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Placental/Decidual Apoptosis in Recurrent Pregnancy Loss:

Immunohistochemical Study

Tarek A Atia1- and !ohamed A"d #l$aher%-&

(Dr Faris for Al-faraby College)

1- Al-Azhar University, Faculty of Medicine, Histology Department; airo, !gypt

"- ollege of Applied Medical #ciences, #alman $in A%del Aziz University, &#A

'- Al-Azhar University, Faculty of Medicine, (%st ) *yn Department; airo, !gypt

+- Faculty of Medicine, #alman $in A%del Aziz University, &#A

• hese authors contri%uted eually to this .or/0

• Author to .hom correspondence are/ A Atia

• !-Mail tare/atiah2"3hotmail0com

t0mohamed3sau0edu0sa

• Tel': (()** +(&%& 1*1,

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A"stract

ackground: 4ecurrent pregnancy loss 54678 is defined as three or more consecutive pregnancy

losses prior to "9 gestational .ee/s0 #everal causes have %een identified in the etiology of 467, %ut

recently, a num%er of studies have sho.n that apoptosis of the chorionic villi and decidua may %e

one of the these etiologies0

."ecti0e: o study the apoptotic changes of the placental:decidual tissues in 467 compared to

those in cases of sporadic miscarriage0

!aterial and !ethods: 6lacental:decidual samples .ere collected from +9 cases .ith 467 5study

group8 and from another '9 cases .ith sporadic a%ortion 5control group80 #amples .ere fied in

19< %uffered formalin and prepared for sections stained .ith mar/ers for apoptotic protein 5$cl",

 p=', Fas80 !pression of these mar/ers .as detected %y using the peroidase la%eled avidin-%iotin

method0 

Result: (ur result indicated that the num%er of D>2 immunopositive decidual macrophages .as

increased significantly in cases of 467 51+&',( ,1'1+,2 compared to those of sporadic a%ortion

5%*',( 1*'*(&8, 6? 999@0 n contrast, the optical density of the D=>B C& cells sho.ed no

significant difference %et.een 467 cases 5('+3(+)*% ('111)&3*2  compared to sporadic

a%ortion cases 5('&3,*3** ('13(,,1338, 6? 90110 Additionally, the num%er of decidual D>2

immunopositive macrophages .as increased significantly in cases of 467 5+(*'( *('+2

compared to those of sporadic a%ortion 51+('&( ,'+%8, 6? 9099@0 Ehile, the optical density of the

D>2B macrophages sho.ed no significant difference %et.een 467 cases 5('&*1&3&&

('11+*)&&2 compared to those of sporadic a%ortion cases 5('&+&)1* ('1(,1+))180

4onclusion: 6lacental:decidual apoptosis and macrophages have a crucial role in  pregnancy

continuation0 Ho.ever, great increase in their num%er could affect the pregnancy outcome, %y

unclear mechanisms leading to 4670

5ey 6ords: 467, placenta:decidual, apoptosis, macrophages, immunohistochemistry

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Introduction

4ecurrent pregnancy loss 54678 is a three or more consecutive losses of clinically recognized

 pregnancies prior to the "9th .ee/ of gestation0 t is a common pregnancy complication, affecting

G'< of pregnant .omen0 #everal causes have %een identified among the etiology of 467 such as

genetic, endocrine, infection and autoimmune disorders, .hich account for G=9< of cases, .hile

the mechanisms for the remaining cases are uneplained170 4ecently, a high level of apoptosis in the

chorionic villi and:or decidual tissues has %een identified in association .ith recurrent a%ortion,

revealing that apoptosis may %e one of the etiologies of uneplained 467%-*0

 Cormal placental development undergoes seuences of cell division and differentiation, follo.ed

 %y invasion of tropho%last cells into the decidua and then remodeling the spiral arteries to increase

 %lood flo. to the placenta and to the developing fetus0 Eith pregnancy continuation, the placenta

develops series of tissue remodeling characterized %y regular loss of tropho%last cells %y apoptosis30

Additionally, many studies have indicated the importance of apoptosis in promoting maternal

immune tolerance to paternal antigens epressed %y the tropho%last cells,7)0 Ehereas, apoptotic cells

have %een noticed in %oth the maternal and fetal interfaces of the placenta during normal pregnancy,

they also identified in tropho%last cells in some complicated pregnancies such as preeclampsia and

intrauterine gro.th retardation1(7 11, suggesting that alterations in the regulation of tropho%last

apoptosis may affect the pregnancy outcome0 

Apoptosis is an interactive and dynamic %iological process involved in degradation of 

undesira%le cells to maintain the normal tissue function0 he interaction %et.een pro- and anti-

apoptotic path.ays can regulate, stimulate or inhi%it cellular apoptosis0 Apoptosis are initiated %y a

family of cysteine proteatses, caspases, that could %e activated through intrinsic 5mitochondrial

mediated8 or etrinsic 5death receptor mediated8 path.ays171%0 n the intrinsic path.ay, the

apoptotic signals are initiated %y the mitochondria in response to cellular stresses such as DCA

damage0 he mitochondrial path.ay can %e activated %y p=', a tumor suppressor protein that in

turn can activate proapoptotic $cl-" family mem%ers0 (n the other hand, in etrinsic path.ay,

apoptosis is initiated %y mem%ers of the CF 5umor Cecrosis Factor8 death receptor family0

Ho.ever, the etrinsic and intrinsic path.ays are not completely independent, as p=' can up-

regulate the epression of certain death receptors, and the mitochondrial path.ay may act to

amplify signals triggered %y the death receptor path.ay, suggesting that crossover can occur 

 %et.een the t.o path.ays1&-1*0

he Fas ligand 5Fas78:Fas path.ay is an important path.ay of apoptosis that controls cell proliferation and

tissue remodeling 1+I0 Fas 5D@=8 is a transmem%rane protein of the CF:nerve gro.th factor super-family

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that is epressed on %oth immune and nonimmune cell types 1=I0 Fas .hen %ound %y Fas7 activates a signal

transduction path.ay that eventually results in cell apoptosis0

Fas 5D@=8 is a cell surface protein %elonging to the CF receptor family0 ross-lin/ing of Fas .ith its

natural ligand 5Fas7; D@=78 induces apoptosis of Fas-%earing cells 5Cagata and *oldstein, 1@@=80 Fas and

Fas7 m4CAs are .idely epressed in several immune and non-immune tissues 5French et al., 1@@>80 and

$ lymphocytes %ecome activated .hen their receptors %ind foreign antigens0 his activation induces Fas

epression .hich delivers an apoptotic signal to the cell .hen %ound %y its ligand, allo.s disposal of 

lymphocytes and suppresses the immune response 5Jau, 1@@=; #cott et al.,1@@>80 Fas7 epression in the

immune-privileged tissues has %een proposed as inducing apoptosis of the target cells, the activated Fas-

epressing lymphocytes, providing a po.erful mechanism to do.n-regulate a deleterious immune response

5#treilein, 1@@=; Jau, 1@@=80 Ee postulate that a similar mechanism mediated %y FasKFas7 interaction may

 play a role in the protection of the semi-allogeneic fetus from maternal immune attac/ and may ma/e a

critical contri%ution to the immune-privileged environment of the placenta0

p53, a negative cell cycle regulator, is important in numerous biological processes, such as the cell cycle, DNA repair,

differentiation and apoptosis (7). p53 has been observed to be expressed abnormally in the chorionic villi and decidua

of females with hydropic, spontaneous or missed abortions; however, the expression of p53 in the chorionic villi from

patients with URSA has, to the best of our knowledge, yet to be investigated (8-10).

As cells undergo apoptosis, macrophages present at the maternal-fetal interface uic/ly remove apoptotic

cells %y phagocytosis to promote tropho%last survival and facilitate invasion and transformation of the spiral

arteries0 n placentas from complicated pregnancies, shallo. tropho%last invasion and inefficient spiral artery

transformation have %een o%served, .hich may %e partly due to the distri%ution and activation state of 

infiltrating macrophages0

10 #hang E, #hu MM, 7iu M, Eang AM, 7v 7$, Lhao , 7i M, an 70 !levated epressions of p=', D&CA1,

and $a in placental villi from patients .ith recurrent spontaneous a%ortion0 !ur 4ev Med 6harmacol

#ci0 "91';15"+8''>-290

"0 #un , Lhang , ang 7, Eang #, 6iao H7, ao , Lhu 4, Du M4 , 7i DN0  hemo/ine 7"2

induces apoptosis of decidual stromal cells via %inding 4':419 in human spontaneous a%ortion0 Mol

Hum 4eprod0 "91';1@5198>>-2>0'0  4ull &, om%erg &, &O/s #, MPnni/ N, MQls M, #irot/ina M, JPrv #, 7aan M0 ncreased placental epression

and maternal serum levels of apoptosis-inducing 4A7 in recurrent miscarriage0 6lacenta0 "91';'+5"81+1-20+0 inar (, &ara F, an A0 6otential role of decidual apoptosis in the pathogenesis of miscarriages0  *ynecol

!ndocrinol0 "91";"25=8'2"-=

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=0 6anzan MR, Mattar 4 , Maganhin , #imOes 4dos #, 4ossi A*, Motta !7, $aracat !, #oares NM Nr 0

!valuation of FA# and caspase-' in the endometrial tissue of patients .ith idiopathic infertility and recurrent

 pregnancy loss0 !ur N (%stet *ynecol 4eprod $iol0 "91';1>518+-="0>0 Eei D, Eu R, #hi H0 Apoptosis and p=' epression in the placental villi of females .ith

uneplained recurrent spontaneous a%ortion0 !p her Med0 "91+;5181@1-1@+0

0 Mayhe. M0 Jillous tropho%last of human placenta a coherent vie. of its turnover, repair and contri%utions

to villous development and maturation0 Histol Histopathol0 "991; 1>1"1'K1""+0

20 Uc/an D, #teele A, herry, Eang $, hamizo E, &outsoni/olis A, *il%ert-$arness !, *ood 4A0ropho%lasts epress Fas ligand a proposed mechanism for immune privilege in placenta and maternal

invasion0 Mol Hum 4eprod0 1@@; '>==K>>"@0 A%rahams JM, #trasze.s/i-havez #7, *uller #, Mor *0 First trimester tropho%last cells secrete Fas ligand

.hich induces immune cell apoptosis0 Mol Hum Reprod 0 "99+; 19==K>'190 shihara C, Matsuo H, Mura/oshi H, 7aoag-Fernandez N$, #amoto , Maruo 0 ncreased apoptosis in the

syncytiotropho%last in human term placentas complicated %y either preeclampsia or intrauterine gro.th

retardation0 Am J Obstet Gynecol 0 "99"; 12>1=2K1>>

110 roc/er 6, ooper #, (ng # , $a/er 6 C0 Differences in apoptotic suscepti%ility of cytotropho%lasts and

syncytiotropho%lasts in normal pregnancy to those complicated .ith preeclampsia and intrauterine gro.th

restriction0 Am J Pathol 0 "99'; 1>">'K>+'1"0 #chimmer A D, Hedley D E, 6enn 7 L, Minden M D0 4eceptor- and mitochondrial-mediated apoptosis in

acute leu/emia a translational vie.0 $lood0 "991; @2 '=+1-'=='1'0 Mer/is , ristofolini A, #anchis !, &oncurat M0 !pression of death cellular receptors FA#:D@= and D4+

during porcine placentation0 nt0 N0 Morphol0, "919; "25'82"@-2'+01+0 $rQ/er 7 !, &ruyt F A !, *iaccone *0 ell Death ndependent of aspases A 4evie.0 Clin Cancer Res.

"99=;11'1==-'1>"0

1=0 #trasze.s/i-havez  # 7, A%rahams J M J, and Mor   *0 he 4ole of Apoptosis in the 4egulation of 

ropho%last #urvival and Differentiation during 6regnancy0 !ndocrine 4evie.s, "99=;">582K 2@

1>0 #harp AC, Heazell A!6, roc/er 6, and Mor *0 6lacental Apoptosis in Health and Disease0 Am N 4eprod

mmunol0 "919; >+5'8 1=@K1>@

10 Atia A and Mohamed A%d !lzaher M0 Catural /iller cells, Macrophages and nflammatory hemo/ines in

4ecurrent 6regnancy 7oss mmunohistochemical #tudy0 7ife #cience Nournal, "91+;115"8"'+-1+"

Decidual macrophages 5DM8 are the second most prominent cells in the maternal-fetal interfaceafter the C& cells0 hey produce more cyto/ines as part of their primary role as antigen presenting

cells1+71*0 Macrophages are involved in mediating %oth normal and a%normal placentation as .ell as

in modulating the placental response to infection via regulating -cell activities0 Additionally,

macrophages accumulate near the  etravillous tropho%lasts, phagocytose the apoptotic decidual

cells and enhance the etravillous tropho%lasts invasion0 Ho.ever, increased num%er of 

macrophages and the associated a%errant apoptosis could adverse the pregnancy outcomes1371,71)0

t is currently %elieved that many adverse pregnancy outcomes are referred to several

 pathological conditions0 ndeed, placental:decidual apoptosis has the potential to illuminate many

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aspects of these conditions0 #o, .e aim to study the apoptotic changes of these cells in relation to

4670

!aterials and !ethods

Patients

his prospective study .as conducted on +9 .omen .ith 467 5study group8 and another '9

.omen .ith spontaneous miscarriage 5control group80 #amples have %een collected from the

(%stetrics and *ynecology departments, at #alman %in A%dulaziz University Hospital and &ing

&halid Hospital, Al-&harS, &#A0 he study protocol .as approved %y the Hospital !thics

ommittee0 Eritten informed consent .as o%tained from all .omen prior to enrollment in the

study0 

Placental specimens 8rom medically terminated normal pregnancies at ,9( 6eeks7 6ith noconcurrent medical illness7 history o8 a"ortions or smoking/alcohol ha"its 6ere included as

age-matched controls n ; %+2' Patient data included gestational age7 gra0idity7 parity  as 6ell

as a"ortion and o"stetrics history

his prospective study .as conducted %et.een Nune "911 to August "91', at the (%stetrics and

*ynecology department, at the #alman $in A%dulaziz University Hospital and &ing &halid Hospital, Al-

&harS, &#A0 he study protocol .as approved %y Hospital !thics ommittee0 Eritten informed consent .as

o%tained from all .omen prior to enrollment in the study0 Five hundred .omen presented .ith spontaneous

miscarriage admitted to our hospital during this period, only =9 .omen .ith 467 .ere eligi%le for study

5study group8, another =9 .omen .ith sporadic miscarriages and matched for age for those .ith 467

recruited in the study as control group0

Decidual:placental samples have %een ta/en from all cases %y dilatation and evacuation .ithout any prior 

 pharmaceutical induction, .ithin the first "+ hours after diagnosis0 All enrolled .omen of the study the

follo.ing data .ere collected motherTs age, parity, %ody mass inde, maternal previous diseases, and family

history0 After that a thorough clinical eamination .as performed, aiming to eclude apart from common

disorders already /no.n as aggravating factors for increased ris/ for miscarriages0 Follo.ing evacuation,

specimens from %oth groups .ere formalin-fied and paraffin-em%edded for further staining0

Tissue Samples

Decidual:placental samples have %een ta/en from all cases %y dilatation and evacuation .ithout

any prior pharmaceutical induction, .ithin the first "+ hours after diagnosis0 All .omen enrolled in

this study the follo.ing data .ere collected motherTs age, parity, %ody mass inde, maternal

 previous diseases, and family history0 After that a thorough clinical eamination .as performed,

aiming to eclude apart from common disorders already /no.n as aggravating factors for increased

ris/ for miscarriages0 Follo.ing evacuation, specimens from %oth groups .ere formalin-fied and

 paraffin-em%edded for further staining0

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Immunohistochemical staining

Decidual:placental samples .ere fied in 19< neutral-%uffered formalin, routinely processed

and em%edded in paraffin .a, and then sectioning and mounting onto A6!# coated slides0

mmunohistochemical assay for decidual macrophages 5D>28, $cl-", p=', and Fas epression .as

 performed on the seuential paraffin-em%edded tissue sections using the peroidase la%eled avidin-

 %iotin method0 ommercially availa%le anti%odies for D>2 5#-"99>98, $cl-" 5#-=9@8, p=' 5#-

211>28, and Fas 5#-299@8 5#anta ruz $iotechnology nc, #anta ruz, A, U#A8 .ere used to

recognize these anti%odies according to the manufacturers instructions0

 #euential slides .ith + Vm-thic/ tissue sections .ere deparaffinized and hydrated in seuential

treatment of ylene, ethanol and .ater0 Heated citrate %uffer  59091 M citric acid, pH >098 .as used

to retrieve antigens0 !ndogenous peroidase activity and non-specific %inding .ere %loc/ed .ith

'< H"("0 #ections .ere then incu%ated .ith primary anti%odies overnight at +W0 he follo.ing

day, %iotinylated secondary anti%ody and streptavidin-horseradish peroidase .ere added0 he

 peroidase reaction .as developed .ith ','-diamino%enzidine 5DA$; #igma hemical o08 to

give a %ro.n product0 ounterstaining .as done lightly .ith hematoylin, and the sections .ere

dehydrated in alcohol %efore mounting0 Appropriate positive controls .ere performed in each

staining run, and negative controls .ere performed for each sample %y replacing the primary

anti%ody .ith mouse g*0

Data registration and Statistical analysis:

he optical density of immunopositive D>2 macrophages, as .ell as the immunoreactivity of 

$cl-", p=', and Fas stained cells .as assessed using an image analysis techniue0 Digital images of 

19 randomly selected high-po.er 5X+998 fields h0p0fI .ere captured and analyzed using !clipsenet

soft.are 5Ci/on80 Also, the num%ers of immunopositive D>2 macrophages, $cl", p=' and Fas

cells in each field .ere counted using the manual select tool in the image soft.are0

he results .ere ta%ulated and statistically analyzed using a computer program #6## 5statistic a

 pac/age for social science8 version 1= soft.are0 he sample mean 5X8, standard deviation 5#D8, and

standard error of the mean as .ell as the range .ere o%tained for numerical varia%les0 For non-

numerical varia%les, the freuency, distri%ution and percentage .ere calculated0

Mann- Ehitney U test 

5nonparametric8 .as used0 he studentTs 5t8 test .as used to test the

significance of the difference %et.een " independent means0 he hi suare test 5XY8 .as used to

test .hether the distri%ution of a certain phenomenon among t.o or more groups .as eual or not0

he pro%a%ility 568 value .as calculated and a 6-value Z 909= .as considered statisticallysignificant0

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Result:

1- !aternal Demographic data

Hundred .omen .ere included in the study; =9 .omen .ith 467 5#tudy group8 and the other =9

.omen .ith sporadic miscarriages and matched for age for those .ith 467 recruited as 5control

group80 he t.o groups .ere similar; there .ere no significant differences .ith respect to age 5p ?

908, parity 5p ? 90@8, %ody mass inde 5p ? 90'8, previous caesarean section 5p ? 9028, and

gestational age 5p ? 90+8 at time of a%ortion, %ut the num%er of a%ortions .as significantly higher 

among the study group 

than among those of control group 56 ?909918 5a%le 180

Ta"le1 Maternal Demographic data; .here $M? $ody Mass nde 0#? 4esarean

Sections

2- !acrophage Population and optical density:

he decidual:placental macrophages .ere immuno-stained against the D>2 mar/er0 t .as

found that the macrophage D>2B population .as significantly increased in 467 cases 58ig' %8

compared to sporadic a%ortion cases 58ig' &8; 5mean? =9>0"9 per 19 h0p0f0; #D [ ">0=""8 vs. 51=90+9

 per 19 h0p0f0; [20='"8 respectively, 56 ? 9099@80

n contrast, there .as no significant difference of the D>2B macrophages\ optical density

 %et.een 467 cases compared to sporadic a%ortion cases 5mean ? 90+>1+++ per 19 h0p0f0; #D [

901"1=>@++8 vs0 5mean ? 90+=+@1">"+ per 19 h0p0f0; #D [ 901921=@@1"8 respectively, 56 ? 90>9"80

Study group n ;+(2 4ontrol groupn ;+(2 P 0alue

!aternal age years SD2 "=0+[ +0@ "=01 [ '0" 90

Parity "0@ [ 10' "02 [ "0' 90@

!I 5g/m<2 "+0= [ '0 "'0 [ "0@ 90'

=estational age at time o8 a"ortion 1'02 [ +01 1'01 [ '0+ 90+

>um"er o8 A"ortions %'+ ('3 1'% ('+ ('((1

Pre0ious 4'S @ 512<8 51+<8 902

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?igure 12:  mmuno-histogram of the decidual immune-stained D>2Bve macrophages 5$ro.n

color cells8; 5A8 in cases of 467, 5$8 in cases of sporadic a%ortion0 X+99I

 

Study group n;+(2 4ontrol group n;+(2 P- 0alue

4D*,-4ell 4ount

D>2-(ptical dens

=9>0"9 [ ">0=""

90+>1+++ [ 901"1=>@++

1=90+9 [ 20='"

90+=+@1">"+ [ 901921=@@1"

('(()

90>9"

Ta"le : #tatistical analysis of all cell population the their optical density in cases of 467 vs cases

of #poradic A%ortion

?igure +2: mmuno-histogram sho.ing the apoptotic mar/er Fas epression in decidual:placental

tissue samples 5$ro.n colour cells80 A placental tissue of 467 and $ placental tissue of sporadic

a%ortionX"99I0

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?igure +2: mmuno-histogram sho.ing the apoptotic mar/er Fas epression in decidual:placental

tissue samples 5$ro.n colour cells80 A placental tissue of 467 and $ placental tissue of sporadic

a%ortionX+99I0

?igure *2: mmuno-histogram sho.ing the anti- $7" anti%odies epression in decidual:tissue

samples 5$ro.n colour cells80 A placental tissue of 467 and $ placental tissue of sporadic

a%ortionX+99I0

Discussion:

According to macrophages, our result indicated that the num%er of decidual immunopositive

D>2 macrophages .as increased significantly in 467 cases compared to sporadic a%ortion cases

5=9>0"9 [ ">0="" vs0 1=90+9 [ 20='"8 respectively, 56?9099@80 n contrast the optical density of the

D>2B cells sho.ed non-significant differences %et.een the t.o groups0 (ur result .as indicated

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 %y others1+7%17%, as they had indicated that there .as mar/ed increase decidual D>2B macrophages

in .omen .ho had suffered 4670 n contrast,  Jassiliadou et al%% had reported that the increased

macrophage cell population .as not significant, .hile Ruac/ et al %& have demonstrated that there

.as no difference in the num%er of macrophages among cases .ith 467 and spontaneous a%ortion0

Ho.ever, ecess macrophages together .ith impaired tropho%lastic cell apoptosis can eaggerate

the maternal inflammatory response to the invading tropho%lasts resulting in pregnancy failure0

Also, macrophages epression of ecess 7-1] and CF-^ associated .ith infections can cause

impaired tropho%lasts invasion and a%normal pregnancy outcome0 Additionally, ecess activated

decidual macrophages as .ell as stimulated first trimester decidual cells %y pro-inflammatory

cyto/ines promote etravillous tropho%last apoptosis%+0 

Macrophages have a .ide dynamic plasticity; their response to eternal stimuli is varia%le and

can produce M1 or M" phenotypes0 M1-phenotype is proinflammatory and activated %y interferon-_; .hile M"-phenotypeis anti-inflammatory alternatively activated %y 71' or 7+%*0 M1 and M"

activated macrophages perform different functions; M" phenotype can %e anticipated during

normally developing pregnancy; .hile, the M1-su%type could %e a part of mechanism causing

 pregnancy failure%30

hemo/ines and chemo/ine receptors are involved in materno-fetal immune regulations,

macrophage migration and  angiogenesis%,0  (ur result noticed an epression of chemo/ines

X71" and 7' in decidual tissues of %oth study groups0 Ehere, the cell population and the

optical density of the immunopositive cells sho.ed no significant difference among %oth groups0

his result .as indicated %y 6ar/ et al1, as he had noticed that the immunopositive cell populations

and the intensity of X71" and 7' reactivity in decidual tissues .ere not correlated to the

num%er of dC& D=>B cells0 Ho.ever, other investigators indicated that invasive tropho%last cells

have the a%ility to produce the chemo/ines 7', 71+, and X71"%); .here their 

corresponding receptors are found on the decidual macrophages and C&-cells0 his in turn

indicated that the epression of these chemo/ines %y tropho%last cells plays an important role in the

recruitment of specific leu/ocytes to the decidua&(7&10 herefore, from this result and others&  .e

have speculated that epression of these chemo/ines regulate leu/ocytes migration to the site of 

implantation regardless the pregnancy complications0

In conclusion, decidual C& cell and macrophages play a crucial role in normal pregnancy

continuation, .hile increasing level of these cells could affect the pregnancy outcome0 n contrast

the decidual chemo/ines epression seems to %e correlated directly to recruitment of leu/ocytes to

the site of implantation regardless the pregnancy outcome or the pathophysiology of the pregnancy

itself0 Additionally, further studies are needed to investigate other possi%le causes of 467 such as

chromosomal aneuploidy as .ell as epression of apoptotic mar/ers in decidual tissues0

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Ackno6ledgment

his study .as supported %y a grant from #alman %in A%delaziz University, Al-&harS, &#A0

Re8erences

10 Ford H$ and #chust DN 4ecurrent pregnancy loss Aetiology, diagnosis, and therapy0 4ev (%stet

*ynecol0 "99@;"5"8>-2'0

"0 #tephenson MD Freuency of factors associated .ith ha%itual a%ortion in 1@ couples0 Fertil

#teril01@@>;>>518"+-@0

'0 7i , %al , Anstie $, *illham N, Amer #, Eood &, 7aird # An analysis of the pattern of 

 pregnancy loss in .omen .ith recurrent miscarriage0 Fertil #teril "99"; 21199K119>0

+0 7ee 4M and #ilver 4M 4ecurrent pregnancy loss summary and clinical recommendations0 #emin

4eprod Med "999; 12+''K++9

=0 Munoz-#uano A, Hamilton A$, $etz A* *imme shelter he immune system during pregnancy0

mmunological 4evie.s "911, 5 "+18 "9K'2

>0 A%rams ! and Miller !M he 4oles of the mmune #ystem in Eomens 4eproduction

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6regnancy0 American Nournal of 4eproductive mmunology, 5"9198; >' >91K>19

20 Nen/ins , 4o%erts N, Eilson 4 ,  Mac7ean MA, #hilito N,  Eal/er NN !vidence of a 5H8 1 type

response associated .ith recurrent miscarriage0 Fertil #teril0 "999;'5>81"9>-20

@0 Eilson 4 , Moor N, Nen/ins , Miller H, Eal/er NN, Mc7ean MA,  Corman N, Mcnnes $  A%normal

first trimester serum interleu/in 12 levels are associated .ith a poor outcome in .omen .ith a

history of recurrent miscarriage0 Am N 4eprod mmunol0 "99+;=15"81=>-@0

190 4odrigues MC, Favaron 6(, Dom%ro.s/i N*, de #ouza 4M, Maria Angelica Miglino MA 4ole of 

natural /iller 5C&8 cells during pregnancy A revie.0 (pen Nournal of Animal #ciences0 "91';'5"8

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110 Dosiou D and *iudice 7 Catural &iller ells in 6regnancy and 4ecurrent 6regnancy 7oss

!ndocrine and mmunologic 6erspectives0 !ndocrine 4evie.s, "99=; ">518++K>"

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6lacentation and in the 6athogenesis of 6reeclampsia0 N (%stet *ynaecol an "992;'95>8+>K+>

1'0 7ash *! and $ulmer NC Do uterine natural /iller 5uC&8 cells contri%ute to female reproductive

disorders` N 4eprod mmunol0; "911;225"81=>->+

1+0 ang AE,  Alfirevic L,  Ruen%y #  Catural /iller cells and pregnancy outcomes in .omen .ith

recurrent miscarriage and infertility a systematic revie.0 Hum 4eprod0 "911;">5281@1-290

1=0 *uenther #, Jre/oussis , Heu%lein #, $ayer $, Anz D, &na%l N, Cavrozoglou N, Dian D, Friese &,

Ma/rigianna/is A and Nesch/e U Decidual Macrophages Are #ignificantly ncreased in #pontaneous

Miscarriages and (ver-!press Fas7 A 6otential 4ole for Macrophages in ropho%last Apoptosis0

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and Nesch/e U0 Macrophages Are hey nvolved in !ndometriosis, A%ortion and 6reeclampsia and

Ho.` N Cippon Med #ch "91'; 29 @19'

120 Houser $7 Decidual Macrophages and heir 4oles at the Maternal-Fetal nterface0 A7! Nournal

of $iology and Medicine; "91", 2=19=-112

1@0 Ruen%y #, Ci/ H, nnes $, 7ash *, urner M, Drury N, and $ulmer N Uterine natural /iller cells and

angiogenesis in recurrent reproductive failure0 Human 4eproduction, "99@, "+518 +=K=+

"90 7achapelle MH, Miron 6, Hemmings 4, 4oy D !ndometrial , $, and C& cells in patients .ith

recurrent spontaneous a%ortion0 Altered profile and pregnancy outcome0 N mmunol 1@@>; 1=>+9"K 

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"10 6ar/ DE, 7ee HN, 6ar/ E, Hong #4, &.a/-&im N, ang &M 6eripheral %lood C& cells reflect

changes in decidual C& cells in .omen .ith recurrent miscarriages0 Am N 4eprod mmunol "919;

>' 1'K1290

""0 JuSa/liSa JD, *ulic , #ucic #, Cagata &, (ga.a &, 7as/arin *, #aito #, Haller H, 4u/avina D0

First trimester pregnancy decidual natural /iller cells contain and spontaneously release high

uantities of granulysin0 Am N 4eprod mmunol "911; >> '>'K'"

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 C& ell Activity in !arly 6regnancy orrelates .ith #u%seuent A%ortion .ith Cormalhromosomes in Eomen .ith 4ecurrent A%ortion0 American Nournal of 4eproductive mmunology;

"991; +>1'"K1'>

"+0 &ing &, #mith #, hapman M, #ac/s * Detailed analysis of peripheral %lood natural /iller 5C&8

cells in .omen .ith recurrent miscarriage0 Hum 4eprod, "919;"=="K=20

"=0 Fu/ui A, Funamizu A, o/ota M, amada &, Ca/amua 4, Fu/uhara 4, &imura H, Mizunuma H0

Uterine and circulating natural /iller cells and their roles in .omen .ith recurrent pregnancy loss,

implantation failure and preeclampsia0 N 4eprod mmunol, "911;@919=K1190

">0 Ruen%y #, $ates M, Doig , $re.ster N, &e.is-Nones D, Nohnson 6M, Jince *0 6re-implantation

endometrial leu/ocytes in .omen .ith recurrent miscarriage0 Hum 4eprod, 1@@@;1+"'2>K"'@10

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systematic revie. and meta-analysis0 Human 4eproduction Update, "91'; 1K190

"20 7aird #0M0, Mariee C0, Eei 70, 7i 00 Measurements of D=>1 cells in peripheral %lood and

endometrium %y flo. cytometry and immunohistochemical staining in situ0 Human 4eproduction,

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"@0 o ! , *ormley M, &apidzic M, 4osen D $, #cott M A, #tolp H A 4, McMaster M, 7anier 7 7, $a

brcena A, Fisher # N Maternal Decidual Macrophages nhi%it C& ell &illing of nvasive

ytotropho%lasts During Human 6regnancy0 $iology of 4eproduction,"91', 225>81==, 1K@

'90 JuSa/liSa DJ, #ucic #, *ulic , Dominovic M, 4u/avina D ell Death Mechanisms at the Maternal-

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'10  Ca/ashima A, #hioza/i A, MyoSo #, to M, atematsu M, #a/ai M, a/amori , (ga.a &,  Cagata &,

#aito  # *ranulysin 6roduced %y Uterine Catural &iller ells nduces Apoptosis of !travillous

ropho%lasts in #pontaneous A%ortion0 Am N 6athol0 "992; 1'5'8 >='K>>+0'"0 Eang EN, Hao F, 7in RD Dysregulation of macrophage activation %y decidual regulatory cells

in uneplained recurrent miscarriage patients0 N 4eprod mmunol0 "911;@"51-"8@-19"0

''0 Jassiliadou C, $ulmer NC mmunohistochemical evidence for increased num%ers of classicD=B

natural /iller cells in the endometrium of .omen suffering spontaneous early pregnancy loss0 Hum0

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'+0 Ruac/ &, Jassiliadou C, 6udney N, Anderson DN, Hill NA 7eu/ocyte activation in the decidua of 

chromosomally normal and a%normal fetuses from .omen .ith recurrent a%ortion0 Hum0 4eprod0

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'=0 Eu LM, ang H, 7ia M, eha , #chatze F, 7oc/.oode N, Di% E, Huanga #N 6ro-inflammatory

cyto/ine-stimulated first trimester decidual cells enhance macrophage-induced apoptosis of 

etravillous tropho%lasts0 6lacenta, "91", ''5'8 122K1@+0'>0 M&, *ilman-#achs A, haouat  *, $eaman  &D 6lacental A6ase !pression s a 7in/ $et.een

Multiple auses of #pontaneous A%ortion in Mice0 $iology of 4eproduction, "911, 2= 5 '8 >">->'+

'0 #ica A, Mantovani A Macrophage plasticity and polarization n vivo veritas0 N0 lin0 nvest0"91",

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'20 4o%ertson MN 4ole of chemo/ines in the %iology of natural /iller cells0 Nournal of 7eu/ocyte

$iology , "99", 11'-12'0

'@0 Dra/e 6M, *unn MD, haro F, sou 7, Lhou , Huang 7, Fisher #N0 Human placental

cytotropho%lasts attract monocytes and D=>5%right8 natural /iller cells via the actions of monocyte

inflammatory protein 1alpha0 N !p Med, "991;1@'11@@-1"1"0

+90 MarSono A$, $ro.n DA, Horton &!, Eallace !M, $reit #C, Manuelpillai U0 Macrophage

inhi%itory cyto/ine-1 in gestational tissues and maternal serum in normal and pre-eclamptic

 pregnancy0 6lacenta "99';"+199-19>0

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+10 Fest #, Aldo 6$, A%rahams JM, Jisintin , Alvero A, hen 4, havez #7, 4omero 4, Mor *0

ropho%last-macrophage interactions a regulatory net.or/ for the protection of pregnancy0 Am0N

4eprod0mmunol0 "99;===->>0

+"0 Freitag 7, von &aisen%erg , Hans &reipe H, Hussein & !pression analysis of leu/ocytes

attracting cyto/ines in chronic histiocytic intervillositis of the placenta0 nt N lin !p 6athol,

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