Association between amino acid turnover and chromosome aneuploidy during human ...

49 Pages·2010·0.71 MB·English

MolecularHumanReproduction,Vol.16,No.8pp.557–569,2010
AdvancedAccesspublicationonJune22,2010 doi:10.1093/molehr/gaq040
ORIGINAL RESEARCH
Association between amino acid
turnover and chromosome aneuploidy
during human preimplantation embryo
development in vitro
D
o
w
n
lo
a
d
Helen M. Picton1,*, Kay Elder2, Franchesca D. Houghton3, e
d
Judith A. Hawkhead4, Anthony J. Rutherford5, Jan E. Hogg1, fro
m
h
Henry J. Leese4, and Sarah E. Harris1 ttp
s
1DivisionofReproductionandEarlyDevelopment,LeedsInstituteforGenetics,HealthandTherapeutics,TheLIGHTLaboratories, ://a
c
UniversityofLeeds,ClarendonWay,LeedsLS29JT,UK2BournHallClinic,Bourn,CambridgeCB232TN,UK3CentreforHuman a
d
Development,StemCellsandRegeneration/DivisionofHumanGenetics,UniversityofSouthampton,SouthamptonGeneralHospital, e
m
TremonaRoad,SouthamptonSO166YD,UK4DepartmentofBiology,UniversityofYork,POBox373,YorkYO105YW,UK5Leeds ic
CentreForReproductiveMedicine,LeedsTeachingHospitalNHSTrust,SeacroftHospital,YorkRoad,LeedsLS146UH,UK .o
u
p
*Correspondenceaddress.E-mail:[email protected] .co
m
SubmittedonFebruary2,2010;resubmittedonApril21,2010;acceptedonMay18,2010 /m
o
le
abstract: This study investigated the relationship between human preimplantation embryo metabolism and aneuploidy rates during hr/a
development in vitro. One hundred and eighty-eight fresh and cryopreserved embryos from 59 patients (33.9+0.6 years) were cultured rtic
le
for 2–5 days. The turnoverof 18 amino acids wasmeasured in spent media by high-performance liquid chromatography. Embryos were -a
b
eitherfixedforinterphasefluorescentinsituhybridizationanalysisofchromosomes13,18,19,21,XorY,orwereassayedformitochondrial s
activity. Amino acid turnover was different (P,0.05) between stage-matched fresh and cryopreserved embryos due to blastomere loss trac
following warming. The proportion of embryos with aneuploid cells increased as cell division progressed frompronucleate- (23%) to late t/16
cleavagestages(50–70%).Asparagine,glycineandvalineturnoverwassignificantlydifferentbetweenuniformlygeneticallynormalanduni- /8/5
5
formlyabnormalembryosonDays2–3ofculture.ByDays3–4,theprofilesofserine,leucineandlysinedifferedbetweenuniformlyeuploid 7
/1
versusaneuploidembryos.Gendersignificantly(P,0.05)affectedthemetabolismoftryptophan,leucineandasparaginebycleavage-stage 0
0
1
embryos.Pronucleatezygoteshadasignificantlyhigherproportionofactive:inactivemitochondriacomparedwithcleavage-stageembryos. 0
2
Furthermore,mitochondrialactivitywascorrelated(P,0.05)withalteredaspartateandglutamineturnover.Theseresultsdemonstratethe 7 b
association between themetabolism,cytogenetic composition and healthof humanembryosin vitro. y g
u
e
Keywords: aneuploidy /mitochondria / metabolomics / embryo development /FISH st o
n
1
7
N
o
Introduction abnormalities (Delhanty et al., 1997; Munne´ et al., 1998; Gianaroli v
e
m
et al., 1999; Kahraman et al., 2000; Marquez et al., 2000). Errors in b
e
Assisted reproduction technologies (ART) are hampered by two both meiotic and mitotic segregation of chromosomes in the oocyte r 2
major problems, namely low success rates and the high incidence of andearlyembryo,respectively,canleadtodifferentpatternsofaneu- 01
8
multiple pregnancies (The ESHRE Capri Workshop Group, 2000; ploidy including polyploidy and chaotic mosaics. Indeed, the latter
Adamson and Baker,2004). A major priority in human assistedcon- account for (cid:2)30% of aneuploidies in which multiple chromosomes
ception clinics is therefore the development of a reliable diagnostic are affected in each cell (Munne´ and Wells, 2003). However,
test which can be used to select those embryo(s) with the greatest despite the lethality of grossly aberrant chromosome complements,
developmental competence for transfer. Although the number of it is rarely possible to distinguish morphologically between healthy
embryos transferred after ART can be limited to reduce the embryosandthosewithabnormalchromosomalcomplements.Con-
number of multiple gestations, more sophisticated methods must be sequently,manyof theseembryosaregradedas suitablefor transfer
employed if the success rates of IVF are to be improved as .40% (Rienzietal.,2005;Nagy,2008;Sturmeyetal.,2008).Overall,theevi-
of ART-derived embryos are known to harbour chromosomal dence suggests that subjective scoring systems based on embryo
&TheAuthor2010.PublishedbyOxfordUniversityPressonbehalfoftheEuropeanSocietyofHumanReproductionandEmbryology.Allrightsreserved.
ForPermissions,pleaseemail:[email protected]
558 Pictonetal.
morphology are an ineffective means of selecting developmentally and the extra time and expense incurred, may not justify the wide-
competent embryos (Munne´ et al., 2009). There is therefore a clear spread application of this technique as a routine method forembryo
need to develop alternative objective approaches which can be used selection across all patients groups. Indeed, the use of aneuploidy
toselectthe best embryos for transferand cyropreservation. screening for this purpose has recently been questioned (Platteau
Aconsiderableamountofresearchhasbeenconductedtodevelop et al., 2006; Anderson and Pickering, 2008; Harper et al., 2008;
techniques which will reliably identify which in vitro-derived embryos Jansenetal.,2008;Mastenbroeketal.,2008;YakinandUrman,2008).
have the potential to produce a successful pregnancy. For many An alternative, non-invasive strategy which has consistently been
years, embryo selection has been based on the subjectiveevaluation shown to be of value in predicting the developmental potential of
ofembryocellnumber,blastomeresize,symmetryandfragmentation, human embryos in vitro is the measurement of different aspects of
andcleavagerate(Puissantetal.,1987;Steeretal.,1992;VanRoyen the metabolomic changes in the embryo’s culture environment
etal.,1999;Hardarsonetal.,2001;Racowskyetal.,2003;Rienzietal., (Brison et al., 2007; Nagy et al., 2008; Sturmey et al., 2008). Non-
D
2005). Recently, these methods have been extended to include pro- invasive metabolic profiling methods are frequently based on the o
w
nucleate scoring of zygotes (Tesarik and Greco, 1999; Scott, 2003), analysis of spent embryo culture media. These include the use of n
lo
cumulative scoring systems (Scott and Smith, 1998; Lan et al., 2003; near infrared and Raman spectroscopy (Seli et al., 2007; Scott et al., ad
e
Cirayetal.,2005;ChenandKattera,2006)andembryorespirometry 2008b) and scanning electrochemical microscopy (Utsunomiya et al., d
(Scottetal.,2008a).Thedevelopmentofnewgeneration,sequential 2008). One metabolomic approach which has proved robust across from
embryo culture media, which promoted blastocyst development in a number of species is the measurement of amino acid turnover in h
ttp
vitro in conjunction with blastocyst grading (Gardner et al., 2000; spent embryo culture media by high-performance liquid chromato- s
Balaban et al., 2006) and elective single blastocyst transfer, has also graphy (HPLC) (Houghton et al., 2002; Brison et al., 2004). On the ://a
c
a
been proposed as a means of improving the success of IVF while at basisoftheturnoverofasmallnumberofkeyaminoacids,itisposs- d
e
the same time reducing multiple births (Gardner et al., 2004; ible to discriminate accurately between morphologically similar fresh m
ic
Cutting et al., 2008). However, despite the advantages of many of or frozen-thawed (Stokes et al., 2007) cleavage-stage embryos .o
u
theseapproachesasresearchtools,theyhavefrequentlyproveddiffi- which have the capacity to form blastocysts (Houghton et al., 2002) p.c
o
culttoincorporateintoroutineclinicalpractice.Forexample,blasto- and achieve pregnancy (Brison et al., 2004). However, although m
cyst generation in vitro has the advantage that it is a non-invasive amino acid turnover appears to be predictive of fresh and cryopre- /m
o
approach; however, it is costly in time and resources and there are served embryo developmental potential in vitro (Houghton et al., leh
concerns about the safety of prolonged culture to the blastocyst 2002), it is unclear whether theregulationof amino acidmetabolism r/a
stage and the risk of potential aberrant epigenetic programming is linked to the genetic health of the embryo. In this context, for rticle
eventsduringextendedinvitrodevelopment(forreview,seeHuntriss example, according to the data available through the NCBI gene -a
b
s
and Picton, 2008). In contrast,as chromosomal aneuploidy is known website,severalaminoacidtransportersarelocalizedonchromosome tra
to affect all stages of preimplantation embryo development 19—a chromosome we have previously shown to be susceptible to ct/1
(Marquez et al., 2000; Bielanska et al., 2002), recent attention has abnormality in in vitro cultured human metaphase II oocytes (Clyde 6
/8
focused on the application of aneuploidy screening of oocytes and et al., 2003). /5
5
embryos as a means of improving embryo developmental potential This study was therefore conducted to quantify the aneuploidy 7/1
0
and pregnancyoutcome. levels of a panel of six chromosomes that are frequently involved in 0
1
Therationaleforusinganeuploidyscreeningasthebasisforembryo early pregnancy loss (Munne´ et al., 1998) in human preimplantation 02
7
selection isthatitwillensurethatthetransferred embryosarechro- embryos in relation to their amino acid metabolism profile and to b
y
mosomally normal (Marquez et al., 2000). Although some of the investigatethepossible roleof mitochondrial potential and activity in g
u
e
observed chromosomal errors such as pre-division of chromosomes this process. Spent culture media were collected from embryos cul- s
in oocytes can be attributed to suboptimal culture environments tured from Days 2 to 5 post-insemination for analysis of amino acid t on
1
andoocyteageinginvitro(Clydeetal.,2003),reviewoftheliterature turnover. Embryos were then disaggregated and chromosomal 7
N
onthegeneticcompositionofARTembryoshasidentifiedtwogroups health of all blastomeres was quantified using fluorescence in situ o
v
of patients who consistently have a high risk of aneuploid embryos hybridization(FISH)analysisforchromosomes13,18,19,21,XandY. em
(Gianarolietal.,1999;Kahramanetal.,2000).Theseincludepatients be
ofadvancedmaternalagewhohaveembryoswiththemostcommon Materials and Methods r 20
1
aneuploidies, such as trisomy 21 and sex chromosome aneuploidies, 8
which have been shown to increase 7- to 70-fold with age (Hook Unless otherwise stated, all chemicals and reagents used were obtained
et al., 1983) and patients with repeated IVF failure (Gianaroli et al., from Sigma Aldrich Chemical Company(Poole, Dorset, UK).Preimplan-
1999; Kahraman et al., 2000). In the latter group, the percentage of tationhumanembryossurplustotherapeuticneedsweredonatedtothis
researchprogrammebypatientsbeingtreatedforinfertilityattheRepro-
chromosomallyabnormalembryoshasbeenfoundtobedirectlypro-
ductive Medicine Unit at Leeds General Infirmary, Leeds, and at the
portionaltothenumberofIVFfailures.Theseobservationsareimpor-
Bourn Hall Clinic, Cambridge. The experimental protocol was approved
tant as an increasing proportion of women undergoing assisted
by the local research ethics committees and licensed by the UK Human
conception are over 35 years old (SART, 1992; Human Fertilisation
Fertilisation and Embryology Authority. Ovarian stimulation was con-
and Embryology Authority, 2007). Although this evidence supports
ducted using conventional GnRH agonist and antagonist protocols
the use of aneuploidy screening in defined patient groups, the (Rutherford et al., 1988; Out et al., 2004), and transvaginal ultrasound-
absence of convincing supporting data from prospective randomized guided oocyte collection and fertilization were performed as described
clinicaltrials,togetherwiththeinvasivenatureofthebiopsyprocedure previously (Tang et al., 2006). Surplus cleavage-stage embryos not
Associationbetweenaminoacidturnoverandchromosomeaneuploidy 559
requiredforcryopreservation onDay2post-inseminationweredonated Embryo chromosome preparation
intheLeedsclinic,whereassurpluscryopreservedtwopronuclear(2PN) and fixation
stage as well as cleavage-stage embryos were donated from the Bourn
Hall Clinic. One-cell embryos(2PN stage)
Embryos were incubated overnight in embryo culture media containing
0.15mg/ml podophyllotoxin and 0.15mg/ml vinblastine sulphate to
Embryo cryopreservation and thawing
induce cell cycle arrest (Angell et al., 1988; Rosenbusch et al., 2001).
Cryopreservedembryoswereslow-cooledinglassampoulesincommer- Embryos were then incubated in 0.1% (w/v) hypotonic sodium citrate
cially available freezing media containing 1,2-propanediol and sucrose for 15–20min at room temperature before transfer to ice-cold 3:1
according to the manufacturer’s instructions (Medicult UK Ltd, Redhill, methanol:glacial acetic acidfor (cid:2)3sbeforebeingspreadonto aSuper-
Surrey,UK).Embryoswerere-warmedbyexposuretoairfor(cid:2)30s,fol- frostPlusslide(VWR,Lutterworth,UK).
lowedbyimmersioninawaterbathat308Cuntilthawinghadoccurred.
D
The contents of the ampoule were expelled into a 30mm Petri dish o
w
(Nunc,Roskilde,Denmark)andembryosweretransferredthroughsequen- n
lo
tialdilutionsofcryoprotectantsinthethawingsolutions(MedicultUKLtd)at Cleavage-stageembryos ad
room temperature according to the manufacturer’s instructions, to pro- The embryos werestrippedof their zona pellucida by brief exposureto ed
gressivelyremovecryoprotectantagents.Embryoswerethentransferred acidified Tyrode’s saline solution (pH 3, room temperature). Each fro
toanembryoculturemediumat378Cfor(cid:2)1htofacilitatefullrehydration. embryo was incubated in 0.1% (w/v) hypotonic sodium citrate for m h
30minat48CbeforebeingtransferredtoDulbecco’sphosphate-buffered ttp
s
Embryo culture saline(Ca2+andMg2+-free)for5min.Finally,theembryowastransferred ://a
to ice-cold 3:1 methanol:glacial aceticacidfor (cid:2)3s beforebeing spread ca
d
Allembryoswerescoredusingestablishedmorphologicalcriteriaandcell ontoaSuperfrostPlusslide.Theslidewasobservedunderphase-contrast e
m
number (Houghton et al., 2002). Embryos were then incubated in 4ml microscopyand,wherenecessary,smalldropletsof5:4:1methanol:glacial ic
dropsof adefined culturemedium with acomposition based on thatof aceticacid:waterwereaddedtoassistcellspreading.Allslideswereaged .ou
p
human tubal fluid (Tay et al., 1997; Houghton et al., 2002) under overnightbeforedehydrationthroughaseriesofethanolsolutions(70%, .c
o
washed, equilibrated paraffin oil (Medicult UK Ltd), at 378C, and 5% 90% and 100%, 5min in each). Slides were stored on desiccant at 48C m
CO2 in air. After 24h of culture, embryos were scored and were then untilFISHanalysis. /mo
eithercontinuedincultureinafreshdropofmediumuptotheblastocyst le
h
stageorfixedandpreparedforchromosomeanalysis.Alldropsofspent Cytogenetic analysis and evaluation r/a
culturemediawereimmediatelyfrozenat2808Cuntilaminoacidanalysis. rtic
Cytoplasmic debris was removed by a 0.5–5min incubation in 0.01% le
-a
(w/v) pepsin in 0.01M HCl at 378C prior to hybridization. Simul- b
Amino acid analysis taneous FISH analysis for five chromosomes (13, 18, 21, X and Y) stra
c
Following thawing, a 2ml aliquot of culture media was removed from was conducted on all blastomeres according to the methodology t/1
described by Clyde et al. (2001) using a multicolour kit (PGT, MultiVy- 6
embryoculturedropsanddiluted 1:12.5inHPLC-gradewater.Embryo- /8
free control drops incubated alongside the embryo drops were treated sion PGT, Abbot, Maidenhead, UK) containing a premixed cocktail of /55
locus-specific probes for chromosomes 13 and 21, and centromeric 7
in the same manner to allow for any non-specific amino acid degra- /1
probes for chromosomes 18, X and Y. The chromosomal constitution 0
dation/appearance. Analysis of 18 amino acids was performed using 0
1
HPLCasdescribedpreviously(Houghtonetal.,2002). of each blastomere nucleus was classified as normal (diploid) and 02
abnormal (haploid, aneuploid, mosaic or chaotic) for the chromosomes 7
b
analysed according to the definitions described by Ziebe et al. (2003). y
g
Mitochondrial activity On the basis of the results of sibling blastomeres, each embryo was u
e
s
5,5′,6,6′-Tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine then classified as uniformly genetically normal (100% normal with all t o
blastomeres diploid for all chromosomes), overall normal (embryos n
iodide (JC-1, Invitrogen, Paisley, UK) is a commonly used probe for the 1
with .50%of analysed cellshavingnormal nuclei forall chromosomes), 7
assessment of mitochondria membrane potential (Reers et al., 1995; N
overall abnormal (≥50% of analysed cells having abnormal nuclei) or o
Jonesetal.,2004;VanBlerkomandDavis,2006).Uponinteractionwith v
mitochondria, the probe exists as a monomer in the presence of low uniformly genetically abnormal (0% normal blastomeres). A subset of em
potential difference (low activity) and fluoresces green, whereas greater fresh, cleavage-stage embryos were re-analysed with telomeric probes be
membrane potential (high activity) causes dimerization of the probe specific for the p and q arms of chromosome 19 (Telvysion, Abbot, r 20
Maidenhead, UK). 1
whichthenfluorescesorange.Bymeasuringtheratioofgreentoorange 8
fluorescence, it is therefore possible to quantify membrane potential
(Dcm) which reflects mitochondrial activity. Embryos were incubated in Statistical analysis
1mg/ml JC-1 in Hanks-balanced salt solution (HBSS containing 1mg/ml
The data were confirmed as being normally distributed using the
BSAand0.01%dimethylsulphoxide)for30minat378Candwashedfour Anderson–Darlingtest.Statisticalanalyseswereconductedusingthex2,
times in HBSS. Embryos were placed in drops of holding medium under
Student’s t-test or Mann–Whitney U-test, as appropriate. A P-value of
oilonastageheatedto378C,whereaswhole-oocytegreenandorangeflu-
,0.05wasconsideredtobesignificant.
orescencewasmeasuredusingaconventionalZeissAxioplanepifluores-
cencemicroscope(VanBlerkometal.,2002)withphotonmultipliertube
and photometer attachments (Photon Technology International, Ford, Results
W. Sussex, UK). Data were processed using Felix 32 software (Photon
Technology International). The JC-1 ratio was calculated from the A total of 66 fresh and 122 cryopreserved embryos were analysed
measuredorangeandgreenfluorescencevalues. from59patientsofmeanage33.9+0.6years(range24–42years).
560 Pictonetal.
Amino acid turnover of cryopreserved
and fresh embryos
OnDays2–3post-fertilization,certainaminoacids(glutamate,aspar-
agine, glycine and arginine) were metabolized differentially by fresh
embryos and those that had been cryopreserved (Fig. 1). On Days
2–3 of culture, embryos that had been cryopreserved on Day 2
had a significantly greater production of glutamate compared with
eitherembryoscryopreservedonDay1orfreshembryos.Turnover
ofasparagine and glycinewassignificantly differentbetweenembryos
cryopreservedonDay1or2post-fertilization,butneitherwassignifi-
cantly different to fresh, control embryos (Fig. 1). Arginine was con- D
o
sumed at significantly greater rates by embryos cryopreserved on w
n
Day 2 (Fig. 1). On Days 2–3, embryos cryopreserved at the 1-cell loa
d
(2PN) stage did not have significantly different amino acid profiles e
d
comparedwithfreshcontrols (Fig. 1). fro
m
BlastomeredamageandcelllossoccurredonthawinginsomeDay h
2 cryopreserved cleavage-stage embryos. The effect of cell loss and Figure 1 Turnover of glutamate (Glu), asparagine (Asn), glycine ttp
dtuervneoloveprmbeynteaml pbrroygorsesosnionDa(ypr2o–gr3espsoinsgt-foerrtailirzraetsiotend)woasnianmveisntoigaatceidd (EGmlyb)ryaonsdwaergrienineeith(eArrgc)ryboypreemsebrrvyeodsoonnDDaayys12(–23PNposstta-fgeer)tiloizratDioany. s://aca
2 (cleavage stage) or were not cryopreserved (Fresh). Values are d
(Fig.2).ThedifferencesinaminoacidturnoveronDays2–3observed mean amino acid turnover per embryo+SEM for the number of em
in cleavage-stage embryos cryopreserved on Day 2 appeared largely replicates shown in parentheses. Negative values indicate amino ic.o
to be associated with cell loss and/or arrested development. acid consumption, whereas positive values indicate production. up
.c
Embryos cryopreserved on Day 2 that lost cells upon thawing were Values with different letters are significantly different (P,0.05) for o
m
also significantly (P,0.05) more likely to arrest during culture (58% thenumberofanalysesshowninparentheses. /m
o
arrested compared with embryos that did not lose cells of which le
h
9%arrested).Celllosswasassociatedwithalteredturnoverofaspar- r/a
agine,glycineandargininecomparedwithDay1embryos,Day2intact orover.Thedatafromfreshembryosandembryoscryopreservedat rtic
embryos and fresh controls (Fig. 2b–d). In this group, embryos that the PN stage were pooled for the analysis of the effect of maternal le-a
arrestedduringtheDays2–3ofculturehadincreasedglutamateturn- age. Embryosthathad been cryopreservedon Day2 were excluded bs
overcomparedwithDay1embryos,Day2intactembryosandfresh from further analysis due to the impact of cryopreservation on trac
controls (Fig. 2a). amino acid turnover in this group. On Days 2–3, aspartate (0.13+ t/1
6
OnDay3–4post-fertilization,theonlyaminoacidaffectedbyprior 0.07 versus 20.11+0.05pmoles/h for embryos from patients /8/5
embryo cryopreservation was arginine. Embryos cryopreserved on under 37 years and 37 years and older, respectively) and glutamate 57
Day 2 showed differential arginine consumption according to (0.15+0.09 versus 20.27+0.15pmoles/h for embryos from /10
0
whether embryos had thawed intact (2.2+1.3pmoles/h; n¼11) patients under 37 years and 37 years and older, respectively) were 10
or had suffered loss of one or more cells (3.8+0.92pmoles/h; consumed in greater quantities by embryos donated from patients 27
b
n¼11; P¼0.03). No other embryos cryopreserved on Day 1 or 2 of advancing maternal age. Tyrosine was produced in greater quan- y g
had alteredaminoacidturnoverduring Days 3–4. tities by embryos on Days 2–3 that were derived from younger ue
Ithasbeenhypothesizedthatmetabolically‘quiet’humanandmam- patients (0.08+0.03 versus 20.01+0.02pmoles/h for patients st o
malian embryos have better developmental potential (Leese, 2002; under 37 years and 37 years and older, respectively). On Days 3–4 n 1
7
Baumann et al., 2007; Leese et al., 2007; Leese et al., 2008). There of culture, there were no significant effects of patient age on amino N
was no significant difference between net amino acid production acid turnover. ove
m
between different embryo groups, but embryos cryopreserved on b
e
Day 2 which subsequently lost cells upon thawing and underwent Cytogenetic analysis r 2
0
developmental arrest during Days 2–3 had consistently and signifi- 1
Clear FISH signals were obtained from 544 cells (Fig. 4) harvested 8
cantly (P,0.05) higher net amino acid consumption, net amino
from 112 embryos of which 85% had corresponding amino acid
acid activityand netturnoverthananyothertissuegroup.
profile data. No significant differences between different-grade
embryos were observed. The demography of the FISH data showed
Effect of maternal patient age on embryo
that there were no statistically significant differences in the number
amino acid turnover
of male and female embryos analysed, such that 53% of embryos
Whenmaternalagewasaccountedfor,significantdifferencesinturn- were female and 47% were male. For those embryos with one or
over of several amino acids were seen in embryos cultured on Days moreabnormalcells,33%ofthechromosomeabnormalitiesinvolved
1–2 (Fig. 3). Aspartate, glutamine, threonine, arginine, tyrosine, onlyoneofthesixchromosomestested,20%involvedtwochromo-
valine and isoleucine were consumed in greater quantities by somes and the remainder (47%) involved three or more chromo-
embryosfrompatientsunder37yearsofage,whereaslysinewascon- somes. Hypoploidies and hyperploidies accounted for 54% and
sumedatgreaterratesbyembryosdonatedbypatientsaged37years 46% of the chromosomal abnormalities, respectively. Numerical
Associationbetweenaminoacidturnoverandchromosomeaneuploidy 561
D
o
w
n
lo
a
d
e
d
fro
m
Figure3 Effectofmaternalageonaminoacidturnoverbyembryos h
culturedfromDays1–2.Valuesaremeans+SEMforthenumberof ttps
embryos shown in parentheses. Significant differences are indicated ://a
byanasterisk(P≤0.05). ca
d
e
m
ic
.o
abnormalities in chromosomes 13 and 21 were the most prevalent, u
p
accounting for 28% and 25% of the genetic errors, respectively. .co
m
Abnormalities in chromosome 18 accounted for 15% of errors, /m
whereaserrorsin Xand/or Yaccounted for 32%.A small subsetof ole
h
thefreshembryosunderwentasecondroundcytogeneticevaluation r/a
forchromosome19.OnDay3,100%oftheembryosthathadnormal rtic
countsforchromosomes13,18,21andX/Yalsohadnormal,diploid, le
-a
counts for chromosome 19 (n¼5). Of all the embryos analysed for b
s
chromosome 19 on Days 3–5, 17.4% had one or more cells with tra
c
an abnormal countforchromosome19. t/1
6
/8
/5
Effect of cryopreservation on incidence of chromosome 5
7
abnormalities /10
0
There was no significant difference in the frequency of chromosome 10
2
abnormalities on Day 3 (after culture from Days 2–3) in embryos 7
b
that had been cryopreserved at the PN stage compared with fresh y g
u
embryos.Celllosspost-thawdidnotaffecttheincidenceofchromo- e
s
someabnormalityinembryoscryopreservedonDay2thatwerepro- t o
n
cessed for FISH onDay4. 1
7
N
o
Stage of embryo development ve
m
Following FISH, the proportion of embryos with chromosome b
e
abnormalities at different stages of development was analysed r 2
0
(Fig. 5). Pronuclear-stage embryos had an abnormality frequency of 18
23% (n¼26). The percentage of genetically abnormal embryos
increasedwithsuccessivedaysofdevelopment(Fig.5).Furtheranaly-
sisrevealedthatthiswasduetoanincreasingoccurrenceofembryos
Figure 2 Turnoverofglutamate(a),asparagine(b),glycine(c)and
arginine(d)byprogressingandarrestedembryosonDays2–3post- withamixtureofnormalandabnormalcells(mosaic)asdevelopment
fertilizationthatwerecryopreservedonDay1(2PNstage)orDay2, progressed(TableI).Theproportionofuniformlyabnormalembryos
compared with fresh embryos. Values are means+SEM for the didnot change significantly.
numberofembryosshowninparentheses,adjustedforanycellloss.
Statistical significance is identified by differences in the histogram Association between amino acid profile and chromosome
labelssuchthatbarslabelledwithdifferentlettersaresignificantlydiffer- abnormalities
ent(P,0.05)forthenumberofanalysesshowninparentheses.Con-
Amino acid turnover on Days 2–3 and 3–4 post-fertilization was
versely,barslabelledwithcommonlabelsarenotsignificantlydifferent.
compared between embryos that were uniformly normal (no
562 Pictonetal.
D
o
w
n
lo
a
d
e
d
fro
m
h
ttp
s
://a
c
a
d
e
m
ic
.o
u
p
.c
o
Figure 4 Interphase FISH of embryo blastomeres for chromosomes: 13 (Spectrum RedTM), 18 (Spectrum AquaTM), 21 (Spectrum GreenTM), m
X(SpectrumBlueTM),Y(SpectrumGoldTM)and19(keyshowninimage).(a)NucleiwerecounterstainedwithCot-1DNAlabelledwithSpectrum /m
o
AquaTM.Images(a)and(b)arenucleifrommaleandfemaleembryos,respectively,withnormalnumberforthechromosomesanalysed.(c)Amale leh
nucleuswithmonosomy21and(d)afemalenucleuswithtrisomy13.(e)Identificationofchromosome19usingtelomericprobesforthep(green) r/a
andq(red)arms,counterstainedwithDAPI. rtic
le
-a
b
s
tra
c
abnormalcells)anduniformlyabnormal(nonormalcells)(Fig.6).On Mitochondrial activity t/1
6
Days 2–3, turnoverof asparagine, glycine and valinewassignificantly /8
Mitochondrial activity, as assessed by JC-1 ratio, was analysed in a /5
deniffceerseontbsbeertvweedeinnntyorromsianleaannddalbysnionremaaplpermoabcrhyeodss(iFgingi.fi6caa)n.cTeh(eFigd.if6fear)-. spuobrstieotnooffemmbitroycohso.nAdrhiaighwivtahluleowforDJcCm-1arnadtiothinedreicfaotreeslaowhigahctpivrioty- 57/100
Asdevelopmentprogressed,therewasashiftintheobserveddiffer- 1
which presumably produce reduced ATP. Conversely, a low JC-1 02
encesuchthatonDays3–4,turnoverofserine,leucineandlysinewas 7
value indicates a low proportion of inactive mitochondria and a b
significantlydifferentbetweennormalandabnormalembryos(Fig.6b). y
high proportion of active mitochondria. Relative mitochondrial g
OnDays2–3and3–4,embryoscontainingamixtureofnormaland activity changed significantly during early development (Table II). ues
abnormalcellsdidnothavesignificantlydifferentaminoacidturnover One-cell embryos had a significantly greater proportion (P,0.05) t on
relative touniformly normal or uniformlyabnormal embryos. ofactivemitochondriacomparedwithallsubsequentstagesofdevel- 17
N
opment. In contrast, cleavage-stage embryos that had arrested had o
Relationship between embryo sex and amino v
proportionally more inactive (P,0.05) mitochondria compared e
m
acid turnover with their counterparts that were still actively progressing. A similar be
The influence of embryo sex, as determined by FISH for chromo- finding was observed in blastocysts (P,0.05) (Table II). Embryos r 2
0
1
somes X and Y, was investigated. On Days 1–2, no significant cryopreserved on Day 2 that were subsequently thawed, cultured 8
differences in amino acid turnover were observed between female and assessed for mitochondrial activity on Day 4 had differences in
(n¼5)andmale(n¼6)embryos.Incontrast,onDays2–3,trypto- JC-1 ratio that approached significance according to whether the
phan was consumed at greater rates by male embryos (20.06+ thawed embryos were cellularly intact or whether they had lost
0.02pmoles/h, n¼12) compared with female embryos (0.002+ one or more cells post-thaw. Embryos that thawed intact had a
0.01pmoles/h, n¼16; P¼0.04). Similarly, leucine was consumed lower JC-1 ratio of 1.7+0.3 (n¼10) and therefore a higher
at greater rates by male embryos (20.38+0.06pmoles/h) number of active mitochondria compared with embryos with cell
compared with female embryos (20.18+0.05pmoles/h; loss (2.9+0.6; n¼9; P¼0.066) which contained proportionally
P¼0.014).ByDays3–4ofculture,onlyasparaginewasdifferentially more inactive mitochondria. Mitochondrial activity was also signifi-
metabolized by female and male embryos (20.14+0.07pmoles/h, cantly correlated with aspartate (P¼0.04, Pearson¼20.32) and
n¼10 versus 0.20+0.11pmoles/h, n¼13 for female and male glutamine turnover (P¼0.041, Pearson¼0.321). At each stage of
embryos, respectively,P¼0.025). embryo development, the proportion of inactive mitochondria was
Associationbetweenaminoacidturnoverandchromosomeaneuploidy 563
D
o
w
n
lo
a
d
e
d
fro
m
h
ttp
s
://a
c
a
d
e
m
ic
.o
u
p
.c
o
m
/m
o
le
h
r/a
rtic
le
Figure 5 Frequency of embryos with one or more abnormalities -a
forchromosomes13,18,21,XorYduringpreimplantationdevelop- bs
ment, with respect to embryo cell number (a) and time post- tra
c
fertilization(b).Thenumbersofembryosanalysedareshowninpar- t/1
6
entheses.Bracketsindicatesignificanttrends(P-valueshowninitalics). /8
Figure6 Aminoacidturnoverbyindividualembryosinrelationto /5
5
abnormalityofchromosomes13,18,21,XandYonDays2–3(a) 7/1
and Days 3–4 (b). Data from embryos cryopreserved on Day 1 00
TableI Incidenceofchromosomeabnormalities 1
were pooled with data from stage-matched fresh embryos for ana- 0
2
duringearlypreimplantationdevelopment. lyses on Days 2–3, whereas data from embryos cryopreserved on 7 b
Day %uniformly %uniformly %mixturenormal/ DEmaybr2yoasndthafrteswheeremburnyifoosrmwleyrechproomoloedsofmorallaynanloysremsalonorDaabynso3rm–4a.l y gue
normal abnormal abnormalcells s
........................................................................................ are shown. Values are means+SEM of the number of embryos t o
1 81.0 19.0 0.0 shown in parentheses. *P,0.05 and a double-asterisk indicates a n 1
2 57.1 14.3 28.6 trend towards significance; P¼0.099 for tyrosine and P¼0.085 7 N
forlysine. o
v
3 51.5 15.2 33.3 e
m
4 44.4 22.2 33.3 b
e
5 30.0 25.0 45.0 Patient age had a significant influence on mitochondrial activity in r 20
1
P 0.007 0.200 0.042 PN-stage embryos. Embryos from young patients of 37 years or less 8
had significantly higher mitochondrial activity (0.59+0.05, n¼8),
P-valuesfortrendanalysisofeachcolumnareshown.
compared with embryos from older patients over 37 years (1.03+
0.09, n¼4; P¼0.014). Maternal age did not significantly influence
higher in embryos that had ≥50% abnormal cells compared with mitochondrial activityatsubsequentstages of embryodevelopment.
embryos with .50% normal cells but the differences at each stage
were not significant. However, when cleavage-stage embryos that
were either uniformly normal or uniformly abnormal were analysed, Discussion
mitochondrial activity was observed to be significantly higher in
normal embryos (1.11+0.16; n¼4) compared with uniformly Itisnowpossibletobaseembryoselectioncriteriaonquantifiablebio-
abnormal embryos which had more inactive mitochondria (2.71+ logicalcharacteristicsofthedevelopingembryoratherthanonsubjec-
0.51; n¼5, P¼0.041). tivemeasures. The resultsof this study confirm and extend previous
564 Pictonetal.
outcomes (Houghton et al., 2002). In the present study, glutamate
TableII Mitochondrialactivityratiosinhuman
was produced in greater quantities by embryos cryopreserved on
embryos,assessedbyJC-1staining.
Day 2 that suffered cell loss and developmental arrest, post-thaw.
Thismightreflectanattempttoneutralizeincreasedammoniumpro-
Embryostage n JC-1ratio SEM
........................................................................................ ductionbytheseembryos(LaneandGardner,1995;OrsiandLeese,
1-cell(2PN) 12 0.74a 0.08 2004). It is pertinent to note that recent preliminary findings suggest
Cleavage—progressing 18 2.25b 0.24 that human embryos which are less likely to result in pregnancy
Cleavage—arrested 22 4.95c 0.93 have elevated glutamate output (Seli et al., 2008). Regardless of the
Blastocyst—progressing 3 1.84b 0.62 differences in amino acid turnover between the embryos as each
Blastocyst—arrested 8 8.89d 1.38 developmental stage, variation in metabolism between the early and
late time points may also reflect the transition from maternally
D
Valuesaremeans+SEMofthenumberofembryosshown.Valueswithdifferentletters sourcedmRNAtoembryo-driventranscriptionandtranslationfollow- o
aresignificantlydifferent(P,0.05).Highervaluesindicateagreaterproportionof w
ing embryonic genome activation. n
inactivemitochondria. lo
Inthecurrentwork,cytogeneticevaluationswereconductedusing ad
e
published definitions of embryo normality (Bielanska et al., 2002; d
measurementsoftheturnoverofaphysiologicalmixtureof18amino Ziebeetal.,2003)suchthatembryoswith≥50%normaldiploidblas- from
acids by human preimplantation embryos (Houghton et al., 2002; tomereswereconsideredtobeviable.Thisdefinitionfacilitatedcom- h
ttp
Stokes et al., 2007). In addition, this is the first report to compare parison between embryos which were 100% genetically normal or s
directly the amino acid turnover of fresh and cryopreserved 100% abnormal with those containing a mixture of cell types. The ://a
c
a
embryos and to link aspects of amino acid profile with cytogenetic analysis of chromosomes presented here correlates with the obser- d
e
assessments of embryo health. Reduced blastocyst formation vations of other studies (Ziebe et al., 2003) and shows that the m
ic
(Houghton et al., 2002; Stokes et al., 2007) and increased DNA chromosome complement of up to 70% of surplus ART embryos is .o
u
damage (Sturmey et al., 2009) have been linked to greater overall compromised by non-disjunction events and mosaicism, depending p.c
o
aminoacidmetabolismwhichsupportsthehypothesisthatmetaboli- onthe stage of development and numberof dayspost-fertilization. m
cally‘quiet’embryoshavebetterdevelopmentalcompetence(Leese, Onthefirstdaypost-fertilization,priortotheonsetofmitoticcell /m
o
2002).Thecurrentdataoncryopreserved-thawedembryosreinforce divisions, the incidence of aneuploidy involving one or more of the leh
this observation, as embryos cryopreserved on Day 2 that sub- six chromosomes tested was (cid:2)20%, which is within the range of r/a
sequentlylostblastomeresonthawinghadreducedratesofdevelop- values reported for good quality oocytes: 8–10% (Smitz et al., rticle
ment and a higher overall amino acid consumption and turnover. 2007), 9–14% (Volarcik et al., 1998) and 35% (Sandalinas et al., -a
b
s
Furthermore, embryo cryopreservation on Day 2 post-insemination 2002).Theproportionofembryoswithoneormorechromosomally tra
was associated with altered amino acid turnover on Days 2–3 of abnormal cells is known to increase during the first few days post- ct/1
culture. In marked contrast, embryos that had been cryopreserved fertilization, largely due to mosaicism, agreeing with the observations 6
/8
on Day 1 (2PN stage) did not have an altered amino acid profile by ofBielanskaetal.(2002).Tofullyassestheconsequencesofchromo- /5
5
Days 2–3 of culture, compared with fresh controls. The differential somealterationsonembryometabolismanddevelopmentalpotential, 7/1
0
aminoacidprofileofembryoscryopreservedonDay2wasassociated itisnecessarytoconductfullkaryotypeanalysesofthecompletecom- 0
1
withshorterrecoverytimecomparedwithembryoscryopreservedon plimentofblastomereswithineachindividualembryo.Thisgoalcanbe 02
7
Day1andwasalsoassociatedwithcellloss.Thissuggeststhatcryo- achieved by achieved by multiparameter FISH analysis of metaphase b
y
preservationpersedoesnotalterembryometabolism,butratherthat spreads of blastomeres using our previously published protocols g
u
e
cellular damage or stress associated with blastomere loss does. (Clyde et al., 2003). However, although the current protocol did s
Additionally, the turnovers of glycine and arginine were exacerbated support the arrest of blastomeres in metaphase, insufficient cells t on
1
by developmental arrest. werearrestedatthisstageofthecellcycletopermitmeaningfulanaly- 7
N
Aminoacidshavenumerousrolesinearlyembryodevelopmentin sisandthegreatermajorityofembryoblastomereremainedininter- o
v
additiontoproteinsynthesis.Glycineisknowntobeanosmoregula- phaseattheendofembryoculture.Consequently,thecurrentstudy em
tor, balancing changes in embryo cell volume (Hammer et al., 2000; in line with accepted practices used interphase FISH on a subset of be
Baltz,2001;SteevesandBaltz,2005).Inthiscontext,glycinewascon- embryo chromosomes (Smith et al., 1998; Marquez et al., 2000; r 2
0
1
sumedingreaterquantitiesbyembryoscryopreservedonDay2that Thum et al., 2008) to analyse all blastomeres and so provide an 8
lostcellsduringthawing.However,itisunclearfromthepresentstudy indexofthecytogenetichealthofeachembryo.Indeed,thechromo-
whether the increased glycine consumption occurred in response to some series analysed in the current work contribute to (cid:2)95% of all
celllossorwhetheritactuallywasthecauseofcellswellingandsub- chromosome disorders observed in live births and 20–27% of the
sequentlysis.Arginineconsumptionhasbeenlinkedtoincreasednitric abnormalities observed in human metaphase II oocytes (chromo-
oxide(NO)productionbymammaliancells(Cendanetal.,1996).NO somes 13, 18, 21 and X; S.E.H. SE and H.M.P., unpublished obser-
is necessary for normal mammalian embryo development (Tranguch vations; Clyde et al., 2003). In support of the proposed close
et al., 2003; Manser et al., 2004) but elevated NO is associated association between early embryo metabolism and karyotype, we
with induction of apoptosis (Brune et al., 1995, 1999; Nicotera havepreviouslyanalysedaminoacidturnoverandenergymetabolism
et al., 1995) and poor human embryo quality (Lee et al., 2004). It inrelationtothefullkaryotypeanalysisofmetaphaseIIhumanoocytes
haspreviously been shown thathigher ratesof arginine consumption and have found these indices of oocyte metabolism to be associated
by human embryos are associated with poorer developmental with patient aetiology and the capacity of the oocytes to progress
Associationbetweenaminoacidturnoverandchromosomeaneuploidy 565
to metaphase II in vitro (H.M.P., S.E.H. and K.E. Hemmings, unpub- and polar body formation. The oocyte and early embryo are thus
lished data). Similarly, in bovine oocytes, the competence of in vitro heavily reliant on mitochondrial respiration of energy metabolites to
matured oocytes to fertilize and progress to the blastocysts in vitro generate energy for subsequent embryo growth and cell replication
canbepredictedbythemeasurementofmetaphaseIIoocytemetab- (Biggers et al., 1967; Hardyet al., 1989; Wilding et al., 2001; Downs
olism(K.E.Hemmings,H.J.L.andH.M.P.,unpublisheddata).Further- et al., 2002; Johnson et al., 2007). Declining oocyte mitochondrial
more,astrongassociationbetweenzygoteandoocytemetabolismis function hasbeen linked to ageing(Tarin, 1996; Wilding et al., 2001;
expected as the cytoplasmic inheritance of the fertilized zygote orig- Eichenlaub-Ritter et al., 2004) and it is thought that this may predis-
inates from the oocyte. With regard to human embryo quality, the poseoocytestomeioticerrors(Schonetal.,2000;Eichenlaub-Ritter
limited cytogenetic data setpresented hereclearlyshowthataltered et al., 2004). The oocyte is especially reliant on pyruvate, but other
metabolic turnover by embryos reflects the common aneuploidies. substrates,includingaminoacids,arealsometabolizedforenergygen-
Furthermore, within the cohort of abnormal embryos analysed in eration viamitochondrial respiration.
D
thecurrentwork,approximatelyone-thirdhadabnormalitiesinvolving This study assessed whether there were any demonstrable links o
w
justonechromosome,whereastheremainingabnormalembryoshad betweenembryoaminoacidprofile,mitochondrialactivityandaneu- n
lo
twoormoreaffectedchromosomes.Onthisbasis,abnormalembryos ploidy. Embryo mitochondrial activity was linked to amino acid turn- ad
e
tendedtohavemorethanoneabnormality,makingitmorelikelythat over; however, although the JC-1 ratio was consistently higher in d
at least one of the abnormal chromosomes will be detected in the embryos with chromosomal abnormalities, the differences between from
current study. It is likely that at least a small proportion of the overall normal (.50% normal cells) and overall abnormal (≥50% h
ttp
embryos that we have classified as ‘uniformly normal’ may have an abnormal cells) embryos were not significant. However, when s
undetected chromosome abnormality which could potentially affect embryos were assessed that had either 100% normal cells or 100% ://a
c
a
amino acid turnover; however, a large proportion can be expected abnormal cells, a significant difference in mitochondrial activity was d
e
tobe normal. observed.Thesedatapointtowardsapossiblelinkbetweenchromo- m
ic
The observed increase in post-zygotic genetic errors in human some abnormality and the quality of the mitochondrial cohort inher- .o
u
embryos grown in vitro is likely to be due to multiple, contributing ited by the embryo from the oocyte. Increased numbers of p.c
o
reasons. Early embryos are also thought to be permissive with embryos needto be analysed toconfirm this preliminary finding. m
respect to mitotic checkpoints (Wells et al., 2005) and spindle Aminoacidturnoverhaspreviouslybeenshowntopredicthuman /m
o
defects are also common (Chatzimeletiou et al., 2005), but whether embryodevelopmentalpotential(Houghtonetal.,2002;Brisonetal., leh
the increase in mosaicism is a natural phenomenon or is related to 2004).Thecurrentpaperrepresentsthefirstreportofanassociation r/a
thesuboptimalenvironmentinvitroorsuboptimalovarianstimulation betweenaminoacidturnoverandhumanembryogenetichealth.Itis rticle
protocolsisnotwellunderstood.Ovarianhyperstimulationisthought ofparticularinterestthatdifferencesweredetectedinasparagineand -a
b
s
to be associated with reduced oocyte quality in humans (Akagbosu glycinemetabolismwhenuniformlynormalversusuniformlyabnormal tra
etal.,1998)andmice(VanderAuweraandD’Hooghe,2001),includ- embryos were compared, as these amino acids have been shown to ct/1
ingchromosomeabnormalities(Simonetal.,1998;Baartetal.,2006; be of value in the prediction of future in vitroembryodevelopmental 6
/8
Dursun et al., 2006). However, even unstimulated cycles produce totheblastocystsstageandsubsequentpregnancypotentialfollowing /5
5
human embryos with high rates of aneuploidy (Verpoest et al., embryo transfer. There are multiple possible explanations for the 7/1
0
2008), suggesting that ovarian hyperstimulation is not the only cause currentobservations.Withextraormissingcopiesofchromosomes, 0
1
of chromosome aberrations. The effect of advancing maternal age nuclear gene dosage becomes perturbed, which can lead to altered 02
7
on incidence of oocyte and embryo chromosome abnormalities is regulation of transcription (Rachidi and Lopes, 2007; Veitia et al., b
y
well documented (Dailey et al., 1996; Pellestor et al., 2005; Munne´ 2008). Also, it has previously been reported that chromosomes g
u
e
et al., 2007). The current study has also highlighted for the firsttime with altered copy numbers have altered nuclear location (McKenzie s
some notable differencesin the turnoverof amino acids byembryos et al., 2004). Additionally, in abnormal nuclei, all chromosomes t on
1
donated from younger compared with older patients, although the (regardlessofcopynumber)tendtohavealterednucleardistribution 7
N
effectsofageonaminoacidturnoverdiminishedasembryodevelop- (McKenzie et al., 2004). Chromosome and nuclear architecture is o
v
mentprogressed.Finally,humanandmammalianembryosareknown known to regulate gene expression (Finlan et al., 2008; Simonis and em
to be susceptible to the in vitro effects of supra-physiological oxygen de Laat, 2008), so it is possible that in abnormal cells, transcription be
tensions(CattandHenman,2000;OrsiandLeese,2001).Aircontain- from multiple chromosomes may be altered. The different elements r 2
0
1
ing5%CO isstillthemostcommonlyusedcultureenvironmentfor of amino acid transport and metabolism are coded by multiple 8
2
in vitro-produced human embryos, although oxygen tension in vivo is genesonmultiplechromosomes(NCBIEntrezGenewebsite).Altera-
in the region of 5% compared with 21% in air. Numerous studies of tion of chromosome content can therefore be hypothesized to alter
animal and human embryos have demonstrated an association the subcellular machinery and these changes would be manifest by
between oxygen tension and reduced embryo quality, likely due to analterationinblastomeremetabolism.Insupportofthisidea,individ-
formation of reactive oxygen species (ROS). Although it is possible uals with trisomy 21 have altered plasma amino acid levels (Lejeune
inthecurrentstudythatROScontributedtotheincreasingincidence etal.,1992;Heggartyetal.,1996),includinglysineandserine.Further-
of chromosomal abnormalities which were observed with embryo more, in trisomic mice, the activity of certain enzymes involved in
developmental progression, this does not explain the reduction in amino acid metabolism is affected (Vaisman et al., 1981). In this
chromosome abnormalitiesrecordedafter Day5of culture. context, for example, chromosome 19 is known to carry several
In oocytes, completion of meiosis involves energy-demanding pro- amino acid transporter genes (NCBI Entrez Gene website) and we
cesses including chromosome congression, segregation, separation have previously shown that (cid:2)6% of human metaphase II oocytes
566 Pictonetal.
haveabnormalcopynumbersofchromosome19(Clydeetal.,2003). apparent around the time of genome activation and may be related
Inthecurrentstudy,17.4%ofcleavage-stageembryoscarriedoneor tocopynumberoftheXchromosome.ChromosomeXcarriesmul-
morecellswithabnormalcopynumberforchromosome19.Although tiple genes related to amino acid turnover and transport, including
thisishigherthanthefrequencyofabnormalityobservedinoocytes,it SLC38A5, a system N transporter which can convey asparagine, one
may be explained by the progressive increase in chromosome of the amino acids differentially metabolized in female and male
abnormalitiesthatoccurascellsreplicateduringearlyembryogenesis, embryos.
asobservedinthecurrentstudyandbyBielanskaetal.(2002).Amino In conclusion, grossly aneuploid embryos demonstrate altered
acid profiles werenot obtainedfromasufficient numberof embryos amino acid turnover in vitro relative to their genetically normal
for turnover to be compared with respect to chromosome 19 counterparts. These data suggest that the non-invasive method of
content. Additional analyses are therefore required to test this aminoacidprofilingwillbeofvalueasatoolforroutineembryoselec-
hypothesis further. tion across all patient groups, as this technology can provide insight
D
Thedatapresentedclearlyshowthataminoacidconsumption/pro- intoembryogenetichealth.Furtherresearchisrequiredtodetermine o
w
duction is predictive of blastocyst potential and the link between how many genetically abnormal blastomeres an embryo can accom- n
lo
amino acidprofileand genetic health in wholly normal versus abnor- modatebeforethedevelopmentalcompetenceoftheembryoiscom- ad
e
malembryosisofclinicalimportance.However,aminoacidturnover promised and whetherit willbe possible to improve embryo culture d
persecannotyetbeusedasanindextopredictthegenetichealthof conditionsand thus reducemitotic errors. from
embryos composed of a mixture of normal and abnormal cells on h
ttp
Days 2–3 post-insemination. This observation is not altogether sur- s
prisingastheframeofreferenceusedforclassificationofembryonor- Authors’ roles ://a
c
a
malityinthecurrentstudywasbasedonthedefinitionusedbyZiebe d
H.M.P.: study design, research lead, grant holder, manuscript prep- e
etal.(2003)tocharacterizewhichembryoswerelikelytodevelopor m
aration. K.E.: supply of resources and comments on manuscript. ic
not.Atthetimeofwriting,thereisstillnoaccuratedatatoinformthe .o
F.D.H.: measurement of amino acids, collaborator, co-author of u
debateonexactlyhowmanyabnormalcellsacleavage-stageembryo manuscript. J.A.H.: experimental work including amino acid analysis p.co
canaccommodatebeforedevelopmentalcompetenceislost.Inclusion m
of the experimental series presented in the present study will help and comments on manuscript. A.J.R.: clinical lead, research consent /m
arenqswuiererdthtisoimapccourrtaatnetlyquaedsdtiroenssbutthisextiessnuseiv.eNfuervtheertrhealneaslsy,sestheasree awnhdichethfuicnsd.eJd.E.sHtu.:dye,msburpyeorlovigsyionwoorfk.YHor.Jk.L-b.:asceod-apppelriscoanntneol,nrgergaunlatsr olehr/a
results, together with the close correlation between the predicted memeebtriynogs ctuoltudreis,cusesmbdraytoa. FSI.SEH.H,.: dpaotast-daoncatlyosriasl arensdearmchanufeslcloriwpt, rticle
developmental potential of embryos by amino acid profiling and -a
preparation. b
s
implantationratesachievedfollowingtransferofembryostypedretro- tra
spectively (Brison et al., 2004), suggest: (i) that human embryos can ct/1
accommodate a proportion of genetically abnormal cells as they Acknowledgements 6
/8
develop; and (ii) that the upper ceiling of pregnancy rate following /5
5
selection based on amino acid profiling will ultimately be set by the WearegratefulforthecontinuedsupportoftheclinicalEmbryologists 7/1
genetichealth of theembryo. at the Reproductive Medicine Unit, Leeds General Infirmary and 00
1
The effect of embryo genetic sex upon amino acid turnover was BournHall Clinic. 02
7
examined. This data set provides the first direct evidence to show b
thatamino acids aredifferentially metabolized by male versus female Conflict of interest: H.J.L., Scientific advisor and shareholder in y g
u
Novocellus—a company which is developing embryo diagnostic e
cleavage-stagehumanembryos.OnDays1–2,thefactthatnosignifi- s
cantdifferenceinmetabolismwasobservedbetweenmaleandfemale methods for usein clinical IVF. F.D.H., shareholderin Novocellus. t on
1
embryos is not surprising as genome activation has not occurred by 7
N
this time and the cellular metabolism of the zygote is largely deter- Funding o
v
mined by the cytoplasmic legacy inherited from the oocyte. In con- em
trast, on Days 2–3 and 3–4, differences in the turnover of 3 of the ThisworkwassupportedbytheUKMedicalResearchCouncil(grant be
18 amino acids analysed, tryptophan, leucine and asparagine, were numbers: G9813925, G9722300, G0800250) and Biotechnology and r 20
1
observed.Recentdatahavedemonstratedthatgender-specificdiffer- Biological Sciences Research Council (grant numbers: BBSRC; BB/ 8
encesexistin7ofthe18aminoacidsand2ofthe18aminoacidsana- C007395/1). F.D.H. was funded by a Wellcome Trust Research
lysedbetweenmaleandfemalebovineblastocystsderivedinvitroand CareerDevelopment Fellowship (grantnumber:WT066492MA).
invivo,respectively(Sturmeyetal.,2010).Furthermore,inthisspecies,
thegenderassociationbetweenaminoaciduptakeandproductionby
References
bovineblastocystsappearedtobedynamicandchangesasblastocyst
developmentadvances.Theidentityoftheaminoacidswhichexhibit
Adamson D, Baker V. Multiple births from assisted reproductive
sex-specificdifferencesappearsnotonlytodifferbetweenhumanand
technologies: a challenge that must be met. Fertil Steril 2004;
bovine embryos but it also changes as embryo development pro-
81:517–522;discussion526.
gresses in vitro. The reason(s) for the observed gender-associated AkagbosuF,MarcusS,AbusheikhaN,AveryS,BrinsdenP.Doesovarian
differences in amino acid turnover is not clear but the division hyperstimulation syndrome affect the quality of oocytes? Hum Reprod
between metabolism in male and female embryos only becomes 1998;13:2583–2584.