Hum. Reprod.-2010-Bellver-humrep-deq098(1)

8/18/2019 Hum. Reprod.-2010-Bellver-humrep-deq098(1)

http://slidepdf.com/reader/full/hum-reprod-2010-bellver-humrep-deq0981 1/15

Hum.

Reprod.

Advance

Access

published

May

24,

2013Human Reproduction, Vol.00, No.0 pp. 1–9, 2013

doi:10.1093/humrep/deq098

R!"!N#$ #R%!&$' !n(ertilit)

* chromo+ome microdeletion+, +perm

N# (ra-mentation and +permoidatie +tre++ a+ cau+e+ o( recur

ent+pontaneou+ aortion o( unnonetiolo-)

. 4eller1,5

, 6. 6e+e-uer1

, $. 6uriel2

, 7. "arcaHerrero1

,6.#.6. 4arreto1, #.$. "arda3, . Remoh1, #. ellicer1,

and N. "arrido1

1!n+tituto Valenciano de !n(ertilidad ;!V!<, =nier+it) o( Valencia, la>a de la olica $ocal, 3, ?@01A Valencia, 7pain

2!V!

Vi-o, la>a Branci+coBernande> del Rie-o, C, 3@203 Vi-o, 7pain

3!V! 6urcia, Nae-ante 6aca+ del o)o, A, 3000C 6urcia, 7pain

5

7umitted on ecemer ?, 2012D re+umitted on Beruar) 2?, 2013D accepted on 6arch 23, 2013

ac-round:

%he aim o( the pre+ent +tud) a+ to ealuate the implication o( male (actor, in term+ o( +perm

# oidation and

(ra-mentation, and * chromo+ome microdeletion+ in recurrent +pontaneou+ aortion ;R7#< o( unnon ori-in in a +trictl) ected cohort.

method+: # pro+pectie cohort +tud) a+ carried out in a priate unier+it)a(Eliated +ettin-. %hree -roup+, each

compri+ed o( 30 male+,

ere compared. %he Er+t a+ (ormed ) health) and (ertile +perm donor+ ;7< ith normal +perm parameter+ ;control -up<, the +econd

) men pre+entin- +eere oli-o>oo+permia ;7< ithout R7# hi+tor), and the third ) men (rom couple+ ho had eper

ced idiopathic

R7#. Brequenc) o( * chromo+ome microdeletion+ and mean +perm N# (ra-mentation and oidation ere determined.

re+ult+: * chromo+ome microdeletion+ ere not detected in an) o( the male+ enrolled in the +tud). 6oreoer, +perm

N# oidation

mea+urement+ ere not demon+trated to e releant to R7#. !ntere+tin-l), +perm N# (ra-mentation a+ hi-her in the -roup than in

the R7# and the 7 -roup+, and al+o hi-her in the R7# -roup compared ith the 7 -roup, ut laced an adequate pred

e poer to e

emplo)ed a+ a di+criminatie te+t o( R7# condition.

conclu+ion+: 7perm N# (eature+ and * chromo+ome microdeletion+ do not +eem to e related to R7# o( unno

ori-in. ther

molecular (eature+ o( +perm +hould e +tudied to determine their po++ile inFuence on R7#.

&linicaltrial+.-o re(erence: n&%00??C39A.

Ge ) ord+: recurrent +pontaneou+ aortion / +emen / * chromo+ome microdeletion+ / +perm N# (ra-mentation / +perm

idatie +tre++

&orre+pondence addre++. %el:

I3?9@30A0900D Ba:

I3?9@30A0999D 'mail:eller ii.e+



!ntroductionRecurrent +pontaneou+

aortion

;R7#<,

deEned

a+ L3

clinic

al

pre-

nanc)

lo++e+ e(ore the

(etu+

ha+

reached

iailit)

;Rai

and Re-an,

200@<, i+ a (ru+tratin- condition that aMect+ 1 o( couple+ o( ch

ildear

in-

a-e ;orter

and

7cott,

200A<.

#lthou-h +ome

cau+e+

h

ae een

identiEed,

other+

remain

mere

+peculation.

%hu+, A0

o(

couple+are

+till

cla++iEed

a+

hain-

uneplained

R7#

+ince

the

u

nderl)in-

mechani+m;+< i+ neer (ound ;orter and 7cott, 200A<.

Gnon cau+e+ include paternal or Ode no/oP chromo+omal aer

ration+

;Rai

and

Re-an,

200@<,

antipho+pholipid

+)ndrome

;'mp+on

et al.,

200A<, +ome inherited thromophilia+, +uch a+ Bactor V $eideand prothromin "20210# -ene mutation ;Re) et al., 2003D Goale

) et al.,

200?D Graendam

et al., 200A<,

con-enital

or

acqui

uterine

anomalie+ ;ei Qold et al., 200@<, endocrine, autoimmune

alloim

mune di+turance+ ;#rredondo and Nole, 200@D &hri+tian+

et al.,

200@< and perhap+ unhealth) li(e+t)le hait+ ;+moin-, oe+it

p+)cho

lo-ical +tre++D $a+hen et al., 200?D ande) et al., 200A<. Hoer, mo+t o(

the+e condition+ are related to the oman, ith the maleP+ c

riution

remainin- relatiel) undereplored.

%he

alteration

o(

+perm

parameter+

ealuated

)

cla+

criteria

;concentration,

motilit),

morpholo-)D

Qorld

Health

r-a

>ation,

%he #uthor 2013. uli+hed ) (ord =nier+it) re++ on ehal( o( the 'uropean 7ociet) o( Human Reproduction and 'mr)olo-). #ll ri-ht+ re+ered.

Bor ermi++ion+, plea+e email: Journal+.permi++ion+Ko(ordJournal+.or-

2 4eller et al

1999<

i+

not clearl) a++ociated ith

the

ri+ o(

either

+poradic

or

recurrent

aortion

;&arrell et

al.,

2003D

4hattachar)a

,

2008<,

althou-h +ome o( the+e +perm deEciencie+ could repre+ent the

mani

(e+tation o( an underl)in- related cau+e. %hi+ i+ the ca+e (or e

mr)o+

ith

chromo+omall) anormalitie+ ori-inatin- in

meiotic +e-r

e-ation

error+ in the +permato>oa o( men ith +eere

oli-oa+then

oterato

>oo+permia ;4ernardini et al., 200AD an- et al., 200A<. !n (a

ct, +ome

author+

hae

propo+ed

per(ormin-

Fuore+cent

in

+itu

h)ri

di>ation

anal)+i+ in +eerel) altered +perm +ample+ to detect ca+e+

that

ma)

eneEt

(rom

preimplantation

-enetic

+creenin-,

thu+

reduc

in- the

aortion rate ;Ruio et al., 2001<. Neerthele++, there i+ no co

n+en+u+

in the current medical literature aout thi+ topic ;Harper et al.,

2008<.

!n

addition,

man)

reported

ca+e+

o(

male

partner+

o(

R7#

+uJect+

+ho normal parameter+ in their eJaculate+ ;#lHa++an et al.,

200A<.

6oreoer, a+ +perm ph)+iolo-) i+ a+certained, an increa+in- n

umer

o( molecular

(actor+

are

implicated

in

male

(ertilit) ;"arrid

o et al.,

2008a,

<,

+uch

a+

oidatie +tre++related molecule+

;6e+e-uer

et

al.,

200?<,

N#

oidation and

(ra-mentation

;#-uilar

et al.,

2009< and mRN# epre++ion proEle+ ;"arrido et al., 2008a,

<, uttheir implication in R7# ha+ receied little or no attention.

&la++ic

parameter+

o(

+perm

ealuation

cannot

e em

)ed to

identi()

+tructural

alteration+

in

the +perm

chromatin

enprei++

et

al.,

200@D

'renprei++ et

al.,

2008D

#endano

et

al.,

2

9D

&ohen

4acrie et al.,

2009<.

7perm

N#

(ra-mentation ha+ een

lated to

male

+u(ertilit),

+poradic aortion

and poorer

re

oductie

outcome, e+peciall) a(ter a++i+ted conception

technolo-ie+

$ar+on&oo

et

al.,

2003D

4orini et

al.,

200@D

$ei+ et

al.,

200

$in

et

al.,

2008D Sini et al., 2008<, ut ith contradictor) re+ult+ ;&o

+ et al.,

2008D

Nicopoullo+

et

al.,

2008D

%aalaee

et

al.,

2009<.

addition,

diMerent

method+ o(

anal)+i+ hae

een

emplo)ed to

-

her data

T&omet

#++a), 7perm

&hromatin

7tructure

#++a) ;7&7#

%=N'$

#++a)D ractice

&ommittee o( #merican

7ociet)

(or Re

ductie

6edicine, 2008D Sini et al., 2008U, and +carce in(ormation iurrentl)

aailale aout their implication

in

R7# ;&arrell et

2003a<.

7imilarl), +ome author+ hae

reported increa+ed

N# d

ma-e

in

+perm cau+ed )

oidatie

+tre++

in in(ertile men ;6e+e

er

et

al.,

2008D #-uilar et al., 2009<, ut nothin- i+ non aout thi+

relation

to R7#.

%o report+ hae al+o de+cried a notal) hi-her preale

e o( *

chromo+ome

microdeletion+

in

the

male

partner+

o(

cou

e+

ith



R7# ;ean et al., 200@D Garaer et al., 2008<, ut thi+ ha+

not een

conErmed ;Gaare et al., 2008D $u et al., 2008<.

%he pre+ent +tud) a+ de+i-ned to pro+pectiel) ealu

ate the

relation+hip o( * chromo+ome microdeletion+, +perm N# (ra-

men

tation and +perm N# oidation cau+ed ) oidatie +tre++ it

h R7#

and to compare their alue+ amon- health) and (ertile +perm

donor+

;7< ith normal +perm parameter+, men ith +eere oli-o>oo+per

mia ;7< and men (rom couple+ ith R7#.

6aterial+ and 6ethod+

7tud) de+i-n %hi+ i+ a pro+pectie ca+e–control +tud), in hich men ere

enrolled

eteen 1 anuar) 2010 and 1 Beruar) 2012. 7uJect+ ere

a++i-ned

to one o( the three -roup+ accordin- to the (olloin- inclu+ion criteria.

%he control -roup included 30 health) 7 o( 18–3A )ear+ oa-e, all&auca+ian, ith normal ar)ot)pe, no (amil) hi+tor) o( di+ea+

normal

+perm anal)+i+ re+ult ;more than 80 million total motile +p

mato>oa

per eJaculate< and preiou+ term pre-nancie+ ithout complicat

con

ceied ith their +perm +ample+.

%he oli-o>oo+permic -roup ;7< con+i+ted o( 30 &auca+ian m

ith

.1 )ear in(ertilit), ,?A )ear+ o( a-e, a+ence o( autoimmu

or endo

crine

di+order+,

normal

ar)ot)pe,

no

hi+tor)

o(

R7#,

norm-enital

eamination, normal B7H, $H, te+to+terone and prolactin alu

and atlea+t

to

+perm

anal)+i+

re+ult+

+hoin-

7

;A

million

+pe

ato>oa/

ml, ut .1 million o( total +perm per eJaculate in order to maea+ilethe +perm te+t+<. %he+e men ere con+idered to repre+en

ca+e+ o(

+eere idiopathic oli-o>oo+permia. No a++i+ted reproduction t

ment

had een per(ormed (or the+e male+ at the time the) entered in

e +tud).

%he R7# -roup a+ compo+ed o( the male partner+ o( 30

uca+iancouple+

ho

had

preiou+l)

eperienced L3

clinical

Er+t

rime+ter

;A–1? ee+< +pontaneou+ aortion+, ith normal ar)ot)pe+( oth

male and (emale and no autoimmune or endocrine di+orde

%he+e

couple+ had not attempted a++i+ted reproduction treatment+ at

time

the) ere accepted (or the +tud). %he (emale partner+ o( the+e

uJect+

ere ,38 )ear+ old, pre+ented normal oarian (unction,

normal

uteru+

conErmed

)

a-inal

ultra+ound

and/or

h)+tero+alpin-

raph)/

h)+tero+cop) and a+ence o( acquired or inherited thromoph

;anti

pho+pholipid +)ndrome, actiated protein & re+i+tance, +eru

(a+tin-

homoc)+tine, protein &, protein 7, antithromin !!!, pr

romin

"20210# -ene mutation, (actor V $eiden<. #ll the men ere

nder ?A

)ear+ old and preiou+ +perm anal)+i+ had -ien a norma

r onl) amildl) altered re+ult ;. 10 million/ml, .3A # I 4 motile +pe

ato>oa,

.

10 o( normal +permato>oa (olloin- Gru-erP+ +trict criteria<

%o en+ure the reliailit) o( the re+ult+ otained, +uJect+ com

eted a

clinical que+tionnaire aout preiou+ con+umption o( alcoho

and/or

dru-+, recent epi+ode+ o( (eer, epo+ure to -onadotoin+ ;e

durin-

chemotherap) or radiotherap)<, pe+ticide or hea) metal+ ;pro(e

onall)<

and itamin intae in the 3 month+ prior to the enrolment in

e +tud).

#ndrolo-ical eamination+ ere per(ormed to rule out the pre

nce o(

aricocele+,

te+ticular

tor+ion,

traumati+m+

and

other

alteratiin

the



-enital tract, +uch a+ -enital tract inFammation or recurrent i

n(ection+

;Viloria et al., 2009<. None o( the men pre+ented an) o( the+e con

dition+.

%he +tud) a+ approed ) the !n+titutional Re+earch 4oard and

'thic+

&ommittee. %he +tud) purpo+e+ and procedure+ ere care(ull) epl

ained,

and the in(ormed con+ent a+ otained (rom tho+e illin- to partic

ipate.

%he +tud) a+ al+o re-i+tered in &linicaltrial+.-o, ith the r

e(erence

N&%00??C39A.

* chromo+ome microdeletion+: i+olationo( N# and +equenceta--ed +ite anal)+i+"enomic N# a+ etracted (rom the peripheral lood leuoc)te+

u+in-

the 6a-N# ure &ompact in+trument ;Roche ia-no+tic+ &orpor

ation<

and the 6a-N# ure &ompact Nucleic #cid !+olation ! method ;

Roche

#pplied 7cience, !ndianapoli+, !nd.< accordin- to the man

cturerP+

in+truction+. 7creenin- (or *q microdeletion+ a+ carried out in

ntrol+

and patient+ u+in- pol)mera+e chain reaction ;&R< technique+

mpli()

in- 20 diMerent +equenceta--ed +ite+ ;7%7+< on the lon- arm

( the *

chromo+ome, corre+pondin- to (our #SB loci +pread oer in

al+ A

and @. %he+e included 7*1? (rom 7R*, 7*81, 7*8?+ and

8@ (rom

#SBaD 7*182 (rom G#$*, 7*121, 7*R3, 7*12?, 7*12C, 7*127*130,

7*133 and 7*13? (rom #SBD 7*1?A and 7*1A2 (rom proim

#SBc/

d locu+D 7*2?2, 7*208, 7*2A? and 7*2AA (rom #SBcD and 7*1A

hetero

chromatic di+tal *q re-ion<.

6ultiple &R a+ per(ormed u+in- the * chromo+ome #SB

nal)+i+

7)+tem ;rome-a, =7#< under the &R condition+ recommende

) the

6ale (actor and idiopathic recurrent mi+carria-e 3

manu(acturer. Bor anal)+i+, the microdeletion+ &R product+ ererun )electrophore+i+ on a 2 a-aro+e -el impre-nated ith ethidium ro

mide (or

i+uali>ation under =V li-ht. Bailure o( ampliEcation (or a -ien 7%7

a+ con

Ermed tice. N# (rom a (ertile male and ater +ered a+ ne-atie

* chromo

+ome microdeletion control+, herea+ N# (rom man ith a * chro

mo+ome

microdeletion +ered a+ a po+itie control (or multiple reaction+.

7perm anal)+i+7emen parameter+ o( eer) eJaculate ere ealuated ) to inde

pendent

o+erer+. #(ter 10 min lique(action o( the +emen at 3C8& and A

&2,the +ample+ ere eamined (or concentration and motilit) in a 6aclerchamer ;7eE $aoratorie+, %el #i, !+rael<, accordin- to th

e QH

-uideline+ ;12<. Re+ult+ ere onl) accepted hen the d

iMerence+

eteen to o+erer+ ere le++ than A.

%o +emen aliquot+ ere taen (rom each +emen +ample to

e pro

ce++ed (or Fo c)tometr) and +perm chromatin di+per+ion ;7&

< te+t+. %he +ample+ ere directl) (ro>en ) immer+ion in liquid nitro-en

;7&<

or in cold ethanol C0 ;(or Fo c)tometr) eperiment+<. %he+e pr

otocol+

hae een alidated preiou+l) and hae een demon+trated to e

+a(e in

re-ard to the maintenance o( +perm characteri+tic+, alloin- the an

al)+i+ o(

all the +ample+ in the +ame eperiment.

%he total numer o( +permato>oa collected (or the eperiment+ r

an-ed

eteen 1 and 10 million/ml in order to proide a +u(Ecient quan

tit) (or

the anal)+i+ a(ter centri(u-ation (or 10 min at ?00- and elimination o( the

+upernatant.

No eJaculate di+pla)ed +i-niEcant ;.

1 million/ml< leel+ o(

uoc)te+

;a non and releant +ource o( R7<, a+ demon+trated ) W

anop

tic ;W&#, 4arcelona, 7pain< +tainin- o( +emen +mear+. %he

n+itiit) o(

the te+t i+ 10 000 leuoc)te+/ml.

N# (ra-mentation ;7&<'ppendor( tue+ ith -elled aliquot+ o( lomeltin- point a-

o+e ere

placed (or A min in a ater ath at 90–1008& to (u+e the

a-aro+e, and +u+equentl) in another ater ath at 3C8&. #(ter A min incuation

3C8& (or

temperature equiliration, @0 ml o( the diluted +emen +ample

added to

the 'ppendor( tue and mied ith the (u+ed a-aro+e.

ml o( the

+emena-aro+e mi ere pipetted onto +lide+ precoated ith

a-aro+e andcoered ith

a

22 X 22 mm

coer+lip.

%he

+lide+

ere

plac

on a cold

plate in the re(ri-erator ;?8&< (or A min to allo the a-aro+e troduce a

micro-el ith the +perm cell+ emedded ithin. %he coer+lip+

re -entl)

remoed and the +lide+ immediatel) immer+ed hori>ontall) in an

cid +olution,

preiou+l) prepared ) miin- 80 ml o( H&l (rom an 'ppendor

ue ith

10 ml o( di+tilled ater and incuated (or C min. %he +lide+ er

ori>ontall)

immer+ed in 10 ml o( the l)+in- +olution (or 2A min. #(ter A min

a+hin- in a

tra) ith aundant di+tilled ater, the +lide+ ere deh)drated

ncrea+in-

concentration+ o( ethanol ;C0–90–100< (or 2 min each and the

airdried.

Bor ri-htEeld micro+cop) in the improed 7& te+t ;H

+perm



it<, +lide+ ere hori>ontall) coered ith a mi o( Qri-htP+ +taini

n- +ol

ution ;6erc, "erman)< and pho+phate uMer +olution

;6erc,

"erman)< ;1:1< (or A–10 min ith continuou+ airFo. 7lid

e+ ere

rieF) a+hed in tap ater and alloed to dr). 7tron- +tainin- i+ p

re(erred

to ea+il) i+uali>e the peripher) o( the di+per+ed N# loop halo+.

Bor thi+ +tud), a minimum o( A00 +permato>oa per +ample ere

+coredunder the

X100 oJectie o( the micro+cope.

N# oidation ;8oo-uanine N# +permmea+urement ) Fo c)tometr)< %he )N# #++a) ;)N# a++a) it, &aliochem, 4arcelona, 7

pain< i+

a+ed on the direct indin- o( a Fuore+cent proe to 8oo-uanine

moietie+

in the N# o( Eed cell+ ;#-uilar et al., 2009<. Bluore+cence can

then e

quantiEed u+in- Fo c)tometr) ;the )N# #++a) i+ per(oed ando+ered directl) in the cell+<. 7perm cell+ ere a+hed tice

47 and

Eedpermeaili>ed ith icecold ethanol C0 at 2208&. 'thano

treatedcell+

ere

pelleted

at

230X -

(or

A min,

and

the

+upern

nt+

ere

remoed and pellet+ ere a+hed tice ith 47. 7perm cell+ e re+u+

pended in 3 ml o( Qa+h 7olution T%ri+uMered 7aline/%Q'

N 20

eter-ent ;%47%<, containin- %himero+alU, and centri(u-ed a

30-

(orA min, a(ter hich the +upernatant a+ remoed and non+peciE

indin-

+ite+ ere loced ith A0 ml (re+hl) prepared 4locin- 7olut

(or 1 h

at 3C8&. %hen 3 ml o( Qa+h 7olution a+ added and a+he+ e

repeated

tice, and then incuated oerni-ht ith 100 ml B!%&&onJu-at

at ?8&.

7ample+ ere a+hed tice ;Qa+h 7olution< and re+u+pend

in 2 ml

47. &ell+ ere maintained in the dar on ice until their re+u+

n+ion in

B#&7 Fuid and ere read in a Fo c)tometer ith B!%& Elter+.

Blo

c)tometr)

anal)+i+

a+

per(ormed

on

an

'pic+

'lite

o &)t

ometer ;&oulter &)tometr), Hialeah, B$, =7#< u+in- an ar-o

ion la+er

tuned at ?88 nm and 1A mQ. eri+ a+ ecluded ) the

nal)+i+ o(

the +catter propertie+. #t lea+t 10 000 eent+ per +ample

e +tored

in li+tmode Ele+. ata ere epre++ed a+ percenta-e o( +

ned cell+

;compared ith ne-atie control+< and Fuore+cence inten+it)

ea+ured

a+ Fuore+cence aritrar) unit+.

7tati+tical anal)+i+7tati+tical anal)+i+ a+ per(ormed u+in- the 7tati+tical aca-

or 7ocial

7cience er+ion 1C.0 ;777 !nc., &hica-o, !$, =7#<. &ate

ical data

ere epre++ed a+ numer and percenta-e, and numeri

data a+

mean+ ith 9A conEdence interal+ ;9A &!<. Qhen a quantita

e anal)

+i+ o( the data a+ per(ormed, -roup+ ere compared ) anal)

o( ar

iance, ith 4on(erroni and 7cheMeP+ po+t hoc anal)+i+ i( mo

than to

-roup+

ere

compared

and

a

7tudentP+

tte+t

hen

onl)

t

-roup+

ere compared. &ro++ta+ and chi+quare te+t+ ere applied (or

the quali

tatie anal)+i+ o( the data. %he predictie poer o( +perm N#

idation

and (ra-mentation to (oreca+t mi+carria-e ere determined

receier

operator cure ;R&< anal)+i+. 7i-niEcance a+ deEned a+ ,

.0A.

Re+ult+

emo-raphic+



%he a+eline compari+on+ o( the three -roup+ are pre+ented in %

ale !.

!n rie(, a-e a+ +i-niEcantl) loer amon- 7 male+ ;ran-in-

(rom

20

to 3?

)ear+<

than

amon-

men

in

the

7 ;ran-e

2@–??

)ear+<

and R7# ;ran-e 30–?2 )ear+< -roup+. 7perm qualit) a+ al+o

+i-niE

cantl) hi-her in the Er+t -roup, in hich the alue+ o( +perm c

oncen

tration

;ran-in-

(rom

29 to 2A0

million/ml<,

+perm motilit) ;r

an-in-(rom

29

to

@C

o(

# I 4

(orm+<

and

the

total

numer

o(

motile

+perm cell+ ;ran-in- (rom 80 to 2C8 million< ere +uperior, particularl)

in compari+on ith the +elected -roup o( 7 ;ran-in- (rom 0.3

to A

million/ml in +perm concentration, (rom 0.09 to C.?2 million

total

motile +perm cell+ and (rom 3 to A3 in # I 4 (orm+<, a+

pected

con+iderin- the inclu+ion criteria.

Qhen +perm qualit) o( the 7 male+ a+ compared ith t

o( the

R7# -roup ;concentration ran-in- (rom 11 to 110 million/m

a +i-

niEcant

diMerence

a+

o+ered in

+perm

concentration,

not

in

other

parameter+.

Hoeer,

in

oth

7

and

R7#,

+permconcen

tration a+ +till normal ;.20 million/ml<.

%he percenta-e+ o( +moer+ ere +tati+ticall) compara

in the

three -roup+, and in oth male+ and (emale+, thu+ ruli

out

an)

con(oundin- inFuence o( thi+ parameter on the re+ult+. Nee

hele++,

? 4eller et al

%al e !

emo-raphic+ o( the +tud) population.

&ontrol donor+ 7eere oli-o+permia Recurrent mi+carria-e alue.....................................................................................................................................................

.....................................6ale

#-e ;)ear+< [email protected] ;2?.A8–28.A2<a,0

3?.00 ;32.38–3A.32<a

3A.2? ;33.8?–3@.@?<0

,0.001

re+ence o( * chromo+ome microdeletion+ 0/30 ;0< 0/30 ;0< 0/30 ;0< n+

7moer ;)e+/total and < 3/30 T10 ;0–20.C?<U 10/30 T33.33 ;[email protected]@–A0.20<U C/30 T23.33

;8.28–38.?@<U n+

6ean numer o( ci-arette+ per da) 0.1A ;0–0.?<a,0

A.21 ;2.0@–8.3A<a

?.28 ;0.8C–C.@9<0

0.019

'Jaculate olume ;ml< ?.02 ;3.22–?.83< ?.38 ;3.A@–A.19< 3.39 ;3.00–3.C8< n+

7perm concentration ;million/ml< C?.C0 ;AA.13–9?.28<a,0

3.1A ;2.0?–?.2A<a,c

A1.CA ;?0.1C–@3.33<0,c

,0.001

N o( # I 4 +perm ?@.@2 ;?2.8@–A0.38<a

[email protected] ;20.@@–32.10<a,0

?3.88 ;38.AA–?9.21<0

,0.001

%otal motile +perm ;million< 119.?9 ;92.C@–[email protected]<a 3.31 ;1.8A–?.CC<a,0 83.91 ;AA.0?

112.C9<0

,0.001

Bemale

#-e ;)ear+< Y 3?.@C ;33.@1–3A.C2< 31.9? ;30.?C–33.?0< n+

7moer ;*e+/%otal and < Y 13/30 ;?3.33 ;2A.@0–@1.0@<<8/30 ;2@.@C ;10.8?–?2.A0<< n+

6ean numer o( ci-arette+ per da) Y 3.@0 ;1.A0–A.C0< 3.21 ;0.AA–A.8@< n+

6ean numer o( mi+carria-e+ per patient Y Y 3.C@ ;3.39–

?.1?< Y

6ean ee at pre-nanc) termination Y Y C.?2 ;@.93–C.?1< Y

ata are epre++ed a+ mean+ or proportion+ ith 9A conEdence interal+. 7uper+cript letter+ ;a, , and c< denote +tati+tical +i-niEcance eteen -roup+ ithin

e +ame ro +harin- the

+uperinde, and n+ denote+ non+i-niEcant diMerence+. alue+ o(

,

0.0A ere con+idered +tati+ticall) +i-niEcant. # I 4 +perm are deEned a+ +perm cell+ ith

pro-re++ie motilit) either

rapid and +trai-ht ;#< or le++ rapid or pre+entin- chan-in- direction ;4<. Normal ran-e+ are con+idered aoe A0 o( # I 4 (orm+.

a trend toard a loer mean numer o( ci-arette+ +moed per da)

a+ o+ered

in

the

7

-roup

;ran-in-

(rom

0

to

3

ci-arette+/da)<

in compari+on ith the 7 ;ran-in-

(rom 0 to 20 ci-arette+/da)<

and R7# ;ran-in- (rom 0 to 30 ci-arette+/da)< -roup+.

%he mean numer and mean -e+tational a-e o( mi+carria-e+ are al+o

pre+ented

in

%ale

!.

%he

numer

o(

pre-nanc)

lo++e+

amon-

the

couple+ o( the R7# -roup

ran-ed (rom 3 to

C, herea+ the mean

-e+tational ee in hich pre-nanc) a+ lo+t ran-ed (rom A to 1?.

6aleP+ -enetic contriution to R7#



Qe ere unale to End an) * chromo+ome microdeletion+ in an) o(

the male+ included in the 7, 7 or R7# -roup+.

Re-ardin- +perm N# qualit),

to parameter+ ere a++e++ed:

+perm N# (ra-mentation and +perm N# oidation. %he re+ult+

can e (ound in Bi-+ 1 and 2.

!n +hort, the mean numer o( +perm cell+ ith (ra-mented N# a+

2?.0@ ;9A &! 20.8C–2C.23< in the 7 -roup, in compari+on ith

[email protected] ;9A &! ?0.?0–A1.@2< in the 7 -roup and 33.?8;9A &!28.2?–38.C2< in the R7# -roup, ith +tati+ticall) +i-niEcant

diMerence+

eteen the 7 and R7# and eteen the 7 and 7 -roup+. %he

re+ult+

o(

the

anal)+i+

o(

+perm

N#

oidation

in

th

e three

-roup+ ere comparale in term+ o( percenta-e o( oidi>e

d cell+,

ith alue+

o( 32.1?

;9A

&! 2?.@2–39.@A<

in the

7

-roup,

2?.01

;9A

&!

[email protected]–31.1C<

in

the

7

-roup

and

32.?3

;9A

&! 2?.@2–?0.2?< in the R7# -roup.

Qhen the

N# oidation +tainin-

inten+it)

a+ compare

d,

the

mean +perm cell N# oidation a+ 8C.2A ;9A &! @1.1@–

113.3?<relatie

Fuore+cence

unit+

;RB=<

in

the

7

-roup,

8C.

A8

RB=

;9A

&! @3.@9–111.?8< in the 7

-roup

and @8.0?

RB=

;9A

&!

A3.AA–82.A3<

in the R7# -roup.

%he diMerence+

et

een the

-roup+ ere not +i-niEcant.

Bi-ure 1 7perm N# (ra-mentation anal)+i+ re+ult+ dependin

onthe ori-in o( the +perm +ample. %he hori>ontal line+ repre+

the

median+,

the

o

idth

the

interquartile

ran-e,

the

loerne

o(

the o the Er+t quartile, the upper line o( the o the third q

rtile,

and the hi+er+ are maimum and minimum.

%he predictie alue o( +perm N# (ra-mentation and N

oi

dation (or (oreca+tin- recurrent mi+carria-e are +hon in Bi-

3. %he

lo alue+ o( the area+ under the cure indicate the a+ence

a pre

dictie

poer

o(

+perm

N#

(ra-mentation

or

oidation

an)+i+ in

+permato>oa ith re+pect to R7#.

!(

onl)

male+

ith

non+eere

+perm

alteration+

;i.e.

me

er+ o(

the

7

and R7#

-roup+< are con+idered, the

capacit) o(

+perm

6ale (actor and idiopathic recurrent mi+carria-e A

aMected

)

thi+

condition

i+

not

adi+ale

+ince their

alte

ion+

are

not currentl) demon+trated to pre+ent a cau+atie role.

N# oidatie dama-e i+ indicatie o( a numer o( conditio

inol

in- the -eneration o( o)-en(ree radical+. %hi+ dama-e cou

e the

primar) +ource o( N# (ra-mentation ;#-uilar et al., 2009<.

(act, a

de(ectie antioidant +)+tem actiit) could e related to an im

airment

o( chromatin paca-in- ; %aro>>i et al., 2009<. Hoeer, thi+

not the

onl) the

mechani+m

o( N#

(ra-mentation

;'renprei++

et

, 200@D

ractice &ommittee o( #merican 7ociet) (or Reproductie 6

icine,

2008<. %hi+ i+ h) e con+idered N# (ra-mentation and

# oi

dation

independentl)

ecau+e, de+pite the a++ociation

t ei+t+

eteen the to, the) are not +)non)mou+.

Qe anal)>ed N# oidation ) rulin- out all the eterna

actor+

that

hae

een

related

to

increa+ed

(ree

radical

dama-

;+ee the

O6aterial+

and

6ethod+P

+ectionD

ractice

&ommittee

o(

merican



7ociet) (or

Reproductie

6edicine,

2008D

Viloria

et

al.,

20

<.

7imi

larl), a

+moin-

hait, hich i+ al+o

a++ociated ith incre

ed N#

dama-e in animal model+ and human+ ;Gun>le et al., 2003D

qualotto

et al., 2008<, a+ identiEed in a +imilar proportion in the t

e +tud)

-roup+. e+pite the (act that almo+t 2C o( omen inclu

d in the

R7# -roup

ere

+moer+

;%ale !<,

the

mean numer o(

-arette++moed

per

da)

a+

lo

;3.2<.

7till,

the

numer

o

ci-arette+

+moed dail) in the R7# and the 7 -roup+ a+ +i-niEca

) hi-her

than

in

the

7 -roup and thi+ ma)

contriute to the

hi-

rate

o(

N# (ra-mentation. Hoeer, a +moin- ha+ not e

clearl)

related

to

uneplained

recurrent

mi+carria-e

in

rece

+tudie+

;Shan- et al., 2010<. %he correlation eteen male a-e

d

+perm

N#

(ra-mentation

i+

controer+ial,

ith

+ome

report+

+hin- an

increa+e

in

+perm N#

dama-e ith

a-e

;la+tira

et

l.,

200CD

Va-nini

et

al.,

200C<

and

other+ Endin-

no

relation+h

;Qinle

et

al.,

2009<,

een

a(ter

?0

)ear+.

!n

our

+tud), e

aim

to aoid

thi+ po++ile con(oundin- (actor ) limitin- the a-e o( the +tu

popu

lation to ?A )ear+ old, althou-h the 7 -roup a+ +i-niEcantl

oun-erBi-ure 2

7perm N# oidation anal)+i+ re+ult+ dependin- on

theori-in o( the +ample. Re+ult+ epre++ed a+ the percenta-e o( oi

di>ed

cell+ ;po+itiel) +tained, top< and mean +tainin- leel+ ;ottom<.

%he

hori>ontal line+ repre+ent the median+, the o idth the interq

uar

tile ran-e, the loer line o( the o the Er+t quartile, the upper l

ine o(

the o the third quartile, and the hi+er+ are maimu

m and

minimum. %he dot+ out+ide the o are anormall) hi-h

alue+.

#+teri++ here denote etreme alue+, ut not +tati+tical diMer

ence+amon- -roup+.

N#

(ra-mentation

and

oidation

mea+urement+

to

di+cri

minate

eteen male+ (rom couple+ ith or ithout recurrent mi+c

arria-e+

can e +een in Bi-. ?. nl) the percenta-e o( oidi>ed cell+ pr

e+ented

a mild capacit) to adequatel) predict R7# occurrence, thou-h

it a+

not

+u(Ecient

(or

it

to

e

applied

a+

a

dia-no+tic

tool

;

here

an

optimal area under the cure +hould e

.0.8<.

i+cu++ion!n the pre+ent +tud), * chromo+ome microdeletion+ and +p

m N#

(ra-mentation

and

oidation

did not

proe

to e

related

R7# o(

unnon ori-in, at lea+t in men ith normal or mildl) alte

d +perm

+ample+.

%here(ore,

the a++e++ment

o(

the+e

parame

+

in

men



compared ith the 7 and the R7# -roup+. %hi+ al+o ma) ha

e con

triuted

to the

hi-her

N#

(ra-mentation rate+ in the latt

er

to

-roup+.

#ccordin-

to

our

re+ult+,

+perm

N#

qualit),

in

term+

o(

N#

(ra-mentation and

oidation,

to

o(

the

main

+)mptom+

o(

r

eactie

o)-en

+pecie+ attac

;"arrido

et

al.,

200?D

#-uilar et

al.,

2009<,

i+

not related to recurrent idiopathic +pontaneou+ aortion+. #lthou-h

+tati+tical

diMerence+ ere

conErmed amon-

the

-roup+,

th

e+e do

not

point to an

increa+e

in

+perm

N#

(ra-mentation or

oi

dation

in men (rom R7# couple+ ith re+pect to the other -roup+ o( pat

ient+.

!ntere+tin-l),

the -roup

ith

the hi-he+t

+perm

N# (ra-men

tation

a+ that

(ormed )

male+

)ieldin-

+eerel) oli-o+permic

+

ample+.

!ncrea+ed leel+ o( impairment to N# inte-rit) in +eere o

o+per

mic male+ hae een reported preiou+l) in the literature

d could

eplain the poor re+ult+ de+cried in a++i+ted reproduction tr

ment+

) +ome author+ ;6e+e-uer

et

al.,

2008<. Hoeer,

a recent

metaanal)+i+ o( 13 +tudie+ inolin- in(ertile couple+, de+pi

a +mall

ut +tati+ticall) +i-niEcant a++ociation eteen

+perm

N#

nte-rit)te+t

re+ult+ and

pre-nanc)

outcome

in

!VB and

!&7!

c)c

,

+perm

N# inte-rit) did not proe to e predictie o( pre-nanc) o

come

and proided no clinical u+e(ulne++ in the

routine practi

;&ollin+

et al., 2008<.

%he inFuence o( +perm N# dama-e on +poradic pre-n

) lo++

ha+

een mainl) ealuated a(ter a++i+ted reproductie

hnolo-).

@ 4eller et al

Bi-ure 3 R& cure anal)+i+ o( the predictie alue o( +perm N# (ra-mentation and oidation to (oreca+t R7#.

%hou-h controer+ial, a recent +)+tematic reie and metaanal)+i+ o(

11 +tudie+ concluded that the ri+ o( pre-nanc) lo++ a(ter !VB a

nd !&7!

a+ 2.A hi-her in men ith N# dama-e, ut diMered dependin- on

the a++a) ;7&7# or %=N'$< emplo)ed ;Sini et al., 2008<.

eer,

onl) to preiou+ report+ hae con+idered couple+ ith une

lained



R7#. !n oth +tudie+ ;&arrell et al., 2003aD Wiu et al., 2008<,

N# (ra-

mentation a+

+i-niEcantl)

hi-her

in

the

R7# -roup

tha

n

amon-

donor+

o(

non

(ertilit)

and

men

(rom

the -eneral

po

pulation.

&la++ic parameter+ o( +perm qualit) correlated ith +perm N

# (ra-

mentation in one o( the +tudie+ ;Wiu et al., 2008<, ut not in t

he other

;&arrell et al., 2003<. ur re+ult+ are +imilar, +ince +perm +a

mple+ o( the R7# -roup ere normal or onl) mildl) altered, and N# (ra

-men

tation in

the +ame -roup

a+

+i-niEcantl)

hi-her

than amo

n- (ertile

donor control+. Hoeer, hen

e a++e++ed

the clinical

pr

edictie

alue o( thi+ Endin- ) mean+ o( R& cure+, it a+ a+e

nt due to

the oerlap

o(

alue+

eteen

men elon-in-

to

oth

-roup+

;Bi-. ?<. %he Endin-+ ere the +ame

hen oli-o>ooper

mic men

ere

taen

into

account

in

the

R&

cure+

;Bi-.

3<.

%here(ore,

de+pite the (act that N# (ra-mentation +eem+ to e incre

a+ed in

the +perm o( men ith uneplained R7#, the clinical alue o

( +perm

N# inte-rit) anal)+i+ a+ a predictie tool i+ ne-li-ile. 7imil

arl), the

cau+atie role o( N# (ra-mentation in R7# cannot e demon+

trated.

%o the e+t o( our noled-e, thi+ i+ the Er+t +tud) in hich

+perm

oidatie

+tre++

ha+ een ealuated

in relation to

idiopat

hic

R7#.

7imilar leel+ o( N# oidation, in term+ o( oth the perca-e o( cell+

ith

oidi>ed

N#

and

mean inten+it) o(

oidat

,

ere

(ound in all the three -roup+

a++e++ed

;7,

7

and m

e+

(rom

couple+ ith

R7#<. nl) the percenta-e o(

oidi>ed cell+

hiited

a

mild capacit)

to adequatel) predict R7#

occurrence

Bi-.

?<,

althou-h thi+

predictie poer doe+ not appear

to e +u(

ent (or

it+

implementation

a+

a

dia-no+tic

tool

;here

an

opal area

under the cure +hould e .0.8<.

ne o( the mo+t +i-niEcant Endin-+ o( thi+ or i+ that

diMer

ence+

in

the

prealence

o(

*

chromo+ome

microdeleti

ere

(ound eteen the -roup+. !n (act, no pre+ence o( * micro

letion+

a+ detected

in an)

o( the 90

+uJect+

included.

%he+e

ult+

are

in complete contra+t to hat a+ initiall) puli+hed ) e

n et al.

;200@<.

!n

their

or,

1?

out

o(

1C

male+

;82<

(rom

R

couple+

pre+ented microdeletion+ in one

or more o( the

(our +e-

ent+

o(

the proimal #SBc re-ion +tudied, herea+ thi+ percenta-e

pped

to 20

in in(ertile male+ ith +eerel)

impaired

+perm

o-ene+i+

and a+ nonei+tent amon-

men ith proen

(ertilit).

ecentl),

Garaer et al. ;2008< al+o detected * chromo+ome microde

on+ in

one

or

more

o(

the

(our

7%7+

a++e++ed

in

the

#SB

d

#SBd

re-ion+

in 1@

o( men

(rom

couple+

ith

R7#

and

re

ted no

ca+e amon- (ertile men. Hoeer, the percenta-e o( micro

letion+

detected diMered hu-el) eteen the+e to +tudie+ ;82 er+

1@<

and the re-ion+ aMected ere mo+tl) diMerent.

6ale (actor and idiopathic recurrent mi+carria-e C



Bi-ure ? R& cure anal)+i+ o( the predictie alue o( +perm N# (ra-mentation and oidation to (oreca+t R7# con+iderin-

ho+e male+ ithnormal +perm parameter+.

!n our or, the increa+ed +ample +i>e +hould hae alloedclariE

cation o( the con+iderale diMerence+ reported preiou+l) )ean

et

al.

;200@<. #t lea+t,

+ome

* chromo+ome

microdeletion+

+hould

hae

een

o+ered

-ien

that

e

hae

con+idered

+imil

ar popu

lation+.

!n addition,

e a++e++ed 20

*lined

7%7+ elon-in-

to (our

#SB

loci

re-ion+

;#SBa, #SB,

#SBc and

#SBd<

rather

th

an

onl)

(our

7%7+

in one

or to

#SB re-ion+

;ean et al., 200@

D Garaer

et al., 2008<.

Burthermore,

ean

et

al.

;200@<

ar-ue

that

their

patien

t+

ere

+elected (rom a tertiar) re(erral center that receie+ a di+pro

portion

ate numer o( R7# patient+ and that the+e patient+ ere liel)

to hae

(ailed

ealuation+

and

treatment+

prior

to re(erral,

thu+ +e

in- the

re+ult+. !n our +tud), patient+ ere +elected (olloin- +trict i

nclu+ion

criteria. %he

couple+

de+cried a+

in(ertile

in

the

e

an

et al.

report

ehiited

+imilar

condition+ o(

+perm

qualit)

a+

tho+e

in

our

+tud),

ith

the

eception

o(

tho+e

male+

pre+entin-

a>oo

+permia,

ho ere not included in our population due to the impo++ii

lit) o(

per(ormin-

the

de+cried

N# (ra-mentation and oidatio

n

te+t+

ith their

+ample+. %hi+

(act

ma) partl)

eplain

the

l

ac

o(

*

chromo+ome microdeletion+ in our 7 -roup, +ince the+e altation+

hae

een

de+cried

in

hi-her

proportion+

in

men

ither)

lo

+perm

count+

;,1 million/mlD

6artine>

et

al.,

2000<.

!

addition,

de+pite

the

+trict

inclu+ion

criteria

emplo)ed,

the

+ample

>e

con

+idered

in

the

+tud)

;90

men<

ma)

al+o

repre+ent

a

li

ation (or

the re+ult+ otained.

7imilarl)

to our

+tud),

other recent

report+

hae not

id

tiEed

*

chromo+ome microdeletion+ in male partner+ o( R7# couple

Gaare

et al. ;2008< ealuated ?0 men (rom couple+ ith at lea+t

ee mi+

carria-e+ and anal)>ed 33 7%7 loci +pannin- the hole * chr

o+ome

plu+ the ? 7%7 loci u+ed ) ean et al. ;200@<. No microde

ion a+

(ound in an) ca+e. # +tud) o( a &hine+e population ) $u et

;2008<

al+o (ailed to detect an) ca+e o( * chromo+ome microdeleti

in 2@

chorionic

illou+

+ample+

o(

aortie

male

emr)o+

or

in

1

lood

+ample+

o(

men

ho+e

ie+

had

eperienced

R7#.

%h

+tudied

12 7%7+ in the #SBa, #SB and #SBc re-ion+ o( *q11.2.

iMerence+ in the pre+ence o( * chromo+ome microdeletio

could

e due to

ariation+ in the

+tud)

population+ ;race and

thnicit)<,

+uJect +election

criteria, the 7%7+ con+idered

and

meth

olo-ical

8 4eller et al

a+pect+

o(

the

anal)+i+.

Neerthele++, no clear

eidence

currentl) ei+t+

re-ardin-

the

relation+hip

eteen

idiopathic

R7and

*

chromo+ome microdeletion+.



%o conclude, there +eem+ to e no clinical u+e (or the a++e

++ment

o(

*

chromo+ome

microdeletion+

and

N#

(ra-mentation

an

d

oi

dation hen dealin- ith couple+ ith idiopathic R7#, at lea+t

hen

the

+perm +ample+ o( the

male partner are

not

+eerel) i

mpaired.

=ntil

+uch

a

clinical u+e

i+ demon+trated,

thi+ a++e++ment

can onl)

+ere re+earch purpo+e+. "ien the continuou+ de+cription o(

+permmolecular

(actor+

related

to

male

in(ertilit),

(urther

re+ea

rch

into

R7# u+in- the nel) di+coered molecule+ a+ marer+ +hould

e con

ducted. Recent adance+ in microarra) technolo-) applied to

+perm

ill undoutedl)

con+titute poer(ul

tool+ in the characteri>a

tion

o(

the+e (actor+.

#uthor+P role+

.4.

de+i-ned

the

+tud),

+elected

the

patient+

and

rote

the paperD

6.6. helped in the de+i-n o( the +tud) and per(ormed the N

# oi

dation

+tudie+D $.6.

per(ormed

the

N#

(ra-mentation

+tudie+D

7.".H.

collaorated

in

the N# oidation +tudie+ and

in

the

ritin- o( the paperD 6.#.6.4. included man) o( the patient+ a

nal)>ed

and helped in the

de+i-n

o( the

paperD

#.$.".

per(orme

d

the

*

chromo+ome

microdeletion anal)+i+D

.R.

helped

in

the de+i

-n

and

monitored the +tud)D #.. helped in the +tati+tical approach and

mon

itored the +tud) and the paperD N.". de+i-ned the +tud), per(

ormed

the

+tati+tical anal)+i+, coordinate

the +perm

and

lood

anal

)+i+

and

helped in the ritin- o( the paper.

#cnoled-ement+ %he author+ ould lie to acnoled-e the #ndrolo-) la tech

nician+

*olanda

6arque>,

&arolina Rico,

aloma Rodr-ue>, #licia

6

enca+,

#licia Surilla, ) Vicente &entelle+ (or their help in the +ample+

mana-e

ment and +tora-e.

Re(erence+#-uilar &, 6e+e-uer 6, "arciaHerrero 7, "il7alom 6, P&on

nor ',

"arrido N. Releance o( te+ticular +perm N# oidation (

or

the

outcome o( oum donation c)cle+. Bertil 7teril 2009D une

9 T'pu

ahead o( printU.

#lHa++an 7, Hellani #, #l7hahrani #, #leer) 6, aroudi G,

&o+un 7.

7perm chromo+omal anormalitie+ in patient+ ith u

plained

recurrent aortion+. #rch #ndrol 200ADA1:@9–C@.

#rredondo B, Nole $7. 'ndocrinolo-) o( recurrent pre-nanc) lo

7emin

Reprod 6ed 200@D2?:33–39.

#endano &, Branchi #, uran H, ehnin-er 7. N# (ra-me

tion

o( normal +permato>oa ne-atiel) impact+ emr)o qualit) and

intrac)topla+mic +perm inJection outcome. Bertil 7teril 2009D

arch 30

T'pu ahead o( printU.

4ernardini $6, &alo-ero #', 4otta>>i &, $anteri 7, Ve

urini $,

4urrello N, e alma #, &onte N, Ra-ni N. $o total norm

motile

count alue+ are a++ociated ith increa+ed +perm di+om) an

iploid)

rate+ in in(ertile patient+. !nt 3 #ndrol 200AD28:328–33@.

4hattachar)a 76. #++ociation o( ariou+ +perm param

er+ ith

uneplained repeated earl) pre-nanc) lo++Yhich i+ mo+t iortantZ

!nt =rol Nephrol 2008D?0:391–39A.



4orini #, %aro>>i N, 4i>>aro , 4onu 6#, Baa $, Blami-ni &, &oticchio ".

7perm N# (ra-mentation: paternal eMect on earl) po+t

implantation

emr)o deelopment in #R%. Hum Reprod 200@D21:28C@–2881.

&arrell %, $iu $, eter+on

&6, one+ G, Hata+aa

HH, 'ric

+on $,

&ampell 4. 7perm N# (ra-mentation i+ increa+ed in

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2003aD

?9:?9–AA.

&arrell %, Qilco #$, $o) $, eter+on &6, one+ G, 'ric

+on

$,&ampell 4, 4ranch Q, Hata+aa HH. 'leated +perm chromo

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pre-nanc) lo++. +tet ")necol 2003D101:1229–123A.

&hri+tian+en 4, Niel+en H7, Golte #6. Buture direction+ o(

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&ohen4acrie , 4elloc 7, 6ene>o *, &lement , Hamidi ,

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&orrelation eteen N# dama-e and +perm paramet

er+:

a

pro+pectie +tud) o( 1,@33 patient+. Bertil 7teril 2009D91:1801–

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&ollin+ #, 4arnhart G%, 7chle-el N. o +perm N# inte-rit

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predict pre-nanc) ith in itro (ertili>ationZ Bertil 7teri

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ei Qold #7, ham N, #rici #. #natomic (actor+ in recurrent pre-

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lo++. 7emin Reprod 6ed 200@D2?:2A–32.

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&ochrane ataa+e 7)+t Re 200AD&0028A9.

'renprei++ , 4un-um 6, 7pano 6, 'l>anat) 7, ridan+ , "ierc

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"arrido N, 6artine>&oneJero #, aure-ui , HorcaJada+ #, 7

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Documents

Hum. Reprod.-2010-Bellver-humrep-deq098(1)