© 2015 Nature America, Inc. All rights reserved. to to E.R.H. ( and Comparative Biology, University of Leeds, Leeds, UK. 5 University of Sussex, Brighton, UK. 1 or to be lacking altogether (non-exchange, E gested to be too far from the centromeres to mediate correct attachment between the two sister chromatids centromeres to close too it conceptions, that has trisomic inferred some been occur crossovers in mother the from inherited SNPsas two such on the only occur in adult women. By following the pattern of genetic markers maintained for be decades, to as the have two rounds linkages of chromosomeThe segregation females. in development fetal during place ( meiosis of stage prophase the during together sister chromatid cohesion, crossovers physically link the homolog pair DNA homologous between chromosomes (homologs). Together with of exchange reciprocal crossovers—the of result the are offspring in chromosomes Recombinant recombination. altered is sexes both in maternal age, one important factor that predisposes to missegregation unclear. are relative contributions still In to and their causes addition aneuploid are conceptions natural of 10–30% that estimates conservative despite but, age, of years ~35 from women in orders dis congenital and loss pregnancy failure, implantation to leading conceptions, aneuploid of cause major a are meiosis female human in divisions meiotic the during segregation in Errors meiosis chromatids in pattern crossovers Genotyping maps () Crossover Alan H Handyside Alan R Thornhill Hrishikesh A Joshi Christian S Ottolini for maternal recombination rates segregation in human and embryos show selection Genome-wide maps of recombination and chromosome Nature Ge Nature Received 11 November 2014; accepted 23 April 2015; published online 18 May 2015; Illumina, Illumina, Inc., Fulbourn, UK. The Bridge Centre, London, UK.

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meiosis II errors in the activated oocytes as compared to embryos 4 of 230; embryos versus of 299 (6 by ICSI to generated compared as oocytes activated the in errors II meiosis (85%, ( PB2 the of and extrusion II meiosis of completion induced which ionophore, calcium a out fertilization by activating mature, meiosis II–arrested oocytes with and 12 Zygote Oocyte Supplementary TableSupplementary 1 n 13 = 40; 40; =

Frequency e 150 50 0 14 Embryo-PB trio 0 Supplementary Table 2 Table Supplementary PB2 (median: 107kb) a PB2 Oocyte -PBtrio Resolution (kb) 500 DVANCE ONLINE PUBLICATION ONLINE DVANCE 15 Embryo culture Fig. 1b Fig. c a in vitro n 1,500 16 =883 , b ) The genotypes of the two maternal maternal two the of genotypes ) The ). ). A 13 further trios were generated with 17 , c 0 ). This method was highly successful successful highly was method This ). (median: 331kb) Resolution (kb) 500 18 Embryos n n e (preimplantation) =1,342(p) =1,149(m) ) Histograms of the resolution resolution the of ) Histograms ) and did not alter the rate of of rate the alter not did and ) Embryo 1,500 19 Table 20

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© 2015 Nature America, Inc. All rights reserved. of both homologs of the chromosome at meiosis I, we observed the the observed we I, meiosis at chromosome the of homologs both of ( events PSSC ent independ two for frequency predicted the than greater 100× than frequency of both homologs separating their sister chromatids is more to be the result of two independent PSSC events because the observed in II meiosis ( I, in first by followed meiosis of chromatids the non-sister separation separated homologs both of chromatids sister that infer we as tion, segrega Weas reverse is normal. pattern new cells to this refer three analysis used previously matids ( chro sister of instead chromatids non-sister had but content some chromo normal contained PB2 the and oocyte the both instances, centromeres; the around fingerprints orange and (green chromatids 2 non-sister contained to that a PB1 rise gave centromere (CEN) are shown in blue. Two examples were found in this egg (chromosomes 4 and 16; mature oocyte and a PB1 each containing two non-sister chromatids. The expected chromosome fingerprints corresponding to heterozygous SNPs around the continuity). ( (euploid and aneuploid; Frequencies are shown in non-canonical segregation patterns (bottom). ( ( ( donors donors and average (av.) age are shown. The age ranges were 25–35 years for FPN-PB (FPN-PB) in a younger donor population embryo-PB trios. Errors detected in MeioMaps generated from the female pronucleus nondisjunction. ( nondisjunction; PSSC, precocious separation of sister chromatids; MII NDJ, meiosis II patterns detected by MeioMaps, see Only examples leading to trisomic conceptions are shown. For all possible segregation (orange and green around the centromeres) in all three products of female meiosis. pattern. ( Figure 2 s e l c i t r A  of intermediates a predicted reverse segregation, mature and oocyte a c S c a ) Chromosome abnormalities resulting in aneuploid oocytes or embryos (top) and all upplementary Frequency Frequency Unexpectedly, the most frequent non-canonical segregation pattern 10 0 1 2 3 4 5 0 1 2 3 4 5 6 7 8 9 1 1 1 0 Normal Abnormal segregation–allevents Abnormal segregation–lossorgaininoocyte

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© 2015 Nature America, Inc. All rights reserved. bination rates might increase the risk of generating vulnerable crossover of individual homolog pairs? We hypothesized outcomes segregation that the loweraffect rates global recombination recom global do How Non-recombinant average). overall the (5.8/41.5, age maternal advanced of women in 14% as much as by rates increasing thirties, their enter in as women they selection may selected rates under be is recombination that are which of many embryo of the events, implantation before against aneuploidy to rise occur, give do they errors When woman. adult chromosome in later in decades errors segregation against protect development, fetal during are determined which frequencies, recombination global higher that influ that segregation chromosome of factors fidelity the other ence of presence the with consistent is groups aneuploid and euploid the for rates recombination of distributions oocyte aneuploid the in chromosomes for crossovers that might not be detected owing to the presence of two accounted we when even significant, was difference This ones. ploid aneu than more events recombination 5.8 average on had embryos respective recombination rates ( into two oocytes groups (euploid or their aneuploid) and determined and embryos the divided we case, the was this whether Toexamine embryos. aneuploid of those than frequencies recombination wide genome- maternal higher exhibit that chromosomes contain should embryos euploid normal then errors, segregation chromosome otic in of the incidence aneuploidy (outlier excluded; permutation test). bination rate is an important factor, accounting for 18% of the variation detectable crossovers among any of the chromosome pairs. The recom we excluded an outlier embryo that contained 12 aneuploidies and no bination of rate was a strong predictor incidence of aneuploidy ( the with aneuploidy in and correlated individual oocytes embryos. Indeed, recomglobal were rates recombination wide humanaffects aneuploidy, we genome-the global, whether addressed rates recombination maternal in variability the how understand To Global vulnerable embryos. ultimately, and, to oocytes rise gives that are to adult propagated individuals oocytes in fetal configurations crossover within and between recombination of events distribution and rates the hypothesis in the variability to the support that lends and approach our validates This ( of a errors proportion standard bars, Error are shown. (two-sided) a from G derived of test heterogeneity R more in are as Statistics trios. and embryo-PB in rates oocyte-PB recombination (R one non-recombinant least ( one-sided. (W), test Wilcoxon Mann-Whitney- Statistics: bar). vertical (magenta the median above is mean shown The arithmetic and embryos. oocytes aneuploid versus ( analyses. statistical subsequent ( significant highly still coefficient the regression was omitted, events no with recombination the When outlier family). binomial lines; (dashed interval and 95% confidence model regression the logistic show lines Black or in rate oocyte. the embryo recombination of global as a function chromosomes of aneuploid of the frequency regression ( line. germ for in female the human and are selected 4 Figure s e l c i t r A  E non-exchange considered first We configurations.

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© 2015 Nature America, Inc. All rights reserved. able, or in fetal oocytes, which arise decades before the segregation segregation the before decades arise which oocytes, fetal in or able, not avail was were information body polar where populations, in studied segregation chromosome and recombination recently, Until DISCUSSION disease proposed mitochondrial of treatment in use for been has PB2 the because relevant particularly is oocyte and PB2 the of between structure health genome in genomic difference the The and children. structure population of terms in both or propagation of segregation distorters single haplotype block, thereby reducing the probabilitya of as inbreeding inherited being from chromosomes entire prevent may matids chro non-recombinant against Selection II. meiosis at segregation chromatid sister during force driving a as acts also but I meiosis at homologs of segregation accurate the for important only not is tion PB2 ( the to compared as oocytes in rates recombination the in elevation meiotic drive against non-recombinant chromatids resulted in a 6.6% chromosomes allele an of inclusion preferential the for ( line germ human the from eliminated thus and PB2 the into driven erentially at tids segregated II, meiosis non-recombinant chromatids were pref ( and PB2 in oocyte the rates were similar dom and recombination the ran was segregation their recombined, chromatids sister both when as, chromatids non-recombinant against to be appears selection The oocyte. the in than PB2 the in found be to likely as twice nearly were R expectation, this to Contrary PB2. the and oocyte the R These ( normally segregated PB2 had and or non-recombinant were oocyte that an in chromatids 135 showed MeioMaps The recombina of tion. lack the by affected also is II meiosis at segregation their but PSSC, of risk at only not are chromatids Non-recombinant Meiotic segregation. chromatid sister of dynamics the influences also but pairs homolog of non-exchange putative and segregation eration arrest dictyate decades-long the during bivalent the of rest the from ciated disso of becoming risk are at elevated chromatids non-recombinant that It I. possible is at meiosis chromatid sister their from separating gray. in shown is chromosome paternal The line. germ female human the in II meiosis at chromatids non-recombinant against drive meiotic of representation ( oocyte. or PB2 the to R of frequencies ( (two-sided). proportions for test G Statistics: oocyte. the to segregate to likely as twice was chromatid recombinant the chromatid, recombinant one and chromatid non-recombinant one contained chromosomes when However, II. meiosis at randomly segregate to expected are ( line. germ female human the in rates recombination increases II meiosis at chromatids 5 Figure Nature Ge Nature the oocytes, adult unselected from MeioMaps studied. being events Supplementary TableSupplementary 5 Supplementary TableSupplementary 5 41 ,

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2 0 10 12 14 16 18 R crmtd ad uaie o-xhne (E non-exchange putative and chromatids ) . . This model predicts that children born to younger 0 2 4 6 8 0 1 Meiosis 8 7 6 5 4 3 2 Oocyte PB2 I ). Centromeric crossovers interfere with with interfere crossovers Centromeric ). 0 Oocyt homologs underwent either PSSC or a or PSSC either underwent homologs 1 0 and embryos now provide a ‘missing ‘missing a provide now embryos and e 0 ) chromatids are a risk factor for for factor risk a are chromatids ) Drosophila melanogaster PB1 5 1 27– . Increased loop number corre number loop Increased . 1 9 Meiosis II Fertilization 2 Chromosome 1 0 9 . Sex-specific differences in in differences Sex-specific . 1 1 1 2 13 1 4 1 5 s e l c i t r A 1 6 0 PB1 PB homologs and and homologs 1 7 2 1 8 2 9 Recombinant chromosome 7 to offspring transmitte and bud 2 0 n =13 2 1 1 0 X 2 29 and and 5 d , 5 2

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© 2015 Nature America, Inc. All rights reserved. Reprints and permissions information is available online at at online available is information permissions and Reprints The authors declare no competing interests.financial manuscript. All authors proofread and accepted the manuscript. wrote the manuscript. A.H.H., C.S.O., D.K.G., A.C., L.J.N. and edited E.R.H. the A.H.H. andE.R.H., L.J.N. generated the A.H.H., figures. L.J.N.E.R.H., and C.S.O. out data analysis and simulations. andE.R.H. E.H. outcarried statistical analyses. L.J.N., C.S.O. and analyzed E.R.H. the data. encoded andE.R.H. A.D.H. carried and D.C. outcarried amplification, SNP array and aCGH experiments. A.H.H., oversaw and ethical legal regulation in Italy and the UK. A.C., C.S.O., S.A.N., H.A.J. consenting, andcollection activation. F.M.U.,L.R., oocyte oocyte A.H.H. and K.S. A.C., C.S.O., D.C., F.M.U.,L.R., K.S., M.C.S. and A.R.T. were responsible for donor OrganizationBiology Young(EMBO) Investigator award to E.R.H. (Senior Research Fellowship to G0902043) and E.R.H.; a European Molecular support for this research was provided by the UK Medical Research Council data and discussion on their study of female pronucleus–PB Financial trios. reading of the manuscript. We thank Y. Hou, W. Fan and S. Xie for freely sharing F. Cole and members of the Hoffmann laboratory for discussion and critical We gratefully acknowledge C. May, A. Eyre-Walker, J. Gruhn, R. Rowsey, J. Turner, online version of the pape Note: Any Supplementary Information and Source Data files are available in the the of in version available are references associated any and Methods M at origin their meiosis. female human in I have meiosis can II meiosis mistakes in to segregation attributed chromosome events in that demonstrate case either in but determined be to remain crossovers centromeric and mechanisms chromosomes between attachment physical on rely not do that mechanisms tion molecules) be could physically attached by unresolved recombination chromatids intermediates (joint non-sister II, meiosis At humans. in case the (refs. I meiosis at chromatids sister segregate preferentially univalents oocytes, mouse In aneuploidies. human underlying component age-related derived, maternally the match events such of effects chromosome-specific and frequencies I oocytes in latter human meiosis for the is There evidence into apart univalents. or falling by bivalents rioration of cohesion between sister chromatids or sister kinetochores by dete occur could I. This at meiosis division to equational dispose pre might arrest dictyate extended the during structure bivalent the P meiosis II (77% compared to 50% at byexpected random chance, segregation chromatid non-sister accurate for preference weak a by followed I, meiosis in fashion equational an in chromatids sister s e l c i t r A  7. 6. 5. 4. 3. 2. 1. reprints/index.htm COMPETING FINANCIAL INTERESTS FINANCIAL COMPETING AUTHOR CONTRIBUTIONS A

< It 0.05). that or is failure to crossovers possible maintain establish ckno

ethods Koehler, K.E. Koehler, S.B. Freeman, T.Hassold, Hassold,T.J.Jacobs,P.A.& Trisomy man. in human of genesis the M.V. human: Zaragoza, is (meiotically) err To P. Hunt, & T. Hassold, T.J.Hassold, P.A.S.I., Hunt, Nagaoka, new & and mechanisms aneuploidy: Human Nat. Genet. Nat. in implementation. Genet. liveborns. and fetuses trisomic 432 of aneuploidy. problem. age-old an into insights rspia melanogaster Drosophila w

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25  , © 2015 Nature America, Inc. All rights reserved. after warming—under Sage mineral oil. Slides were placed in the EmbryoScope slides (Unisence Fertilitech) in cleavage medium—the medium used for culture culture. activation culture for was performed were min. 40–120 The then oocytes moved to post- activation medium under Sage mineral oil, numbered appropriately. Activation 35- to transferred were Oocytes 1:40. diluted (Sigma-Aldrich) DMSO in solution stock a from HSA, 10% with supplemented medium age 100 activation. and biopsy PB1 with 10% HSA under mineral oil Surgical) (Cooper and cultured for 2 h before 35- numbered vidually experiment. the throughout traceability allow to numbered were wells and drops culture and performed was culture CO 6% in activation. and PB1 of biopsy before cultured were oocytes surviving the and discarded was were oocytes warming degenerated until warming, oocyte nitrogen After performed. liquid in submerged stored were oocytes All (Kitazato tool vitrification BioPharma) with a plastic cap for cryotop protection during storage a in liquid nitrogen. on stored were Oocytes hCG. of tion administra after h 40 of maximum a performed was procedure vitrification The and kits were BioPharma). warming (Kitazato used vitrification available Kuwayama by described as performed were dures vitrification. for identified then were oocytes II Meiosis hCG. of administration after h 40 and 37 between performed was CO 6% controlled a in Medical) radiata with the use of plastic ‘denuding’ pipettes of diameters defined (COOK corona the of removal mechanical by albu followed Surgical), serum Cooper (HSA; human min 10% with supplemented medium fertilization Sage solution in hyaluronidase IU/ml 40 to exposure brief by performed was mass cumulus the of Removal (hCG). gonadotropin chorionic human of istration treatment. patient influence not and did of Valle the Clinic Board Giulia Review approved by Institutional the consent was obtained from the patients. The study and informed consent were and were meiosis II later vitrified oocytes included in the study after informed remaining The a patient. per inseminated collected, be could were oocytes three of maximum oocytes these when force in law Italian to proto According col. GnRH-antagonist the and protocol long (GnRH)-agonist hormone gonadotropin-releasing the protocols: different two using performed lation and 2008 hyperstimu 15 ovarian 2 May controlled September following 2009 between Rome in GENERA Medicine treat ICSI Reproductive for Centre undergoing the in patients ment from obtained were study the for oocytes trios. body Oocyte–polar SNPs informative analysis. data for used with were B and A as coded data secondary Only B. and A as represented were SNPs informative that such encoded were data primary All practice. of code (HFEA) Authority Embryology and Fertilisation Human the by covered was and analysis genetic clinical of validation or was follow-up clinical genotyping as SNP performed result. clinical the on depending discarded, or stored transferred, were embryos after samples biopsy clinical of reanalysis or tests clinical in outcome abnormal previous a following analysis for (destroyed) samples for the study were by obtained either tubing in entirety embryos their embryo All treatment. patient influence not did and stored were oocytes the where Clinic ValleGiulia the of was Board study Review the Institutional for the by approved oocytes the of Use treatment. IVF for retrieval oocyte of at time the law place in Italian with accordance in vitrified were used oocytes The analysis. for destroyed were and treatment IVF their of completion after informed patient consent. All foroocytes the study were obtained from donors statement. Ethics ONLINE Nature Ge Nature Post-activation culture was performed in separate wells of EmbryoScope EmbryoScope of wells separate in performed was culture Post-activation comprising medium, activation to exposure by activated were Oocytes indi to allocated were oocytes surviving the warming, after Immediately activation. and culture Oocyte warming. and vitrification Oocyte collection. Oocyte µ M calcium ionophore (A23187, C7522, Sigma-Aldrich) in Sage cleav Sage in Sigma-Aldrich) C7522, (A23187, ionophore calcium M 2

and 5% O 5% and METHODS n etics All material for the study was ethically sourced with fully fully with sourced ethically was study the for material All Oocyte collection was performed 35 h after the admin the after h 35 performed was collection Oocyte 2 . To enable tracking of the oocytes and PBs, individual individual PBs, and oocytes the of To. tracking enable µ l microdrops l of microdrops Sage supplemented medium cleavage

Patient participation and consent. and participation Patient All oocyte culture was performed at 37 °C °C 37 at performed was culture oocyte All 2 and 37 °C environment. This procedure procedure This environment. °C 37 and The vitrification and warming proce warming and vitrification The et al. et 58 , 5 9 . Commercially Commercially . All meiosis II II meiosis All µ l drops of the the of drops l ------

in in the zona pelucida using laser pulses. The was oocyte removed from the zona created was and a aperture pipette, larger to holding the anchored was oocyte removed from using each the oocyte same setup for the biopsy procedure. The was pelucida zona The removal. pelucida zona full for micromanipulator the to returned then was oocyte The microdrop. the in still oocyte the with tion, amplifica DNA for tube PCR a to transferred was immediately PB2 was it the biopsied, Once amplification. DNA for Sigma-Aldrich) (Corning, tube for immediate transfer a 0.2-ml RNase- and microdrop flat-cap DNase-free PCR thin-walled, the in PB1 biopsied the leaving culture, activation to moved body polar was the oocyte the biopsied, was PB1 the and Once suction. gentle with removed was opening, the through inserted then was TPC) pipette; pelucida–drilling (zona pipette aspiration The pelucida. zona the of surface outer the from inward working Instruments), Research Laser, (Saturn pulses pipette (TPC). An aperture was made in the zona pelucida with a series of laser holding the with by suction and secured body of polar the to view a give clear For both the PB1 and PB2 biopsies, oocytes were positioned on the microscope oil for tractability. mineral Sage under HSA 10% with supplemented medium 10- numbered individually in performed were sies after extrusion activation as by previously described Capalbo PB1 The Instruments). its following biopsied was PB2 and the activation, Scientific oocyte before biopsied was (Linkam stage heating °C 37 a and trast on (Nikon) with an Hoffman equipped Modulation con microscope inverted (Narishige) micromanipulators using meiosis, of products three the between biopsy. PB2 before pronuclei of appearance the and extrusion PB2 of assessment for Fertilitech) (Unisence incubator time-lapse genome amplification products (SurePlex) from both polar bodies were SNP SNP were bodies polar both from (SurePlex) products amplification genome whole- the bodies, polar both of analysis number copy by aneuploidy detect trios. Embryo-PB trios. oocyte-PB the for described as performed then were PB1 and oocyte the for and amplification tubing whole-genome PB1, of Biopsy oil. mineral under HSA with10% supplemented mature to allowed trios. the oocyte-PB for the of processing described those to methods HFEA similar under using L0070-14-a, embryos license the clinic of testing genetic and biopsy purposes, clinical control following quality for performed was genotyping SNP from patients. consent the informed with (London) Centre Bridge the at protocols IVF standard following aneuploidy, for PGS or defects single-gene of PGD either for cases clinical four from analyzed were aneuploid and/or as affected nosed trios. embryo-PB and Embryos incubation. 2-h short a with performed was amplification Multiple-displacement instructions. manufacturer’s the to according per formed was (Illumina) amplification SurePlex library-based PCR or Illumina) (SureMDA, amplification kit multiple-displacement and Single-Cell REPLI-g 4 of volume final a to brought were samples containing tubes meiosis PCR of The amplification. whole-genome products and cells the of lysis by three obtained was all from DNA instructions. manufacturer’s the 30 of volume final a for Projects) Cell (Isohelix, kit extraction Extraction of genomic DNA from the swabs Projects). wasCell performed(Isohelix, swabs using cell a buccal proteinaseusing obtained was K donors oocyte amplification. whole-genome before °C at −20 and stored nitrogen in liquid snap frozen centrifuged, briefly were then tubes 4 than more no of 1 around in vinyl alcohol (Sigma-Aldrich). Individual samples were expelled into the DPBS with 2 ing pipette with a 130- DNAfor amplification. tube PCR a to transferred was oocyte the pelucida, zona the from free Once pipette. aspiration the with using pelucida both displacement and zona manipulation pelucida techniques biopsy. body Polar In one of the PGD cases, two surplus denuded meiosis I oocytes were were oocytes I meiosis denuded surplus two cases, PGD the of one In amplification whole-genome and extraction DNA Transfer of the samples to using a denud PCR plastic was tubes performed µ l l of Dulbecco’s PBS Gibco, (DPBS, Life Technologies) with 0.1% poly µ l of the medium containing the sample, leaving a final volume volume final a leaving sample, the containing medium the of l µ In another PGS case, in which aCGH had been used to to used been had aCGH which in case, PGS another In in vitro in l of medium with the sample in each PCR tube. The PCR PCR The tube. PCR each in sample the with medium of l Polar bodies were biopsied sequentially to discriminate discriminate to sequentially biopsied were bodies Polar µ m m lumen. Individually labeled PCR tubes were primed by overnight culture in Sage fertilization medium medium fertilization Sage in culture overnight by

Embryo samples. Embryo Thirty-five embryos diag embryos Thirty-five µ . Genomic DNA from all all from DNA Genomic . l microdrops of HEPES HEPES of microdrops l doi: et et al. 10.1038/ng.3306 µ µ 1 l, following following l, l with PBS, PBS, with l 6 . . All biop ------

© 2015 Nature America, Inc. All rights reserved. SNPs (blue regions) cannot be phased. be cannot regions) SNPs (blue heterozygous because undetectable are events these but PB1, the in present to be be expected would proportion A similar in PB2 mapped the and oocyte. distances chromosomes. along and crossovers between chromosomes among distribution crossover Simulation: shown). not (data >99% was comparison. In all cases, the concordance of recombination rates and positions direct for embryo same the from blastomeres individual child 10 the assessing by of and DNA genomic the to donor a from cells individual 15 in maps above. described as editing, final and display processing, further for spreadsheet into a second and imported tagged were manually phase transitions the case, either In only. haplotype maternal the for informative were that genotypes parental of combinations all identified algorithm This used. with a algorithm, karyomapping PB2 as or the modified reference, was oocyte 4.0) v Multi (BlueFuse dedicated software using or Excel in either haplotypes parental four all for loci SNP were karyomapped using the standard algorithm, which informativeidentifies samples the available, was embryo sibling a cases, or, some in relative close a crossovers. all double-check to and trio reference the in chromosomes aneuploid any MeioMap to references two least at with run were trios oocyte-PB All necessary. as manually edited further and checked removed them from all the other samples. The MeioMaps were then displayed, identified these common crossovers, restored them to the reference sample and and the PB2 or in oocyte that trio). Macros in the second spreadsheet therefore reference the between any of crossover exception the (with analyzed samples other all in positions identical in appear sample particular that in crossovers phasing was achieved using a reference sample, any phase transitions caused by SNPs were imported into a second spreadsheet for processing. further Because closest SNP calls bracketing each crossover, and the type and the positions of copying these by Excel in tagged manually then were crossovers at transitions Phase haplotypes. maternal of both presence the indicating (blue), erozygous het or (green) reference the to opposite (orange), reference the as same the as displayed and loci SNP informative these of each at interrogated then was reference, the including samples, the of each of genotype The chromosome. homozygous SNP loci (haplotype; AA or BB) for the whole genome across each of set a reference defined This reference. as the sample or PB2 oocyte haploid ple was applied algorithm to SNP maternal phase all heterozygous using loci a macros written in Visual Basic for Applications. For the oocyte-PB trios, a sim and displayed by the importing data into Excel and it processing using custom analysis. MeioMap Excel Microsoft in analysis for file text a as exported or Multi v (BlueFuse 4.0) for program karyomapping software a using dedicated data were analyzed The SNPs, protocol. genotype a using ~300,000 shortened (PB1, samples Human CytoSNP-12 or the Human on Karyomapping array for BeadChip embryo (Illumina) processed was and embryo) whole or blastomere single single-cell oocyte, and PB2, the from products amplification Illumina). 4.0, v Multi (BlueFuse software dedicated using analyzed and imported were Data Illumina). (24Sure, slides microarray on processed were blastomere) or and PB2, oocyte PB1, DNAthe (from products amplified single-cell SurePlex aCGH, SNP bead array and data analysis. diagnosed been all had that embryos aneuploid. as fertilized corresponding (SureMDA) products nine of amplification whole-genome the patients, the from consent informed with and, DNA genomic parental with along genotyped, doi: We validated our workflow on single cells by comparing recombination recombination comparing by cells single on workflow our validated We of SNP Where the genotype were used. two methods trios, For embryo-PB ng 400 DNAof For genomic SNP or genotyping, 8 10.1038/ng.3306 Following SNP genotyping, MeioMaps were constructed constructed were MeioMaps genotyping, SNP Following 11 , 1 2 . Alternatively, to improve resolution, a resolution, improve to Alternatively, . For aCGH analysis, 4- Twelve percent of crossovers were Twelveof crossovers percent µ l of the whole-genome l of whole-genome the 1 2 . µ l l aliquots of - -

The simulation reported the total number of crossovers per chromosome chromosome per crossover from outermost the The distance crossovers distances. and inter-crossover the of number total the reported simulation The position. crossover existing an from kb) (107 distance minimum the within not positions possible all included locations available The location. available an by selecting iteratively randomly was determined crossover of an allocated one crossover per chromosome (obligate). For each chromosome, the position of allocation following random or (non-obligate) random completely either was Allocation crossovers. more received chromosomes longer thus, length; chromosome the using probability weighted with to chromosomes randomly data were Crossovers set. allocated mapped the within experimental locations crossover the between distances maximum and minimum the using length specified a allocated were Chromosomes chromosomes. of set the to events weak very is interference) chromatid (negative in a crossover involvement first their given crossover that second a in reports re-engage to with chromatids sister consistent two for is preference This the detected. was first the in as crossover second the in engaging chromatids two same the of probability decreased or homolog pairs had engaged in crossing over twice, no evidence of an increased when Indeed, chromatids. among randomly non-sister occurs recombination ( bodies polar the in discerned those with well correlated embryo or oocyte the in rates bination of in went crossovers the ( two undetected polar bodies from young women trios similar to those reported for fetal oocytes 60. 59. 58. library ggplot2 the or R in functions basic using rendered were Graphics R. in libraries psperman and lmPerm lme4, the We used indicated. otherwise unless employed, were tests the if using rejected was hypothesis tested null the and were freedom of degrees and variance Residual R. in function plot(model) the using assessed were variances of error distributions regression, we used the AIC to the choose appropriate Binomial link function. logistic For manuscript. the throughout indicated as analysis, regression tic out or tests in logis Perl or and non-parametric were R. tests All permutation and graphics. modeling Statistics, weak is interference chromatid negative that reports with consistent crossover ( both the interference chromatid no of reject in hypothesis null to unable involved were We be participation. to random to compared likely as more events or less same the were whether chromatids asked we 2 and crossovers 2 with pairs chromosome 134 interference. Chromatid kb. at 200 1% and kb at 150 kb, 0.75% at 107 kb, at 0.52% 30 were at 0.04% 10 to frequencies adjacent The kb,cumulative telomeres. 0.15% crossover distances was constructed, ignoring distances for crossover that were between kb 0 A them (Ottolini_Scripts_CrossoverData.pl). cumulative distribution of of inter- distance minimum a with chromosomes along crossovers public GNU and copyright under license. available freely are (Ottolini_Scripts_ scripts CrossoverData.pl) The distributions. the create to performed simulations were thousand Ten included. not was terminus chromosome the to Simulations were performed to allocate a specified number of crossover crossover of number specified a allocate to performed were Simulations were trios and embryo-PB oocyte-PB in the rates recombination Maternal To of we 125 crossovers, estimate the missed randomly distributed fraction

Wickham, H. Wickham, Kuwayama, M., Vajta, G., Kato, O. & Leibo, S.P. Highly efficient vitrification method humanoocytes of for cryopreservation vitrification efficient Highly M. Kuwayama, (2005). oocytes. human of cryopreservation for method. Cryotop the embryos: and 1 0 . ggplot2: Elegant Graphics for Data Analysis Data for Graphics Elegant ggplot2: Supplementary Fig. 3 Fig. Supplementary 1 0 6 To detect chromatid interference, we identified identified we interference, chromatid detect To . When only the embryo or oocyte was used, >50% was used, . or When oocyte only embryo the 0 . Statistical tests Statistical and were modeling carried Theriogenology 27– erd Boe. Online Biomed. Reprod. P 2 9 ). This supports the notion that that notion the supports This ). value was below 5%. Two-sided Two-sided 5%. below was value as well as female pronucleus–PB P > 0.5, 0.5, >

67 , 73–80 (2007). 73–80 , t Fig. Fig. 3a (Springer, 2009). (Springer, test for proportions), proportions), for test Nature Ge Nature , b

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