Evidence of Selection against Complex Mitotic-Origin Aneuploidy during Preimplantation Development

English version České info

By day 3 of development, more than half of human embryos contain at least one cell that deviates from the typical 46-chromosome complement. These whole-chromosome abnormalities include polyploidies, which affect the entire chromosome set, as well as aneuploidies, which involve gains and losses of particular chromosomes. The rate of aneuploidy increases with maternal age, primarily due to chromosome segregation errors arising during egg formation (maternal meiosis). While some forms of aneuploidy, such as Trisomy 21, are compatible with live birth, most aneuploid embryos do not survive to term. Our study applied genetic techniques to screen early embryos from in vitro fertilization cycles, demonstrating that while diverse whole-chromosome abnormalities can be observed at early developmental stages, these errors are strongly filtered during preimplantation development. Specifically, errors occurring during the initial post-fertilization cell divisions often result in the simultaneous loss of multiple chromosomes, a pattern consistent with abnormal cell division. Our data provide evidence of selection against this class of aneuploidy before day 5 of development, thus reducing fertility. Patients referred for genetic screening due to previous IVF failure had higher rates of mitotic error, highlighting its clinical relevance and indicating that patient-specific genetic and environmental factors influence error rates.

Vyšlo v časopise: Evidence of Selection against Complex Mitotic-Origin Aneuploidy during Preimplantation Development. PLoS Genet 11(10): e32767. doi:10.1371/journal.pgen.1005601
Kategorie: Research Article
prolekare.web.journal.doi_sk: https://doi.org/10.1371/journal.pgen.1005601

Souhrn

Zdroje

1. Macklon NS, Geraedts JP, Fauser BC. Conception to ongoing pregnancy: the ‘black box’ of early pregnancy loss. Human Reproduction Update. 2002;8(4):333–343. doi: 10.1093/humupd/8.4.333 12206468

2. Edmonds DK, Lindsay KS, Miller JF, Williamson E, Wood PJ. Early embryonic mortality in women. Obstetrical & Gynecological Survey. 1983;38(7):433–434.

3. Hassold T, Hunt P. To err (meiotically) is human: the genesis of human aneuploidy. Nature Reviews Genetics. 2001;2(4):280–291. doi: 10.1038/35066065 11283700

4. Levy B, Sigurjonsson S, Petterson B, Maisenbacher M, Hall M, Demko Z, et al. Genomic imbalance in products of conception: SNP chromosomal microarray analysis. Obstetrics & Gynecology. 2014;124(2pt1):202–209.

5. Vanneste E, Voet T, Le Caignec C, Ampe M, Konings P, Melotte C, et al. Chromosome instability is common in human cleavage-stage embryos. Nature Medicine. 2009;15(5):577–583. doi: 10.1038/nm.1924 19396175

6. Johnson D, Gemelos G, Baner J, Ryan A, Cinnioglu C, Banjevic M, et al. Preclinical validation of a microarray method for full molecular karyotyping of blastomeres in a 24-h protocol. Human Reproduction. 2010;25(4):1066–1075. doi: 10.1093/humrep/dep452 20100701

7. Penrose LS, et al. The relative effects of paternal and maternal age in mongolism. Journal of Genetics. 1933;27(2):219–23. doi: 10.1007/BF02984413

8. Angell RR. Predivision in human oocytes at meiosis I: a mechanism for trisomy formation in man. Human Genetics. 1991;86(4):383–387. doi: 10.1007/BF00201839 1999340

9. Kuliev A, Zlatopolsky Z, Kirillova I, Spivakova J, Janzen JC. Meiosis errors in over 20,000 oocytes studied in the practice of preimplantation aneuploidy testing. Reproductive Biomedicine Online. 2011;22(1):2–8. doi: 10.1016/j.rbmo.2010.08.014 21115270

10. Handyside AH, Montag M, Magli MC, Repping S, Harper J, Schmutzler A, et al. Multiple meiotic errors caused by predivision of chromatids in women of advanced maternal age undergoing in vitro fertilisation. European Journal of Human Genetics. 2012;20(7):742–747. doi: 10.1038/ejhg.2011.272 22317970

11. Capalbo A, Bono S, Spizzichino L, Biricik A, Baldi M, Colamaria S, et al. Sequential comprehensive chromosome analysis on polar bodies, blastomeres and trophoblast: insights into female meiotic errors and chromosomal segregation in the preimplantation window of embryo development. Human Reproduction. 2013;28(2)509–518. doi: 10.1093/humrep/des394 23148203

12. Ottolini CS, Newnham LJ, Capalbo A, Natesan SA, Joshi HA, Cimadomo D, et al. Genome-wide maps of recombination and chromosome segregation in human oocytes and embryos show selection for maternal recombination rates. Nature Genetics. 2015;47:727–735. doi: 10.1038/ng.3306 25985139

13. Mantikou E, Wong KM, Repping S, Mastenbroek S. Molecular origin of mitotic aneuploidies in preimplantation embryos. Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease. 2012;1822(12):1921–1930. doi: 10.1016/j.bbadis.2012.06.013

14. van Echten-Arends J, Mastenbroek S, Sikkema-Raddatz B, Korevaar JC, Heineman MJ, van der Veen F, et al. Chromosomal mosaicism in human preimplantation embryos: a systematic review. Human Reproduction Update. 2011;17(5):620–627. doi: 10.1093/humupd/dmr014 21531753

15. Mertzanidou A, Wilton L, Cheng J, Spits C, Vanneste E, Moreau Y, et al. Microarray analysis reveals abnormal chromosomal complements in over 70% of 14 normally developing human embryos. Human Reproduction. 2013;28(1):256–264. doi: 10.1093/humrep/des362 23054067

16. Chow JF, Yeung WS, Lau EY, Lee VC, Ng EH, Ho PC. Array comparative genomic hybridization analyses of all blastomeres of a cohort of embryos from young IVF patients revealed significant contribution of mitotic errors to embryo mosaicism at the cleavage stage. Reproductive Biology and Endocrinology. 2014;12(1):105. doi: 10.1186/1477-7827-12-105 25420429

17. Huang J, Yan L, Fan W, Zhao N, Zhang Y, Tang F, et al. Validation of multiple annealing and looping-based amplification cycle sequencing for 24-chromosome aneuploidy screening of cleavage-stage embryos. Fertility and Sterility. 2014;102(6):1685–1691. doi: 10.1016/j.fertnstert.2014.08.015 25241375

18. Taylor TH, Gitlin SA, Patrick JL, Crain JL, Wilson JM, Griffin DK. The origin, mechanisms, incidence and clinical consequences of chromosomal mosaicism in humans. Human Reproduction Update. 2014;20(4):571–581. doi: 10.1093/humupd/dmu016 24667481

19. Harrison R, Kuo HC, Scriven P, Handyside A, Ogilvie CM. Lack of cell cycle checkpoints in human cleavage stage embryos revealed by a clonal pattern of chromosomal mosaicism analysed by sequential multicolour FISH. Zygote. 2000;8(03):217–224. doi: 10.1017/S0967199400001015 11014501

20. Barbash-Hazan S, Frumkin T, Malcov M, Yaron Y, Cohen T, Azem F, et al. Preimplantation aneuploid embryos undergo self-correction in correlation with their developmental potential. Fertility and Sterility. 2009;92(3):890–896. doi: 10.1016/j.fertnstert.2008.07.1761 18829021

21. Bielanska M, Tan SL, Ao A. Chromosomal mosaicism throughout human preimplantation development in vitro: incidence, type, and relevance to embryo outcome. Human Reproduction. 2002;17(2):413–419. doi: 10.1093/humrep/17.2.413 11821287

22. Fragouli E, Lenzi M, Ross R, Katz-Jaffe M, Schoolcraft W, Wells D. Comprehensive molecular cytogenetic analysis of the human blastocyst stage. Human Reproduction. 2008;23(11):2596–2608. doi: 10.1093/humrep/den287 18664475

23. Santos MA, Teklenburg G, Macklon NS, Van Opstal D, Schuring-Blom GH, Krijtenburg PJ, et al. The fate of the mosaic embryo: chromosomal constitution and development of Day 4, 5 and 8 human embryos. Human Reproduction. 2010;25(8):1916–1926. doi: 10.1093/humrep/deq139 20519247

24. Pinkel D, Albertson DG. Comparative genomic hybridization. Annual Review of Genomics and Human Genetics. 2005;6:331–354. doi: 10.1146/annurev.genom.6.080604.162140 16124865

25. Franasiak JM, Forman EJ, Hong KH, Werner MD, Upham KM, Treff NR, et al. The nature of aneuploidy with increasing age of the female partner: a review of 15,169 consecutive trophectoderm biopsies evaluated with comprehensive chromosomal screening. Fertility and Sterility. 2014;101(3):656–663. doi: 10.1016/j.fertnstert.2013.11.004 24355045

26. Sandalinas M, Sadowy S, Alikani M, Calderon G, Cohen J, Munné S. Developmental ability of chromosomally abnormal human embryos to develop to the blastocyst stage. Human Reproduction. 2001;16(9):1954–1958. doi: 10.1093/humrep/16.9.1954 11527904

27. Adler A, Lee HL, McCulloh DH, Ampeloquio E, Clarke-Williams M, Wertz BH, et al. Blastocyst culture selects for euploid embryos: comparison of blastomere and trophectoderm biopsies. Reproductive Biomedicine Online. 2014;28(4):485–491. doi: 10.1016/j.rbmo.2013.11.018 24581980

28. Erickson JD. Down syndrome, paternal age, maternal age and birth order. Annals of Human Genetics. 1978;41(3):289–298. doi: 10.1111/j.1469-1809.1978.tb01896.x 147046

29. Hassold T, Chiu D. Maternal age-specific rates of numerical chromosome abnormalities with special reference to trisomy. Human Genetics. 1985;70(1):11–17. doi: 10.1007/BF00389450 3997148

30. Baart EB, Martini E, Eijkemans MJ, Van Opstal D, Beckers NG, Verhoeff A, et al. Milder ovarian stimulation for in-vitro fertilization reduces aneuploidy in the human preimplantation embryo: a randomized controlled trial. Human Reproduction. 2007;22(4):980–988. doi: 10.1093/humrep/del484 17204525

31. Verpoest W, Fauser B, Papanikolaou E, Staessen C, Van Landuyt L, Donoso P, et al. Chromosomal aneuploidy in embryos conceived with unstimulated cycle IVF. Human Reproduction. 2008;23(10):2369–2371. doi: 10.1093/humrep/den269 18621755

32. Yakut T, Karkucak M, Sher G, Keskintepe L. Comparison of aneuploidy frequencies between in vitro matured and unstimulated cycles oocytes by metaphase comparative genomic hybridization (mCGH). Molecular Biology Reports. 2012;39(5):6187–6191. doi: 10.1007/s11033-011-1436-4 22207182

33. Hassold TJ. Chromosome abnormalities in human reproductive wastage. Trends in Genetics. 1986;2:105–110. doi: 10.1016/0168-9525(86)90194-0

34. for Disease Control C, Prevention SfART American Society for Reproductive Medicine. 2011 Assisted Reproductive Technology National Summary Report. US Department of Health and Human Services, CDC, Atlanta, Ga, USA. 2013;.

35. Fonseka K, Griffin D. Is there a paternal age effect for aneuploidy? Cytogenetic and Genome Research. 2011;133(2–4):280–291. doi: 10.1159/000322816 21212646

36. Rabinowitz M, Ryan A, Gemelos G, Hill M, Baner J, Cinnioglu C, et al. Origins and rates of aneuploidy in human blastomeres. Fertility and Sterility. 2012;97(2):395–401. doi: 10.1016/j.fertnstert.2011.11.034 22195772

37. McCoy RC, Demko Z, Ryan A, Banjevic M, Hill M, Sigurjonsson S, et al. Common variants spanning PLK4 are associated with mitotic-origin aneuploidy in human embryos. Science. 2015;348(6231):235–238. doi: 10.1126/science.aaa3337 25859044

38. Jacobs P, Angell R, Buchanan I, Hassold T, Matsuyama A, Manuel B. The origin of human triploids. Annals of Human Genetics. 1978;42(1):49–57. doi: 10.1111/j.1469-1809.1978.tb00930.x 686684

39. Campbell A, Fishel S. Atlas of Time Lapse Embryology. CRC Press; 2015.

40. Kim YJ, Ku SY, Jee BC, Suh CS, Kim SH, Choi YM, et al. Increased responsiveness may be associated with tripronuclear embryos in both conventional in vitro fertilization and intracytoplasmic sperm injection cycles using gonadotropin-releasing hormone agonist long protocols: A self-matched observational study. Journal of Obstetrics and Gynaecology Research. 2011;37(1):37–44. doi: 10.1111/j.1447-0756.2010.01313.x 21040208

41. Figueira RC, Setti AS, Braga DP, Iaconelli A Jr, Borges E Jr. Prognostic value of triploid zygotes on intracytoplasmic sperm injection outcomes. Journal of Assisted Reproduction and Genetics. 2011;28(10):879–883. doi: 10.1007/s10815-011-9610-0 21805146

42. Rosenbusch B. The Chromosomal Constitution of Embryos Arising from Monopronuclear Oocytes in Programmes of Assisted Reproduction. International Journal of Reproductive Medicine. 2014;2014. doi: 10.1155/2014/418198 25763399

43. Kuliev A, Cieslak J, Ilkevitch Y, Verlinsky Y. Chromosomal abnormalities in a series of 6733 human oocytes in preimplantation diagnosis for age-related aneuploidies. Reproductive Biomedicine Online. 2003;6(1):54–59. doi: 10.1016/S1472-6483(10)62055-X 12626143

44. Fragouli E, Wells D, Whalley K, Mills J, Faed M, Delhanty J. Increased susceptibility to maternal aneuploidy demonstrated by comparative genomic hybridization analysis of human MII oocytes and first polar bodies. Cytogenetic and Genome Research. 2005;114(1):30–38. doi: 10.1159/000091925

45. Forman E, Treff N, Stevens J, Garnsey H, Katz-Jaffe M, Scott R, et al. Embryos whose polar bodies contain isolated reciprocal chromosome aneuploidy are almost always euploid. Human Reproduction. 2013;28(2):502–508. doi: 10.1093/humrep/des393 23169867

46. Lathi RB, Westphal LM, Milki AA. Aneuploidy in the miscarriages of infertile women and the potential benefit of preimplanation genetic diagnosis. Fertility and Sterility. 2008;89(2):353–357. doi: 10.1016/j.fertnstert.2007.02.040 17509575

47. Ljunger E, Cnattingius S, Lundin C, Annerén G. Chromosomal anomalies in first-trimester miscarriages. Acta Obstetricia et Gynecologica Scandinavica. 2005;84(11):1103–1107.

48. Yusuf RZ, Naeem R. Cytogenetic abnormalities in products of conception: a relationship revisited. American Journal of Reproductive Immunology. 2004;52(1):88–96. doi: 10.1111/j.1600-0897.2004.00196.x 15214948

49. Rolnik DL, Carvalho MHBd, Catelani ALPM, Pinto APAR, Lira JBG, Kusagari NK, et al. Cytogenetic analysis of material from spontaneous abortion. Revista da Associação Médica Brasileira. 2010;56(6):681–683. doi: 10.1590/S0104-42302010000600017 21271135

50. Menasha J, Levy B, Hirschhorn K, Kardon NB. Incidence and spectrum of chromosome abnormalities in spontaneous abortions: new insights from a 12-year study. Genetics in Medicine. 2005;7(4):251–263. doi: 10.1097/01.GIM.0000160075.96707.04 15834243

51. Nagaishi M, Yamamoto T, Iinuma K, Shimomura K, Berend SA, Knops J. Chromosome abnormalities identified in 347 spontaneous abortions collected in Japan. Journal of Obstetrics and Gynaecology Research. 2004;30(3):237–241. doi: 10.1111/j.1447-0756.2004.00191.x 15210050

52. Templado C, Vidal F, Estop A. Aneuploidy in human spermatozoa. Cytogenetic and Genome Research. 2011;133(2–4):91–99. doi: 10.1159/000323795 21282942

53. Rius M, Daina G, Obradors A, Ramos L, Velilla E, Fernández S, et al. Comprehensive embryo analysis of advanced maternal age–related aneuploidies and mosaicism by short comparative genomic hybridization. Fertility and Sterility. 2011;95(1):413–416. doi: 10.1016/j.fertnstert.2010.07.1051 20797709

54. Munné S, Sandalinas M, Escudero T, Márquez C, Cohen J. Chromosome mosaicism in cleavage-stage human embryos: evidence of a maternal age effect. Reproductive Biomedicine Online. 2002;4(3):223–232. doi: 10.1016/S1472-6483(10)61810-X 12709271

55. Thomas NS, Ennis S, Sharp AJ, Durkie M, Hassold TJ, Collins AR, et al. Maternal sex chromosome non-disjunction: evidence for X chromosome-specific risk factors. Human Molecular Genetics. 2001;10(3):243–250. doi: 10.1093/hmg/10.3.243 11159943

56. Katz-Jaffe MG, Trounson AO, Cram DS. Chromosome 21 mosaic human preimplantation embryos predominantly arise from diploid conceptions. Fertility and Sterility. 2005;84(3):634–643. doi: 10.1016/j.fertnstert.2005.03.045 16169396

57. Antonarakis SE, Avramopoulos D, Blouin JL, Talbot CC, Schinzel AA. Mitotic errors in somatic cells cause trisomy 21 in about 4.5% of cases and are not associated with advanced maternal age. Nature Genetics. 1993;3(2):146–150. doi: 10.1038/ng0293-146 8499948

58. Daphnis D, Fragouli E, Economou K, Jerkovic S, Craft I, Delhanty J, et al. Analysis of the evolution of chromosome abnormalities in human embryos from Day 3 to 5 using CGH and FISH. Molecular Human Reproduction. 2008;14(2):117–125. doi: 10.1093/molehr/gam087 18222960

59. Delhanty JD, Harper JC, Ao A, Handyside AH, Winston RM. Multicolour FISH detects frequent chromosomal mosaicism and chaotic division in normal preimplantation embryos from fertile patients. Human Genetics. 1997;99(6):755–760. doi: 10.1007/s004390050443 9187668

60. Coonen E, Derhaag JG, Dumoulin JC, van Wissen LC, Bras M, Janssen M, et al. Anaphase lagging mainly explains chromosomal mosaicism in human preimplantation embryos. Human Reproduction. 2004;19(2):316–324. doi: 10.1093/humrep/deh077 14747173

61. Hardy K, Winston R, Handyside A. Binucleate blastomeres in preimplantation human embryos in vitro: failure of cytokinesis during early cleavage. Journal of Reproduction and Fertility. 1993;98(2):549–558. doi: 10.1530/jrf.0.0980549 8410824

62. Holland AJ, Cleveland DW. Chromoanagenesis and cancer: mechanisms and consequences of localized, complex chromosomal rearrangements. Nature Medicine. 2012;18(11):1630–1638. doi: 10.1038/nm.2988 23135524

63. Daphnis D, Delhanty J, Jerkovic S, Geyer J, Craft I, Harper J. Detailed FISH analysis of day 5 human embryos reveals the mechanisms leading to mosaic aneuploidy. Human Reproduction. 2005;20(1):129–137. doi: 10.1093/humrep/deh554 15567885

64. Chatzimeletiou K, Morrison EE, Prapas N, Prapas Y, Handyside AH. Spindle abnormalities in normally developing and arrested human preimplantation embryos in vitro identified by confocal laser scanning microscopy. Human Reproduction. 2005;20(3):672–682. doi: 10.1093/humrep/deh652 15689349

65. Kalatova B, Jesenska R, Hlinka D, Dudas M. Tripolar mitosis in human cells and embryos: Occurrence, pathophysiology and medical implications. Acta Histochemica. 2015;117(1):111–125. doi: 10.1016/j.acthis.2014.11.009 25554607

66. Ganem NJ, Godinho SA, Pellman D. A mechanism linking extra centrosomes to chromosomal instability. Nature. 2009;460(7252):278–282. doi: 10.1038/nature08136 19506557

67. Scriven P, Handyside AH, Ogilvie CM. Chromosome translocations: segregation modes and strategies for preimplantation genetic diagnosis. Prenatal Diagnosis. 1998;18(13):1437–1449. doi: 10.1002/(SICI)1097-0223(199812)18:13%3C1437::AID-PD497%3E3.0.CO;2-P 9949444

68. Mackie OC, Scriven P. Meiotic outcomes in reciprocal translocation carriers ascertained in 3-day human embryos. European Journal of Human Genetics: EJHG. 2002;10(12):801–806. doi: 10.1038/sj.ejhg.5200895

69. Pehlivan T, Rubio C, Rodrigo L, Romero J, Remohi J, Simon C, et al. Impact of preimplantation genetic diagnosis on IVF outcome in implantation failure patients. Reproductive Biomedicine Online. 2003;6(2):232–237. doi: 10.1016/S1472-6483(10)61715-4 12676006

70. Gandolfi TB, Gandolfi F. The maternal legacy to the embryo: cytoplasmic components and their effects on early development. Theriogenology. 2001;55(6):1255–1276. doi: 10.1016/S0093-691X(01)00481-2 11327683

71. O’Brien KL, Varghese AC, Agarwal A. The genetic causes of male factor infertility: a review. Fertility and Sterility. 2010;93(1):1–12. doi: 10.1016/j.fertnstert.2009.10.045

72. Krausz C. Male infertility: pathogenesis and clinical diagnosis. Best practice & research Clinical endocrinology & metabolism. 2011;25(2):271–285. doi: 10.1016/j.beem.2010.08.006

73. Fragouli E, Wells D, Thornhill A, Serhal P, Faed M, Harper J, et al. Comparative genomic hybridization analysis of human oocytes and polar bodies. Human Reproduction. 2006;21(9):2319–2328. doi: 10.1093/humrep/del157 16704993

74. Pellestor F, Andréo B, Arnal F, Humeau C, Demaille J. Maternal aging and chromosomal abnormalities: new data drawn from in vitro unfertilized human oocytes. Human Genetics. 2003;112(2):195–203. 12522562

75. Franasiak JM, Forman EJ, Hong KH, Werner MD, Upham KM, Treff NR, et al. Aneuploidy across individual chromosomes at the embryonic level in trophectoderm biopsies: changes with patient age and chromosome structure. Journal of Assisted Reproduction and Genetics. 2014;31(11):1–9. doi: 10.1007/s10815-014-0333-x

76. Steiner B, Masood R, Rufibach K, Niedrist D, Kundert O, Riegel M, et al. An unexpected finding: younger fathers have a higher risk for offspring with chromosomal aneuploidies. European Journal of Human Genetics. 2014;23:466–472. doi: 10.1038/ejhg.2014.122 25005732

77. Ruangvutilert P, Delhanty JD, Serhal P, Simopoulou M, Rodeck CH, Harper JC. FISH analysis on day 5 post-insemination of human arrested and blastocyst stage embryos. Prenatal Diagnosis. 2000;20(7):552–560. doi: 10.1002/1097-0223(200007)20:7%3C552::AID-PD871%3E3.0.CO;2-F 10913953

78. Bielanska M, Jin S, Bernier M, Tan SL, Ao A. Diploid-aneuploid mosaicism in human embryos cultured to the blastocyst stage. Fertility and Sterility. 2005;84(2):336–342. doi: 10.1016/j.fertnstert.2005.03.031 16084874

79. Rubio C, Rodrigo L, Mercader A, Mateu E, Buendía P, Pehlivan T, et al. Impact of chromosomal abnormalities on preimplantation embryo development. Prenatal Diagnosis. 2007;27(8):748–756. doi: 10.1002/pd.1773 17546708

80. Treff NR, Levy B, Su J, Northrop LE, Tao X, Scott RT. SNP microarray-based 24 chromosome aneuploidy screening is significantly more consistent than FISH. Molecular Human Reproduction. 2010;16(8):583–589. doi: 10.1093/molehr/gaq039 20484246

81. Vega M, Breborowicz A, Moshier EL, McGovern PG, Keltz MD. Blastulation rates decline in a linear fashion from euploid to aneuploid embryos with single versus multiple chromosomal errors. Fertility and Sterility. 2014;102(2):394–398. doi: 10.1016/j.fertnstert.2014.04.026 24890269

82. Johnson DS, Cinnioglu C, Ross R, Filby A, Gemelos G, Hill M, et al. Comprehensive analysis of karyotypic mosaicism between trophectoderm and inner cell mass. Molecular Human Reproduction. 2010;16(12):944–949. doi: 10.1093/molehr/gaq062 20643877

83. Liu J, Wang W, Sun X, Liu L, Jin H, Li M, et al. DNA microarray reveals that high proportions of human blastocysts from women of advanced maternal age are aneuploid and mosaic. Biology of Reproduction. 2012;87(6):148. doi: 10.1095/biolreprod.112.103192 23136294

84. Hlinka D, Kalatova B, Uhrinova I, Dolinska S, Rutarova J, Rezacova J, et al. Time-lapse cleavage rating predicts human embryo viability. Physiological Research. 2012;61(5):513–25. 22881225

85. R Core Team. R: A Language and Environment for Statistical Computing. Vienna, Austria; 2013. Available from: http://www.R-project.org/