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. 36bs_bs_query © 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. 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 43bs_bs_query 1472-6483/© 2017 Reproductive Healthcare Ltd. Published by Elsevier Ltd. All rights reserved. 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