analysis of human blastocysts with the use of FISH, CGH and aCGH: scientific data and technical evaluation. Hum Reprod 26:480–490 doi: 10.1093/humrep/deq344. pmid:21147821
[31]
Wells D, Kaur K, Grifo J, Glassner M, Taylor JC, et al. (2014) 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 51: 553–562. doi: 10.1136/jmedgenet-2014-102497. pmid:25031024
[32]
Zhang X, Wu XQ, Lu S, Guo YL, Ma X (2006) Deficit of mitochondria- derived ATP during oxidative stress impairs mouse MII oocyte spindles. Cell Res 16: 841–850. pmid:16983401 doi: 10.1038/sj.cr.7310095
[33]
Choi WJ, Banerjee J, Falcone T, Bena J, Agarwal A, et al. (2007) Oxidative stress and tumor necrosis factor-induced alterations in metaphase II mouse oocyte spindle structure. Fertil Steril 88:1220–1231. pmid:17601599 doi: 10.1016/j.fertnstert.2007.02.067
[34]
Johnson MT, Freeman EA, Gardner DK, Hunt PA (2007) Oxidative metabolism of pyruvate is required for meiotic maturation of murine oocytes in vivo. Biol Reprod 77: 2–8. pmid:17314311 doi: 10.1095/biolreprod.106.059899
[35]
Yu Y, Dumollard R, Rossbach A, Lai FA, Swann K (2010) Redistribution of mitochondria leads to bursts of ATP production during spontaneous mouse oocyte maturation. Journal of Cellular Physiology 224: 672–680. doi: 10.1002/jcp.22171. pmid:20578238
[36]
Wang Q, Ratchford AM, Chi MM, Schoeller E, Frolova A, et al. (2009) Maternal diabetes causes mitochondrial dysfunction and meiotic defects in murine oocytes. Mol Endocrinol 23: 1603–1612. doi: 10.1210/me.2009-0033. pmid:19574447
[37]
Wilding M, De Placido G, DeMatteo L, Marino M, Alviggi C, et al. (2003) Chaotic mosaicism in human preimplantation embryos is correlated with a low mitochondrial membrane potential, Fertil Steril 79: 340–346. pmid:12568843 doi: 10.1016/s0015-0282(02)04678-2
[38]
Capalbo A, Wright G, Elliott T, Ubaldi FM, Rienzi L et al. (2013) FISH reanalysis of inner cell mass and trophectoderm samples of previously array-CGH screened blastocysts shows high accuracy of diagnosis and no major diagnostic impact of mosaicism at the blastocyst stage. Hum Reprod 28: 2298–2307. doi: 10.1093/humrep/det245. pmid:23739221
[39]
Leese HJ (2002) Quiet please, do not disturb: a hypothesis of embryo metabolism and viability. Bioessays 24: 845–849. pmid:12210521 doi: 10.1002/bies.10137
[40]
Harton GL, Munné S, Surrey M, Grifo J, Kaplan B, McCulloh DH, et al. (2013) Diminished effect of maternal age on implantation after preimplantation genetic diagnosis with array comparative genomic hybridization. Fertil Steril 100: 1695–1670 doi: 10.1016/j.fertnstert.2013.07.2002. pmid:24034939
[41]
Magli MC, Montag M, Koster M, Muzi L, Geraedts J, et al. (2011) Polar body array CGH for prediction of the status of the corresponding oocyte. Part II: technical aspects. Hum Reprod 26:3181–3185. doi: 10.1093/humrep/der295. pmid:21908464
[42]
Gutierrez-Mateo C, Colls P, Sanchez-Garc？a J, Escudero T, Prates R, et al. (2011) Validation of microarray comparative genomic hybridization for comprehensive chromosome analysis of embryos. Fertil Steril 95:953–958. doi: 10.1016/j.fertnstert.2010.09.010. pmid:20971462
[43]
Christopikou D, Tsorva E, Economou K, Shelley P, Davies S, et al. (2013) Polar body analysis by array comparative genomic hybridization accurately predicts aneuploidies of maternal meiotic origin in cleavage stage embryos of women of advanced maternal age. Hum Reprod 28: 1426–1434. doi: 10.1093/humrep/det053. pmid:23477909
[44]
Mertzanidou A, Spits C, Nguyen HT, Van de Velde H, Sermon K (2013) Evolution of aneuploidy up to Day 4 of human preimplantation development. Hum Reprod 28: 1716–1724. doi: 10.1093/humrep/det079. pmid:23526301
[45]
Fregel R, Almeida M, Betancor E, Suarez NM, Pestano J (2011) Reliable nuclear and mitochondrial DNA quantification for low copy number and degraded forensic samples. Forensic Science International: Genetics Supplement Series 3: e303–e304. doi: 10.1016/j.fsigss.2011.09.014
[46]
Li H, Durbin R (2009) Fast and accurate short read alignment with Burrows—Wheeler transform. Bioinformatics 25: 1754–1760. doi: 10.1093/bioinformatics/btp324. pmid:19451168
[47]
Raczy C, Petrovski R, Saunders CT, Chorny I, Kruglyak S et al. (2013) Isaac: ultra-fast whole-genome secondary analysis on Illumina sequencing platforms. Bioinformatics 29:2041–2043 doi: 10.1093/bioinformatics/btt314. pmid:23736529
[48]
Quinlan AR, Hall IM (2010) BEDTools: A flexible suite of utilities for comparing genomic features, Bioinformatics 26: 841–842. doi: 10.1093/bioinformatics/btq033. pmid:20110278
[49]
Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, et al. (2009) The Sequence alignment/map (SAM) format and SAMtools. Bioinformatics 25: 2078–2079. doi: 10.1093/bioinformatics/btp352. pmid:19505943
[50]
Baslan T, Kendall J, Rodgers L, Cox H, Riggs M, et al. (2012) Genome-wide copy number analysis of single cells. Nat Protoc 7: 1024–1041. doi: 10.1038/nprot.2012.039. pmid:22555242
[51]
Zhidkov I, Nagar T, Mishmar D, Rubin E (2011) MitoBamAnnotator: A web-based tool for detecting and annotating heteroplasmy in human mitochondrial DNA sequences. Mitochondrion 11: 924–928. doi: 10.1016/j.mito.2011.08.005. pmid:21875693
[52]
Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative CT method. Nat Protoc 3: 1101–1108. pmid:18546601 doi: 10.1038/nprot.2008.73
