Comprehensive Chromosomal and Mitochondrial Copy Number Profiling in Human IVF Embryos

ARTICLE IN PRESS

1bs_bs_query Q2 Article

2bs_bs_query

3bs_bs_query Comprehensive chromosomal and mitochondrial copy

4bs_bs_query

5bs_bs_query number proﬁling in human IVF embryos

6bs_bs_query Q1 a,1, a,1 a a 7bs_bs_query Wei Shang *, Yunshan Zhang , Mingming Shu , Weizhou Wang , a a b b, b b 8bs_bs_query Likun Ren , Fu Chen , Lin Shao , Sijia Lu *, Shiping Bo , Shujie Ma , b 9bs_bs_query Yumei Gao

a 10bs_bs_query Assisted Reproductive Centre of the Department of Gynaecology and Obstetrics, PLA Naval General Hospital,

11 bs_bs_query Haidian District, Beijing 100048, China b 12bs_bs_query Yikon Genomics, Fengxian District, Shanghai 201400, China

13bs_bs_query

14bs_bs_query

15bs_bs_query Wei Shang has been the Associate Chief Physician at the Department of Gynaecology and Obstetrics, PLA Naval

16bs_bs_query General Hospital, Beijing, since 2005. Her research interests focus on assisted reproduction technology, repro-

17bs_bs_query ductive endocrinology and ovary dysfunction.

18bs_bs_query

19bs_bs_query KEY MESSAGE

20bs_bs_query Using a validated approach called MALBAC-NGS, a comprehensive chromosomal and mitochondrial copy number

21bs_bs_query proﬁling in human embryos was conducted, and correlations of mitochondria quantity with maternal age and

22bs_bs_query embryo stage were observed. The strategy might be used to perform an advanced PGS targeting both chro-

23bs_bs_query mosomal and mitochondria copy numbers.

24bs_bs_query

25bs_bs_query ABSTRACT

26bs_bs_query

27bs_bs_query Single cell whole genome sequencing helps to decipher the genome heterogeneity within a cell population and facilitates the analysis of trace amounts

28bs_bs_query of genetic material, such as is found in human embryos. The mitochondrial genome, although an important part of the genetic composition of eukary-

29bs_bs_query otic cells, is often neglected in single cell genome analysis. A recently developed single cell whole genome ampliﬁcation method was used, known as

30bs_bs_query multiple annealing and looping based ampliﬁcation cycles (MALBAC-NGS), for simultaneous analysis of chromosomal and mitochondrial genomes at

31bs_bs_query the single cell level. The platform was validated by a series of technical and biological replicates and used for chromosomal and mitochondrial copy β 32bs_bs_query number analysis in 399 in-vitro fertilized embryos from 81 couples. A positive correlation of maternal age with increased mitochondria quantity ( =

33bs_bs_query 0.176, P = 0.001) was observed after adjusting for the impact of cell type. Lower numbers of mitochondria were detected in successfully implanted

34bs_bs_query embryos, although the difference was not signiﬁcant. It is proposed that MALBAC-NGS could potentially be used for an advanced pre-implantation genetic

35bs_bs_query screening procedure with both chromosomal constitution and mitochondrial copy number being evaluated.

37bs_bs_query

38bs_bs_query

39bs_bs_query * Corresponding authors.

40bs_bs_query E-mail addresses: [email protected] (W Shang); [email protected] (S Lu). 1 41bs_bs_query These authors contributed equally to the manuscript.

42bs_bs_query https://doi.org/10.1016/j.rbmo.2017.10.110

Please cite this article in press as: Wei Shang, et al., Comprehensive chromosomal and mitochondrial copy number proﬁling in human IVF embryos, Reproductive BioMedicine Online (2017), doi: 10.1016/j.rbmo.2017.10.110 ARTICLE IN PRESS

2 REPRODUCTIVE BIOMEDICINE ONLINE ■■ (2017) ■■– ■■

44bs_bs_query

cells were pooled and genomic DNA was isolated following the pro- 104bs_bs_query

45bs_bs_query Introduction tocol provided by the manufacturer (KW Biotech, China). 105bs_bs_query

46bs_bs_query 106bs_bs_query

47bs_bs_query Single cell genomic studies have been carried out to reveal indi- Human samples 107bs_bs_query

48bs_bs_query vidual cellular behaviours that are not observable in bulk 108bs_bs_query

49bs_bs_query measurements (Kalisky and Quake, 2011; Macaulay and Voet, 2014), Eighty-one couples were recruited from the Assisted Reproductive 109bs_bs_query

50bs_bs_query which necessitates the development of whole genome ampliﬁcation Centre of the Department of Gynaecology and Obstetrics, PLA Navy 110 bs_bs_query

51bs_bs_query (WGA) and genotyping methods. Various single cell WGA and se- General Hospital during 2015–2016. The use of human samples in this 111 bs_bs_query

52bs_bs_query quencing methods have been developed to facilitate genome analysis study was approved by the Ethics Committee of PLA Navy General 112 bs_bs_query

53bs_bs_query of trace amounts of starting materials, such as degenerate Hospital in March 2015. Written consent forms were signed by all the 113 bs_bs_query

54bs_bs_query oligonucleotide-primed PCR (DOP-PCR) (Carter et al., 1992), mul- participants before the embryos were used for pre-implantation genetic 114 bs_bs_query

55bs_bs_query tiple displacement ampliﬁcation (MDA) (Dean et al., 2002), and multiple screening purposes. The ages of the women were available from 64 115 bs_bs_query

56bs_bs_query annealing and looping based ampliﬁcation cycles (MALBAC) (Zong couples. The parent karyotypes were known for 36 couples. 116 bs_bs_query

57bs_bs_query et al., 2012). These methods have successfully achieved analysis of 117 bs_bs_query

58bs_bs_query chromosomal copy number variation (CNV) and single nucleotide varia- IVF, embryo biopsy and cryopreservation 118 bs_bs_query

59bs_bs_query tion (SNV) at the single cell level in various downstream applications, 119 bs_bs_query

60bs_bs_query such as in studying tumour evolution (Navin et al., 2011; Wang et al., IVF embryo biopsy procedures were performed following the stan- 120bs_bs_query

61bs_bs_query 2012), meiotic recombination (Lu et al., 2012; Wang et al., 2012), and dard published protocol (Hou et al., 2013). In brief, fertilization of MII 121bs_bs_query

62bs_bs_query performing pre-implantation genetic screening (PGS) of in-vitro fer- oocytes was achieved by intracytoplasmic sperm injection (ICSI). Fol- 122bs_bs_query

63bs_bs_query tilized embryos (Hou et al., 2013; Wells et al., 2014). lowing the ICSI procedure (day 0), MII oocytes were incubated in G1 123bs_bs_query

64bs_bs_query Mitochondria are crucial subcellular organelles in maintaining medium (Vitrolife, Sweden). Embryos were cultured for 48 h to the 124bs_bs_query

65bs_bs_query energy metabolism and normal function of eukaryotic cells. The human cleavage stage and then transferred to G2 medium (Vitrolife, Sweden) 125bs_bs_query

66bs_bs_query mitochondrial genome is a circular DNA molecule of ~16 kilobases, for 48–72 h. Single cells were biopsied from the 8-cell stages or 3–5 126bs_bs_query

67bs_bs_query encoding important genes involved in respiratory function (Krebs et al., trophectoderm cells (TE cells) from the hatching blastocysts. Biopsied 127bs_bs_query

68bs_bs_query 2009). Point mutations and structural alterations of these genes often blastomeres and TE cells were then cryopreserved by the vitriﬁca- 128bs_bs_query

69bs_bs_query result in severe diseases such as cerebella ataxia, seizures, cardio- tion method according to the recommended protocol (Cook, USA). 129bs_bs_query

70bs_bs_query myopathy, optic atrophy or bilateral deafness (McFarland et al., 2002; Embryos were ﬁrst equilibrated for 5 min at room temperature fol- 130bs_bs_query

71bs_bs_query Schaefer et al., 2008; Zeviani et al., 1995), and therefore it is of sig- lowed by incubation for 1 min in the vitriﬁcation solution, and then 131bs_bs_query

72bs_bs_query niﬁcant clinical importance to analyse these mitochondrial variations. loaded into a carrier and immediately plunged into liquid nitrogen for 132bs_bs_query

73bs_bs_query With the rapid development of next-generation sequencing (NGS) tech- long-term storage. 133bs_bs_query

74bs_bs_query niques, previous studies have shown that mitochondrial genome 134bs_bs_query

75bs_bs_query information, including SNV and CNV, can be retrieved from the NGS WGA and sequencing 135bs_bs_query

76bs_bs_query data (Castle et al., 2010; Fragouli et al., 2015; Zaragoza et al., 2010). 136bs_bs_query

77bs_bs_query In this study, a recently developed single cell WGA protocol called The MALBAC method (Zong et al., 2012) was used for WGA on a 1:100 137bs_bs_query

78bs_bs_query multiple annealing and looping based ampliﬁcation cycles (MALBAC) dilution of the genomic DNA from 100 AFP cells, single AFP cells as 138bs_bs_query

79bs_bs_query (Zong et al., 2012) was used to establish and validate the procedure well as on the biopsies of the in-vitro fertilized embryos, following 139bs_bs_query

80bs_bs_query for single cell chromosomal and mitochondrial copy number proﬁl- the standard protocol provided by the manufacturer (Yikon Genom- 140bs_bs_query

81bs_bs_query ing. The validated approach was then used to perform PGS with in- ics, China). In brief, the ampliﬁcation was initiated by annealing the 141bs_bs_query

82bs_bs_query vitro fertilized embryos, with copy number proﬁling on both DNA to a pool of random primers, each having a common 27- 142bs_bs_query

83bs_bs_query chromosomal and mitochondrial genomes. nucleotide sequence and eight variable nucleotides. A quasi-linear 143bs_bs_query

84bs_bs_query pre-ampliﬁcation step was performed before exponentially amplify- 144bs_bs_query

bs_bs_query µ 85 ing the DNA to ~2 g for NGS. 145bs_bs_query

bs_bs_query 86bs_bs_query Materials and methods About 400 ng puriﬁed DNA was sonicated to ~150–250 bp frag- 146

bs_bs_query 87bs_bs_query ments with the Covaris S2 system (Covaris, USA) and puriﬁed with 147

88bs_bs_query Human ﬁbroblast cell line culture, single cell isolation and the Qiagen PCR Puriﬁcation Kit (Qiagen, Germany), and then was used 148bs_bs_query

89bs_bs_query genomic DNA extraction to construct sequencing libraries using the NEBNext Ultra DNA Library 149bs_bs_query

90bs_bs_query Prep Kit (NEB, USA). The constructed libraries were sequenced on 150bs_bs_query

91bs_bs_query The human ﬁbroblast cell line was kindly provided by Haidian Ma- an Illumina HiSeq 2500 platform (Illumina, USA). 151bs_bs_query

92bs_bs_query ternal and Child Health Hospital. The cells were cultured with 152bs_bs_query

93bs_bs_query Dulbecco’s modiﬁed Eagle’s medium (DMEM) supplemented with 10% Data analysis 153bs_bs_query

94bs_bs_query heat-inactivated fetal bovine serum (FBS, Invitrogen, USA), 1% 154bs_bs_query

95bs_bs_query penicillin-streptomycin (PS, Invitrogen, USA), at 37°C in a humidi- Using an Illumina HiSeq 2500 platform, the MALBAC-ampliﬁed genome 155bs_bs_query

96bs_bs_query ﬁed incubator containing 5% CO2. When the cells were 90% conﬂuent, of each sample was sequenced for a total of ~1.5 million reads. Chro- 156bs_bs_query

97bs_bs_query they were washed three times, then detached by 0.25% trypsin with mosomal CNV analysis was performed following the procedure 157bs_bs_query

98bs_bs_query 0.1% ethylenediaminetetraacetic acid (EDTA; Invitrogen, USA), and were previously described (Zong et al., 2012). In brief, after the removal 158bs_bs_query

99bs_bs_query centrifuged at 150 g for 5 min. The supernatant was discarded, and of the lllumina adaptors and low-quality bases, high-quality reads were 159bs_bs_query

100bs_bs_query the cells were resuspended with fresh DMEM + FBS medium. Cells mapped to the hg19 reference genome using the BWA (Burrows- 160bs_bs_query

101bs_bs_query with more than eight passages were discarded. Detached cells were Wheeler Alignment Tool, version 0.7.12-r1039; Li and Durbin, 2010) 161bs_bs_query

102bs_bs_query resuspended and diluted in PBS buffer and single cells were iso- with default parameters. The duplicate reads were then removed. 162bs_bs_query

103bs_bs_query lated under the microscope via mouth pipetting. A total of 100 single Uniquely mapped reads were extracted from the alignment reads. The 163bs_bs_query

REPRODUCTIVE BIOMEDICINE ONLINE ■■ (2017) ■■– ■■ 3

164bs_bs_query Table 1 – Details of the sequencing reads, duplication rate and the mitochondrial copy numbers (MCN) of three technical replicates.

165bs_bs_query Sample Mapped Duplicate Reads mapped Duplicate rate of reads MCN log2(MCN)

166bs_bs_query reads rate, % to Mt mapped to Mt, %

167bs_bs_query Tech-Rep 1 1,401,598 0.34 730 2.74 160 7.3

168bs_bs_query Tech-Rep 2 1,463,620 0.34 789 2.03 170 7.4

169bs_bs_query Tech-Rep 3 1,473,476 0.88 773 2.46 167 7.4

170bs_bs_query

203bs_bs_query

171bs_bs_query whole reference genome was divided into non-overlapping observa-

Results 204bs_bs_query 172bs_bs_query tion windows (bins) with a size of 1 Mb. Read numbers and GC content

205bs_bs_query 173bs_bs_query were calculated in each bin. In order to remove bias, the bin read count

MALBAC method generated reproducible results for both 206bs_bs_query 174bs_bs_query was normalized based on GC content and a reference dataset to rep-

chromosomal and mitochondrial copy numbers 207bs_bs_query 175bs_bs_query resent the relative copy number. Copy number gain from two to three

208bs_bs_query 176bs_bs_query copies results in a 50% increase in read count in a particular genome

Three technical replicates of single cell-equivalent genomic DNA ma- 209bs_bs_query 177bs_bs_query segment, while copy number loss from two to one results in a 50%

terials from the human ﬁbroblast cell line were performed. These 210bs_bs_query 178bs_bs_query decrease in read count in a genome segment. Mitochondrial content

samples were sequenced on an Illumina HiSeq 2500 platform for ~1.5 211bs_bs_query 179bs_bs_query was quantiﬁed in the form of copy number/nuclear genome. Se-

million reads each (Table 1). In order to calculate the relative copy 212bs_bs_query 180bs_bs_query quencing reads mapped to mitochondrial genome were counted and < number of the mitochondria, all duplicate reads ( 1%) were removed 213bs_bs_query 181bs_bs_query normalized by the read count mapped to the autosomal chromo-

and sequencing reads that were aligned to the mitochondrial genome 214bs_bs_query 182bs_bs_query somes, and mitochondrial copy numbers per nuclear genome (MCN)

were counted and normalized with the reads mapped to the nuclear 215bs_bs_query 183bs_bs_query were calculated and displayed according to the formula:

chromosomes. Notably, the duplicate rate of the reads mapped to the 216bs_bs_query 184bs_bs_query

mitochondrial genome is ~2%. Details of the reads and copy numbers 217bs_bs_query 185bs_bs_query ∗∗ = autosome mappable region2 mitochondrion mapped reads MCN of all chromosomes and mitochondria are shown in Figure 1 and 218bs_bs_query auutosome mapped reads∗ mitochondrion mappable region 186bs_bs_query Table 1. The result conﬁrmed a normal karyotype of the cell line (46, 219bs_bs_query

XY) and highly consistent MCN among replicates, showing the re- 220bs_bs_query 187bs_bs_query Q3 Scripts can be found at https://github.com/bspatYK/mitochondrion

peatability of MALBAC ampliﬁcation and sequencing on both 221bs_bs_query 188bs_bs_query _copy_number.

chromosomal and mitochondrial DNA at the single cell level. 222bs_bs_query 189bs_bs_query

223bs_bs_query 190bs_bs_query Statistical analysis

191bs_bs_query MALBAC method revealed a concordance in chromosomal 224bs_bs_query

192bs_bs_query Student’s t-test and two-way ANOVA were carried out to investigate copy number, but a slight variation in mitochondria copy 225bs_bs_query

193bs_bs_query the differences in MCN between different groups. A multivariable linear number between different subcultured lines 226bs_bs_query

194bs_bs_query regression was performed to study the impact of embryo type, chro- 227bs_bs_query

195bs_bs_query mosomal abnormality as well as female age on the mtDNA copy Two groups of biological triplicates were performed. Three single 228bs_bs_query

196bs_bs_query number. Spearman’s test was carried out to study the correlation of human ﬁbroblast cells were collected from each of the two subcul- 229bs_bs_query

197bs_bs_query the implantation outcome with embryo morphology. All the statisti- tured cell lines and the MALBAC ampliﬁcation and subsequent 230bs_bs_query

198bs_bs_query cal analysis was performed using SPSS 22.0 (IBM Corp., USA). sequencing was performed. Copy numbers of chromosomes and 231bs_bs_query

199bs_bs_query

200bs_bs_query Figure 1 – Chromosomal and mitochondrial copy number analysis of technical replicates. Technical replicates were performed with

201bs_bs_query genomic DNA from a 1:100 dilution of 100 human ﬁbroblast cells. Both chromosomal copy numbers and log2 of mitochondrial copy

202bs_bs_query numbers were calculated and displayed.

4 REPRODUCTIVE BIOMEDICINE ONLINE ■■ (2017) ■■– ■■

232bs_bs_query Figure 2 – Chromosomal and mitochondrial copy number analysis of biological replicates. Two groups of biological replicates of single

233bs_bs_query human ﬁbroblast cells were ampliﬁed and sequenced. Both chromosomal and log2 of mitochondrial copy numbers were calculated and

234bs_bs_query displayed.

235bs_bs_query

236bs_bs_query mitochondria were obtained by the method described previously, and the duplicate rate varied dramatically from 1% to 70% for reads 265bs_bs_query

237bs_bs_query shown in Figure 2 and Table 2. Among the triplicates within each popu- mapped to mitochondria. After the removal of duplicate reads, the 266bs_bs_query

238bs_bs_query lation, both chromosomal and mitochondrial copy numbers showed data generated an average coverage of ~85% for the mitochondrial 267bs_bs_query

239bs_bs_query highly concordant results, further conﬁrming the consistency of the genome (see also the demonstration in Supplementary Figure S1 and 268bs_bs_query

240bs_bs_query MALBAC-NGS method. A signiﬁcant difference in the MCN was ob- Supplementary Table S1). Chromosomal abnormalities were found 269bs_bs_query

241bs_bs_query served between the two subcultured populations (P = 0.001). in over 50% of the IVF-PGS embryos in this study (Supplementary 270bs_bs_query

242bs_bs_query Figure S2 and Supplementary Table S2), and the maternal age showed 271bs_bs_query

bs_bs_query 243bs_bs_query Comprehensive chromosomal and mitochondrial copy number a signiﬁcant positive correlation with embryo aneuploidy (r = 0.204, 272 < bs_bs_query 244bs_bs_query proﬁling on in-vitro fertilized embryos P 0.01; Supplementary Table S2). Therefore, performing PGS could 273

245bs_bs_query help to reduce the chances of miscarriage and repeated implanta- 274bs_bs_query

246bs_bs_query By using the method validated by both technical and biological rep- tion failure by selecting the embryos with normal karyotype. 275bs_bs_query

247bs_bs_query lications, PGS was performed on biopsied samples from in-vitro The data were extracted from 399 embryos. A total of 149 cleav- 276bs_bs_query

248bs_bs_query fertilized embryos of both the cleavage blastomere and D5 blasto- age stage embryos and 250 blastocysts were examined using NGS 277bs_bs_query

249bs_bs_query cyst stages. Approximately 1–2 million reads were obtained from each methodology. The quantity of mtDNA was evaluated based on the NGS 278bs_bs_query

250bs_bs_query sample with a duplication rate of ~1.3% for all mapped reads; however, analysis on a single blastomere (cleavage stage) or 3–5 TE cells (blas- 279bs_bs_query

251bs_bs_query

252bs_bs_query

253bs_bs_query Table 2 – Details of sequencing reads, duplication rate and the mitochondrial copy numbers (MCN) of the two groups of biological

254bs_bs_query replicates.

255bs_bs_query Sample Mapped Duplicate Reads mapped Duplicate rate of reads MCN Mean ± 256bs_bs_query reads rate, % to Mt mapped to Mt, % MCN SE* ± 257bs_bs_query Bio-Rep 1–1 1,659,418 0.46 802 2.74 140 142.33 8.45

258bs_bs_query Bio-Rep 1–2 1,262,363 0.39 656 2.44 158

259bs_bs_query Bio-Rep 1–3 1,139,987 0.46 504 2.58 129 ± 260bs_bs_query Bio-Rep 2–1 1,211,780 0.22 247 0.81 68 67.66 0.88

261bs_bs_query Bio-Rep 2–2 1,248,464 0.25 260 1.92 66

262bs_bs_query Bio-Rep 2–3 1,242,116 0.17 269 2.97 69

263bs_bs_query * P-value from Student’s t-test: 0.001.

264bs_bs_query

REPRODUCTIVE BIOMEDICINE ONLINE ■■ (2017) ■■– ■■ 5

280bs_bs_query Table 3 – The effect of karyotype and cell type on mitochondrial copy numbers (MCN) in embryo cells. Two-way ANOVA was performed.

281bs_bs_query Karyotype Cell type Mean SD N Tests of between-subject effects

282bs_bs_query MCN 283bs_bs_query Source F Sig.

284bs_bs_query Normal Blastomere 1124.46 765.390 61 Karyotype 0.001 NS

285bs_bs_query TE cell 556.55 684.488 128

286bs_bs_query Total 739.84 757.852 189 < 287bs_bs_query Abnormal Blastomere 1180.68 764.867 88 Cell type 75.823 0.000

288bs_bs_query TE cell 495.68 584.252 122

289bs_bs_query Total 782.73 745.606 210

290bs_bs_query Total Blastomere 1157.66 762.996 149 Karyotype *Cell type 0.662 NS

291bs_bs_query TE cell 526.84 637.003 250

292bs_bs_query Total 762.41 750.792 399

293bs_bs_query NS = not statistically signiﬁcant.

294bs_bs_query * Indicates the interaction between karyotype and cell type.

295bs_bs_query

296bs_bs_query β 297bs_bs_query tocysts). The data showed a signiﬁcant correlation of the cell type with hibited: (i) a positive association between age and MCN ( = 0.143); 339bs_bs_query β 298bs_bs_query mitochondria copies, with the TE cells harvesting fewer copies (ii) TE cells carried signiﬁcantly fewer MCN than blastomeres ( = 340bs_bs_query < 299bs_bs_query (Table 3). On the other hand, chromosome abnormality did not appear −0.672, P 0.001). Neither embryonic nor parental karyotype were 341bs_bs_query

300bs_bs_query to be correlated with the embryo MCN regardless of the cell type. associated with MCN signiﬁcantly. 342bs_bs_query

301bs_bs_query The information on maternal age was available for 333 embryos The association between the embryonic cell morphology and MCN 343bs_bs_query

302bs_bs_query out of 399. The linear regression analysis revealed a signiﬁcant cor- was also studied but was not statistically signiﬁcant (data not shown). 344bs_bs_query

303bs_bs_query relation of elevated MCN with advanced maternal age (ß = 0.176, P 345bs_bs_query

304bs_bs_query = 0.001, Table 4) after adjusting for the impact of the cell type. The association of the outcome of implanted embryos with 346bs_bs_query

305bs_bs_query Parental karyotype was known for 174 embryos out of 399 derived MCN and embryonic morphology 347bs_bs_query

306bs_bs_query from 36 couples. To study how all the factors (including the paren- 348bs_bs_query

307bs_bs_query tal and embryonic karyotype, maternal age, cell type) were correlated Thirty-one embryos with normal chromosomal copy number were 349bs_bs_query

308bs_bs_query with MCN, linear regression was carried out. Maternal age and cell transferred as single embryos. Two embryos out of 31 were at the 350bs_bs_query

309bs_bs_query type still showed signiﬁcant associations with MCN after adjusting cleavage stage and did not generate viable pregnancies. Among 351bs_bs_query

310bs_bs_query for the embryonic and parental karyotype (Table 5). The results ex- 29 embryos at the blastocyst stage, 11 did not result in a viable 352bs_bs_query

311bs_bs_query

312bs_bs_query

313bs_bs_query Table 4 – The effect of maternal age and cell type on mitochondrial copy numbers (MCN) in embryos was assessed by multivariable

314bs_bs_query linear regression. Age was treated as a continuous variable. Other independent variables were bi-categorical.

a 315bs_bs_query Model Coefﬁcients tP-value

316bs_bs_query Unstandardized coefﬁcients Standardized coefﬁcients

317bs_bs_query B SE Beta

318bs_bs_query (Constant) 203.976 306.215 0.666 NS

319bs_bs_query Age (continuous) 29.387 8.600 0.176 3.417 0.001

320bs_bs_query Karyotype (abnormal versus normal) −61.450 78.414 −0.040 −0.784 NS < 321bs_bs_query Cell type (TE cell versus blastomere) −591.677 81.798 −0.365 −7.233 0.001

322bs_bs_query NS = not statistically signiﬁcant. a 323bs_bs_query Dependent variable: MCN.

324bs_bs_query

325bs_bs_query

326bs_bs_query Table 5 – The association of age, cell type, embryonic karyotype and parental karyotype with mitochondrial copy numbers (MCN) by

327bs_bs_query multivariable linear regression. Age was treated as a continuous variable. Other independent variables were bi-categorical.

a 328bs_bs_query Model Coefﬁcients tP-value

329bs_bs_query Unstandardized coefﬁcients Standardized coefﬁcients

330bs_bs_query B SE Beta

331bs_bs_query (Constant) 458.335 182.691 2.509 0.013

332bs_bs_query Age (continuous) 13.322 5.418 0.143 2.459 0.015

333bs_bs_query Karyotype (abnormal versus normal) 36.489 46.989 0.043 0.777 NS < 334bs_bs_query Cell type (TE cell versus blastomere) −609.838 54.148 −0.672 −11.262 0.001

335bs_bs_query Parental karyotype (abnormal versus normal) −36.087 54.988 −0.041 −0.656 NS

336bs_bs_query NS = not statistically signiﬁcant. a 337bs_bs_query Dependent variable: MCN.

338bs_bs_query

6 REPRODUCTIVE BIOMEDICINE ONLINE ■■ (2017) ■■– ■■

353bs_bs_query Table 6 – The association of mitochondrial copy numbers (MCN) with the implantation outcome of blastocysts and embryonic

354bs_bs_query morphology.

a b 355bs_bs_query Embryo outcome N MCN Morphology

356bs_bs_query Mean SE FP-value Coefﬁcient P-value

357bs_bs_query Non-viable pregnancy 11 543.73 120.197 1.358 NS −0.238 NS

358bs_bs_query Ongoing pregnancy 18 395.67 67.336

359bs_bs_query Total 29 451.83 62.050

360bs_bs_query NS = not statistically signiﬁcant. a 361bs_bs_query ANOVA was performed to compare the MCN difference in two groups with different outcomes. b 362bs_bs_query Spearman correlation was performed to study the relationship between the outcome and the embryonic morphology.

363bs_bs_query

364bs_bs_query

365bs_bs_query pregnancy and 18 embryos generated successful ongoing preg- tendency to a larger mtDNA quantity with advanced maternal age in 411bs_bs_query

366bs_bs_query nancy or parturition. A greater MCN was observed in the embryos of both blastomeres and TE cells, but a decrease in PB (polar body) 412bs_bs_query

367bs_bs_query non-viable pregnancy than in the successfully implanted ones (543 mDNA with age (Konstantinidis et al., 2014). Using MALBAC-NGS this 413bs_bs_query

368bs_bs_query versus 395), but not signiﬁcantly so (Table 6). All the embryos at the study showed a concordant result. The association of mtDNA content 414bs_bs_query

369bs_bs_query blastocyst stage that implanted harboured MCN less than 1200 and with female age identiﬁed in this study was signiﬁcant, independent 415bs_bs_query

370bs_bs_query the 42 blastocysts with MCN greater than 1200 accounted for ~17% of embryo stage, embryonic karyotype and parental karyotype. A 416bs_bs_query

371bs_bs_query of the total 250 blastocysts being studied. The correlation between decline in the integrity of ‘older’ mitochondria and a consequent com- 417bs_bs_query

372bs_bs_query the embryonic morphology rank and the implantation outcome was promise in ATP production have been observed in animal models 418bs_bs_query

373bs_bs_query also investigated but did not display any signiﬁcance. (Duran et al., 2011; Piko and Taylor, 1987). It is conceivable that the 419bs_bs_query

up-regulation of mtDNA levels is a consequence of a compensatory 420bs_bs_query 374bs_bs_query

mechanism to normalize the ATP generation in response to growing 421bs_bs_query

375bs_bs_query

numbers of defective organelles with reduced function (Fragouli et al., 422bs_bs_query

376bs_bs_query Discussion 2015). Therefore, an increase in MCN might be necessary in the 423bs_bs_query

377bs_bs_query embryos of older women to maintain sufﬁcient ATP levels. However, 424bs_bs_query

378bs_bs_query MALBAC-NGS has successfully been used in a variety of aspects at an alternative idea has also been proposed – that embryos pro- 425bs_bs_query

379bs_bs_query single cell level, such as detecting SNV and CNV (Zong et al., 2012), duced by older oocytes have larger energy requirements due to some 426bs_bs_query

380bs_bs_query analysing the recombination and aneuploidy of single spermatozoa type of stress (Fragouli et al., 2015). Taking into account the decline 427bs_bs_query

381bs_bs_query (Lu et al., 2012) and investigating the genome of human oocytes (Hou of PB mtDNA levels (Konstantinidis et al., 2014), as well as the 428bs_bs_query

382bs_bs_query et al., 2013). The present study demonstrates that MALBAC-NGS is decrease/no change in oocyte mtDNA along with advancing age (Barritt 429bs_bs_query

383bs_bs_query also applicable to study mtDNA copy numbers in single cells with good et al., 2000; Chan et al., 2005; Konstantinidis et al., 2014), it is con- 430bs_bs_query

384bs_bs_query repeatability. Our results suggest that this approach is sensitive enough ceivable that the increase in mtDNA content in embryos of older 431bs_bs_query

385bs_bs_query to detect the minor difference even among the subcultured popula- females might partially originate from the redistribution of mito- 432bs_bs_query

386bs_bs_query tions derived from the same cell line (Table 2). One phenomenon chondria during oocyte meiosis. Overall, compared with younger 433bs_bs_query

387bs_bs_query observed in in-vitro fertilized embryos was that the duplicate rate of females, eggs of older females might be prone to acquiring larger 434bs_bs_query

388bs_bs_query the reads mapped to the mitochondrial genome was mainly higher numbers of mitochondria, probably triggered by certain metabolic 435bs_bs_query

389bs_bs_query than reads mapped to the nuclear genome (average ~20% versus ~1%), stress-responsive mechanisms. However, this hypothesis deﬁnitely 436bs_bs_query

390bs_bs_query and the duplication is signiﬁcantly positively related to the MCN (r = needs further functional investigation. 437bs_bs_query

391bs_bs_query 0.711, P = 0.000). This might be attributed to the inner bias/limitation This study also found blastomeres carrying a much higher quan- 438bs_bs_query

392bs_bs_query in the process of library construction and subsequent sequencing when tity of mtDNA than TE cells, which might be explained by the lower 439bs_bs_query

393bs_bs_query an extremely large copy number of template DNA is handled as start- cytoplasmic volume of TE cells (Fragouli et al., 2015) and by the fact 440bs_bs_query

394bs_bs_query ing material, which, however, is likely to be attenuated by removing that signiﬁcant mitochondria replication does not start until blasto- 441bs_bs_query

395bs_bs_query duplicates. cyst stage (Eichenlaub-Ritter et al., 2011; John et al., 2010). Regarding 442bs_bs_query

396bs_bs_query Mitochondria are indispensable for embryo development, a process the karyotype, there was no signiﬁcant correlation with mtDNA level. 443bs_bs_query

397bs_bs_query requiring a large energy supply (Chan et al., 2005; Van Blerkom, 2011). The published reports investigating this same issue from different 444bs_bs_query

398bs_bs_query The integrity and the quantity of mitochondria are thought to be im- groups also show some discrepancies (Fragouli et al., 2015; Tan et al., 445bs_bs_query

399bs_bs_query portant for the execution of corresponding functions. It is known that 2014; Victor et al., 2017), which remain to be explained. 446bs_bs_query

400bs_bs_query female fertility declines with age. The question of whether mitochon- The demand for an efﬁcient approach to identify embryos of high 447bs_bs_query

401bs_bs_query dria play a role in this decline is worth addressing. Notably the quality is urgent, in order to increase the success rate of assisted re- 448bs_bs_query

402bs_bs_query difference in mtDNA quantity in early stage embryos is prone to origi- productive techniques. It is known that chromosomal aneuploidy is 449bs_bs_query

403bs_bs_query nate from corresponding oocytes. Previous studies on the mtDNA of the main cause of implantation failure, miscarriage and birth defects. 450bs_bs_query

404bs_bs_query oocytes have revealed inconsistent results in relation to female re- Other elements including mtDNA number and accompanying effects 451bs_bs_query

405bs_bs_query productive age, although either a decline or no change in mtDNA levels on ATP content are also indispensable for embryo viability (Van 452bs_bs_query

406bs_bs_query with advancing age was reported in most studies (Barritt et al., 2000; Blerkom et al., 1995). Studies have indicated that the mitochondria 453bs_bs_query

407bs_bs_query Chan et al., 2005; Konstantinidis et al., 2014). A recently study has copy number, as well as the blastomere volume, are associated with 454bs_bs_query

408bs_bs_query reported an increase in mtDNA content with female age in blasto- embryo developmental capability and pregnancy outcome (Fragouli Q4 455bs_bs_query

409bs_bs_query cysts but an opposite trend in cleavage stage by using real-time PCR and Wells, 2015; Murakoshi et al., 2013). Attempts have been made 456bs_bs_query

410bs_bs_query (Fragouli et al., 2015). Another study using microarray revealed a to elucidate the feasibility of using mtDNA quantity as a biomarker 457bs_bs_query

REPRODUCTIVE BIOMEDICINE ONLINE ■■ (2017) ■■– ■■ 7

458bs_bs_query to select embryos for implantation (Diez-Juan et al., 2015; Fragouli transmission of mitochondria DNA mutation in the offspring is prob- 518bs_bs_query

459bs_bs_query et al., 2015; Seli, 2016). Recently, a large retrospective study analysing ably a future applicable aspect of this technology as well. 519bs_bs_query

bs_bs_query 460 1505 blastocysts from 490 couples with 282 being implanted also dem- 520bs_bs_query

461bs_bs_query onstrated the efﬁcacy of mtDNA quantiﬁcation as a tool to assess 521bs_bs_query

462bs_bs_query embryo viability and showed a signiﬁcantly lower rate of successful Acknowledgement 522bs_bs_query

463bs_bs_query implantation in embryos with increased mtDNA level. The authors

523bs_bs_query 464bs_bs_query showed that ~10% of the euploid blastocysts carrying abnormally el-

The authors would like to thank Dr Jun Ren for his help with data 524bs_bs_query 465bs_bs_query Q5 evated mitochondrial quantities failed to implant (Ravichandran et al.,

analysis and annotation. This study was supported by the National 525bs_bs_query 466bs_bs_query 2017). The preliminary results of our study also exhibited a trend of

Key Technology Support Program [2012BAI32B01, 2011] and the Capital 526bs_bs_query 467bs_bs_query fewer mitochondria copy numbers in successfully implanted embryos,

Characteristic Clinical Application Research and Popularization 527bs_bs_query 468bs_bs_query although the difference was not statistically signiﬁcant (Table 6), which

[Z151100004015214, 2015]. 528bs_bs_query 469bs_bs_query might be attributed to the small number of implanted embryos. Also,

bs_bs_query 470bs_bs_query those embryos with ~17% highest MCN were not included, which could 529

471bs_bs_query have already excluded those abnormal embryos carrying extremely 530bs_bs_query

472bs_bs_query high mitochondrial quantity. Notably, the model or threshold gener- Appendix: Supplementary material 531bs_bs_query

473bs_bs_query ated in previous studies were mainly based on real-time PCR and 532bs_bs_query

474bs_bs_query should be validated via different platforms and in prospective studies

Supplementary data to this article can be found online at 533bs_bs_query

475bs_bs_query as well. Besides, the question as to whether the use of both mtDNA

doi:10.1016/j.rbmo.2017.10.110. 534bs_bs_query

476bs_bs_query and chromosomal copy number as combined markers improves the

535bs_bs_query 477bs_bs_query pregnancy rate also needs to be addressed (Seli, 2016). The MALBAC-

bs_bs_query 478bs_bs_query NGS approach used in this study allows simultaneous study of the ARTICLE INFO 536

bs_bs_query 479bs_bs_query CNV and mtDNA copy number at a ﬁner resolution. Taking into account 537

Article history: 538bs_bs_query 480bs_bs_query the high-throughput characteristics of NGS, it is feasible to handle

Received 22 March 2017 539bs_bs_query 481bs_bs_query with numerous samples. All these features make MALBAC-NGS a po-

Received in revised form 11 October 2017 540bs_bs_query 482bs_bs_query tential candidate for further investigation and validation on the

Accepted 17 October 2017 541bs_bs_query 483bs_bs_query efﬁciency of mtDNA as an indicator for the PGS process.

Declaration: The authors report no 542bs_bs_query 484bs_bs_query It has been reported that MALBAC-NGS is an effective approach

ﬁnancial or commercial conﬂicts of 543bs_bs_query 485bs_bs_query for PGD to prevent chromosomal monogenic disease (Yan et al., 2015).

interest. 544bs_bs_query 486bs_bs_query The detection of pathogenic mutations and chromosomal abnormal- 545bs_bs_query

487bs_bs_query ity in embryonic cells can be accomplished in one run. Unlike 546bs_bs_query

bs_bs_query 488 chromosomal monogenic disease, the diseases caused by mitochon- Keywords: 547bs_bs_query

bs_bs_query 489 dria DNA mutation have a more complex heredity and are more difﬁcult IVF 548bs_bs_query

bs_bs_query 490 to interpret. The heterogeneity of mitochondria leads to distinct phe- mitochondria copy number 549bs_bs_query

bs_bs_query 491 notypes among individuals even carrying the same mitochondria next-generation sequencing 550bs_bs_query

bs_bs_query 492 mutation. The quantity of the mutated DNA is one of the factors de- pre-implantation genetic screening 551bs_bs_query

493bs_bs_query termining the severity of the phenotype. Generally speaking, disease

552bs_bs_query

494bs_bs_query symptoms are likely to arise only when the mutation rate exceeds a

REFERENCES 553bs_bs_query 495bs_bs_query certain threshold (Lightowlers et al., 2015). Several PGD cases have

554bs_bs_query 496bs_bs_query been reported by using real-time PCR or a capillary electrophore-

555bs_bs_query 497bs_bs_query sis based technique to calculate mtDNA mutation loads in TE cells

Barritt, J.A., Cohen, J., Brenner, C.A., 2000. Mitochondrial DNA point bs_bs_query 498bs_bs_query of ICSI blastocysts in patients with mitochondrial encephalomyopathy, 556

mutation in human oocytes is associated with maternal age. Reprod. 557bs_bs_query 499bs_bs_query lactic acidosis and stroke-like episodes (MELAS) syndrome (Heindryckx

Biomed. Online 1, 96–100. 558bs_bs_query 500bs_bs_query et al., 2014; Sallevelt et al., 2013; Treff et al., 2012). Mutation-free

Carter, N.P., Bebb, C.E., Nordenskjo, M., Ponder, B.A., Tunnacliffe, A., 559bs_bs_query

501bs_bs_query embryos or embryos with low mutation load were chosen to trans- 1992. Degenerate oligonucleotide-primed PCR: general 560bs_bs_query

502bs_bs_query fer in order to reduce the risk of transmitting the mtDNA disorder ampliﬁcation of target DNA by a single degenerate primer. 561bs_bs_query

bs_bs_query 503 into offspring, giving birth to healthy babies. However, the children Genomics 13, 718–725. 562bs_bs_query

504bs_bs_query born must be followed up to validate the efﬁcacy and safety. The Castle, J.C., Biery, M., Bouzek, H., Xie, T., Chen, R., Misura, K., Jackson, 563bs_bs_query

S., Armour, C.D., Johnson, J.M., Rohl, C.A., Raymond, C.K., 2010. 564bs_bs_query 505bs_bs_query MALBAC-NGS approach is capable of analysing the mutation quali-

DNA copy number, including telomeres and mitochondria, assayed 565bs_bs_query 506bs_bs_query tatively (Yan et al., 2015) and theoretically also quantitatively. However,

using next-generation sequencing. BMC Genomics 11, 244. 566bs_bs_query 507bs_bs_query the feasibility and efﬁcacy of preventing diseases caused by mtDNA Chan, C., Liu, V., Lau, E., Yeung, W.S., Ng, E.H., Ho, P.C., 2005. 567bs_bs_query

508bs_bs_query mutations by this strategy must be investigated and evaluated cau- Mitochondrial DNA content and 4977 bp deletion in unfertilized 568bs_bs_query

509bs_bs_query tiously before use, which is beyond the scope of the current study. oocytes. Mol. Hum. Reprod. 11, 843–846. 569bs_bs_query

510bs_bs_query In conclusion, a comprehensive chromosomal and mitochon- Dean, F.B., Hosono, S., Fang, L., Wu, X., Faruqi, A.F., Bray-Ward, P., 570bs_bs_query

bs_bs_query 511bs_bs_query drial copy number proﬁling of human embryo biopsies using a validated Sun, Z., Zong, Q., Du, Y., Du, J., Driscoll, M., Song, W., Kingsmore, 571

S.F., Egholm, M., Lasken, R.S., 2002. Comprehensive human 572bs_bs_query 512bs_bs_query approach called MALBAC-NGS was conducted. A positive correla-

genome ampliﬁcation using multiple displacement ampliﬁcation. 573bs_bs_query 513bs_bs_query tion of maternal age with increased mitochondria quantity was

Proc. Natl. Acad. Sci. U.S.A. 99, 5261–5266. 574bs_bs_query

514bs_bs_query observed. The mtDNA content was also correlated to embryo stage. Diez-Juan, A., Rubio, C., Marin, C., Martinez, S., Al-Asmar, N., Riboldi, 575bs_bs_query

515bs_bs_query It is proposed that use of the MALBAC-NGS approach presented here M., Díaz-Gimeno, P., Valbuena, D., Simón, C., 2015. Mitochondrial 576bs_bs_query

bs_bs_query 516 might be used to perform PGS targeting both chromosomal and mi- DNA content as a viability score in human euploid embryos: less is 577bs_bs_query

517bs_bs_query tochondrial copy numbers. Moreover, a PGD procedure to prevent better. Fertil. Steril. 104, 534–541, e1. 578bs_bs_query

8 REPRODUCTIVE BIOMEDICINE ONLINE ■■ (2017) ■■– ■■

579bs_bs_query Duran, H.E., Simsek-Duran, F., Oehninger, S.C., Jones, H.W., Jr., Navin, N., Kendall, J., Troge, J., Andrews, P., Rodgers, L., McIndoo, J., 638bs_bs_query

580bs_bs_query Castora, F.J., 2011. The association of reproductive senescence with Cook, K., Stepansky, A., Levy, D., Esposito, D., Muthuswamy, L., 639bs_bs_query

581bs_bs_query mitochondrial quantity, function, and DNA integrity in human Krasnitz, A., McCombie, W.R., Hicks, J., Wigler, M., 2011. Tumour 640bs_bs_query

582bs_bs_query oocytes at different stages of maturation. Fertil. Steril. 96, evolution inferred by single-cell sequencing. Nature 472, 90–94. 641bs_bs_query

583bs_bs_query 384–388. Piko, L., Taylor, K.D., 1987. Amounts of mitochondrial DNA and 642bs_bs_query

584bs_bs_query Eichenlaub-Ritter, U., Wieczorek, M., Lüke, S., Seidel, T., 2011. Age abundance of some mitochondrial gene transcripts in early mouse 643bs_bs_query

585bs_bs_query related changes in mitochondrial function and new approaches to embryos. Dev. Biol. 123, 364–374. 644bs_bs_query

586bs_bs_query study redox regulation in mammalian oocytes in response to age or Sallevelt, S.C., Dreesen, J.C., Drüsedau, M., Spierts, S., Coonen, E., van 645bs_bs_query

587bs_bs_query maturation conditions. Mitochondrion 11, 783–796. Tienen, F.H., van Golde, R.J., de Coo, I.F., Geraedts, J.P., de Die- 646bs_bs_query

588bs_bs_query Fragouli, E., Wells, D., 2015. Mitochondrial DNA assessment to Smulders, C.E., Smeets, H.J., 2013. Preimplantation genetic 647bs_bs_query

589bs_bs_query determine oocyte and embryo viability. Semin. Reprod. Med. 33, diagnosis in mitochondrial DNA disorders: challenge and success. J. 648bs_bs_query

590bs_bs_query 401–409. Med. Genet. 50, 125–132. 649bs_bs_query

591bs_bs_query Fragouli, E., Spath, K., Alfarawati, S., Kaper, F., Craig, A., Michel, C.E., Schaefer, A.M., McFarland, R., Blakely, E.L., He, L., Whittaker, R.G., 650bs_bs_query

592bs_bs_query Kokocinski, F., Cohen, J., Munne, S., Wells, D., 2015. Altered levels Taylor, R.W., Chinnery, P.F., Turnbull, D.M., 2008. Prevalence of 651bs_bs_query

593bs_bs_query of mitochondrial DNA are associated with female age, aneuploidy, mitochondrial DNA disease in adults. Ann. Neurol. 63, 35–39. 652bs_bs_query

594bs_bs_query and provide an independent measure of embryonic implantation Seli, E., 2016. Mitochondrial DNA as a biomarker for in-vitro fertilization 653bs_bs_query

595bs_bs_query potential. PLoS Genet. 11, e1005241. outcome. Curr. Opin. Obstet. Gynecol. 28, 158–163. 654bs_bs_query

596bs_bs_query Heindryckx, B., Neupane, J., Vandewoestyne, M., Christodoulou, C., Tan, Y., Yin, X., Zhang, S., Jiang, H., Tan, K., Li, J., Xiong, B., Gong, F., 655bs_bs_query

597bs_bs_query Jackers, Y., Gerris, J., Van den Abbeel, E., Van Coster, R., Deforce, Zhang, C., Pan, X., Chen, F., Chen, S., Gong, C., Lu, C., Luo, K., Gu, Y., 656bs_bs_query

598bs_bs_query D., De Sutter, P., 2014. Mutation-free baby born from a Zhang, X., Wang, W., Xu, X., Vajta, G., Bolund, L., Yang, H., Lu, G., Du, 657bs_bs_query

599bs_bs_query mitochondrial encephalopathy, lactic acidosis and stroke-like Y., Lin, G., 2014. Clinical outcome of preimplantation genetic 658bs_bs_query

600bs_bs_query syndrome carrier after blastocyst trophectoderm preimplantation diagnosis and screening using next generation sequencing. 659bs_bs_query

601bs_bs_query genetic diagnosis. Mitochondrion 18, 12–17. doi:10.1016/ Gigascience 3, 30. 660bs_bs_query

602bs_bs_query j.mito.2014.08.005. Treff, N.R., Campos, J., Tao, X., Levy, B., Ferry, K.M., Scott, R.T., Jr., 661bs_bs_query

603bs_bs_query Hou, Y., Fan, W., Yan, L., Li, R., Lian, Y., Huang, J., Li, J., Xu, L., Tang, F., 2012. Blastocyst preimplantation genetic diagnosis (PGD) of a 662bs_bs_query

604bs_bs_query Xie, X.S., Qiao, J., 2013. Genome analyses of single human oocytes. mitochondrial DNA disorder. Fertil. Steril. 98, 1236–1240. 663bs_bs_query

605bs_bs_query Cell 155, 1492–1506. Van Blerkom, J., 2011. Mitochondrial function in the human oocyte and 664bs_bs_query

606bs_bs_query John, J.C.S., Facucho-Oliveira, J., Jiang, Y., Kelly, R., Salah, R., 2010. embryo and their role in developmental competence. Mitochondrion 665bs_bs_query

607bs_bs_query Mitochondrial DNA transmission, replication and inheritance: 11, 797–813. 666bs_bs_query

608bs_bs_query a journey from the gamete through the embryo and into Van Blerkom, J., Davis, P.W., Lee, J., 1995. Fertilization and early 667bs_bs_query

609bs_bs_query offspring and embryonic stem cells. Hum. Reprod. Update embryolgoy: ATP content of human oocytes and developmental 668bs_bs_query

610bs_bs_query 16, 488–509. potential and outcome after in-vitro fertilization and embryo 669bs_bs_query

611bs_bs_query Kalisky, T., Quake, S.R., 2011. Single-cell genomics. Nat. Methods 8, transfer. Hum. Reprod. 10, 415–424. 670bs_bs_query

612bs_bs_query 311–314. Victor, A.R., Brake, A.J., Tyndall, J.C., Grifﬁn, D.K., Zouves, C.G., Barnes, 671bs_bs_query

613bs_bs_query Konstantinidis, M., Alfarawati, S., Hurd, D., Paolucci, M., Shovelton, J., F.L., Viotti, M., 2017. Accurate quantitation of mitochondrial DNA 672bs_bs_query

614bs_bs_query Fragouli, E., Wells, D., 2014. Simultaneous assessment of reveals uniform levels in human blastocysts irrespective of ploidy, 673bs_bs_query

615bs_bs_query aneuploidy, polymorphisms, and mitochondrial DNA content in age, or implantation potential. Fertil. Steril. 107, 34–42, e3. 674bs_bs_query

616bs_bs_query human polar bodies and embryos with the use of a novel microarray Wang, J., Fan, H.C., Behr, B., Quake, S.R., 2012. Genome-wide single- 675bs_bs_query

617bs_bs_query platform. Fertil. Steril. 102, 1385–1392. cell analysis of recombination activity and de novo mutation rates in 676bs_bs_query

618bs_bs_query Krebs, J.E., Goldstein, E.S., Kilpatrick, S.T., 2009. Lewin’s Genes X. human sperm. Cell 150, 402–412. 677bs_bs_query

619bs_bs_query Jones and Bartlett Publishers. Wells, D., Kaur, K., Grifo, J., Glassner, M., Taylor, J.C., Fragouli, E., 678bs_bs_query

620bs_bs_query Li, H., Durbin, R., 2010. Fast and accurate long-read alignment with Munne, S., 2014. Clinical utilisation of a rapid low-pass whole 679bs_bs_query

621bs_bs_query Burrows-Wheeler transform. Bioinformatics 26, 589–595. genome sequencing technique for the diagnosis of aneuploidy in 680bs_bs_query

622bs_bs_query Lightowlers, R.N., Taylor, R.W., Turnbull, D.M., 2015. Mutations causing human embryos prior to implantation. J. Med. Genet. 51, 553–562. 681bs_bs_query

623bs_bs_query mitochondrial disease: what is new and what challenges remain? Yan, L., Huang, L., Xu, L., Huang, J., Ma, F., Zhu, X., Tang, Y., Liu, M., 682bs_bs_query

624bs_bs_query Science 349, 1494–1499. Lian, Y., Liu, P., Li, R., Lu, S., Tang, F., Qiao, J., Xie, X.S., 2015. Live 683bs_bs_query

625bs_bs_query Lu, S., Zong, C., Fan, W., Yang, M., Li, J., Chapman, A.R., Zhu, P., Hu, X., births after simultaneous avoidance of monogenic diseases and 684bs_bs_query

626bs_bs_query Xu, L., Yan, L., Bai, F., Qiao, J., Tang, F., Li, R., Xie, X.S., 2012. chromosome abnormality by next-generation sequencing with 685bs_bs_query

627bs_bs_query Probing meiotic recombination and aneuploidy of single sperm cells linkage analyses. Proc. Natl. Acad. Sci. U.S.A. 112, 15964–15969. 686bs_bs_query

628bs_bs_query by whole-genome sequencing. Science 338, 1627–1630. Zaragoza, M.V., Fass, J., Diegoli, M., Lin, D., Arbustini, E., 2010. 687bs_bs_query

629bs_bs_query Macaulay, I.C., Voet, T., 2014. Single cell genomics: advances and future Mitochondrial DNA variant discovery and evaluation in human 688bs_bs_query

630bs_bs_query perspectives. PLoS Genet. 10, e1004126. Cardiomyopathies through next-generation sequencing. PLoS ONE 689bs_bs_query

631bs_bs_query McFarland, R., Taylor, R.W., Turnbull, D.M., 2002. The neurology of 5, e12295. 690bs_bs_query

632bs_bs_query mitochondrial DNA disease. Lancet Neurol. 1, 343–351. Zeviani, M., Mariotti, C., Antozzi, C., Fratta, G.M., Rustin, P., Prelle, A., 691bs_bs_query

633bs_bs_query Murakoshi, Y., Sueoka, K., Takahashi, K., Sato, S., Sakurai, T., Tajima, 1995. OXPHOS defects and mitochondrial DNA mutations in 692bs_bs_query

634bs_bs_query H., Yoshimura, Y., 2013. Embryo developmental capability and cardiomyopathy. Muscle Nerve 18, S170–S174. 693bs_bs_query

635bs_bs_query pregnancy outcome are related to the mitochondrial DNA copy Zong, C., Lu, S., Chapman, A.R., Xie, X.S., 2012. Genome-wide detection 694bs_bs_query

636bs_bs_query number and ooplasmic volume. J. Assist. Reprod. Genet. 30, 1367– of single-nucleotide and copy-number variations of a single human 695bs_bs_query

637bs_bs_query 1375. cell. Science 338, 1622–1626. 696bs_bs_query