Indian Juurnal of Experimental Biology Vol. 41. July 2003. pp. 748-755

Expression and regulation of integrin receptors in human trophoblast cells: Role of estradiol and cytokines

Chandan<l Das * & Sayantani Basak

Department of Biochemi stry . All India Inst itu te or Medical Sciences, An sari agar. New Ddhi 11 0029. India

Embryo implantation and placentation are dynamic cellula r eVl:nt s that require not only synchron y between the maternal environment and the eillbryo. bu t also complex cell -cell comillu nication aillongst thc implanting blastocys t and the recepti ve cndoillctriulll through integrins. a largc fa illil y of proteins involved in the attachillent. migration. invas ion and control of ce llular functions. Integrin s display dynamic tempora l and spatial patterns of exprcssion by the trophobl as t cell s duri ng earl y pregnancy in humans. Howe ver. the precise mechanism of eillbryo implantati on and the Illodula tion or the integ rin receptors during blastocy st attachment and I'urther implantation relll 'lin elusive in the humans. The present study elucidates the expression and hormonal modulat ion of I'ibronectin , vit ronectin and laminin integrin receptors by estrad iol and IL-I a in human trophoblast cells . HUlllan first trimester trophoblast cell s showed the induction of the class ical es trogen

receptor (ER)-a hy its own li gand. estradiol. Trea tillen t with eit her estradi ol or IL- I a induced the expressions of a .j . as. CI.()

and a , integrin receptor subunits at both the mR A and protein leve ls. while ex pression or ~ I remained unaltered. furthermore. estrad iol upregu lated the express ion of I L- I a. thereby suggesting the poss ibi I ity that estrogen lIlay eitber direc tl y or via the proinilamillatory cy lOkine induces the expression of the cell surface integrin receptors. The I'indings del ineate the ro le or horillones and the cy tokines in lIlodulating the adhesiveness and attachillent or the trophoblast celis. Thi s may rcfkct the ill i'il'll scenario whe re the implanting embryo is surrounded by a hormone-cy tokinc ri ch IIterine microenvironillent that may pn:e isel y regulate the expn:s~ion of intcgri ns and therehy l'acilil<Jtc illlplantation .

Keywords : Blastocys t, Cytokinc . Estrogen. Implantation. Integrin

Imp lan tati on of the developing embryo into the recepti ve endometrium is a criti ca l and key event in the estab li shment of early pregnancy in humans. The unique process of adhes ion of trophob las t to the endometri um at the time of imp lantat ion and its subsequent invasion into the matern al tissue fo rms an essential element of embryo implantation. Severa l signalling molecules have been impl ica ted that are known to pl ay cru cial ro le during implan tation which includes Ce ll Adhesion Molecules (CA M s), integrins, hormones, growth factors and cytok ines 1.5.

Incidences of failure in implantat ion and placental development are clini ca ll y importan t. The rate of normal pregnancy loss in human is about 15- 19% of all clinica ll y diagnosed pregnancies6

, the true fi gure is probab ly much higher because of the high incidence of pregnancy loss even before the clini ca l detecti on of pregnanc/ . Implan tat ion rai lures in women after ill \Iil ro ferti li za ti on (IYF)/embryo transfer (ET) are also

* For correspondcnce: Phone: 2659-44832. 265R9617 Fax : 91 - 11 -26588663, 2658864 1 E-Illai I: challciana_d@hotmai l.com

high. Miscarr iage rates after ass isted reproduct ion are much higher than in spontaneous pregnanc iesh

.

The window of implantation represents the peri od of maxtmu m uterine recepti vi ty for blastocyst implan tation and is characteri sed by the regulated ex press ion of severa l au tocri ne-paraeri ne-endocrine factors including intcgrin fam il y or cell adhesion molecules' . Integrin s appear to participate in embryo­endometrial recogn ition and their aberrant express ion in the endometrium o f women with reproducti ve fai lure and unex plained in fertility suggests their critical ro le in implan tation. T he fibronectin receptor, a~~I' which recogni ses fibronectin present on the trophob las ts, is reported to be persisten tl y absent from both the endometria l glands and surface epithelial ce ll s of in ferti le women throughout the lutea l phase of menstrual cyc lex. The suboptimal express ion of integrins primaril y a~ ~1 and a\' ~ .\ in the endometrium of women suffering fro m reproducti ve fa ilure and unexp lai ned in fert i lity , endometri os is or luteal phase defect , are probably indica ti e of defecti ve uterine receptivity and possible implantation failure.

Most o f our understanding o f the implantation process has come from studi es in the mouse and

DAS &BASA K: INTEGRIN EX PR ESSION IN TROPHOBLAST CELLS 749

substantiated in the non-human primates. Using an ill vil'o delayed implantati on mouse model system we have recentl y demonstrated that estrogen modulates the expression of integrins and cy tokines during blastocyst implantation in mice' - ~ . Further, we have

delinea ted a criti ca l role of a 4 integrin in the process of implantation in mice by show ing that intrauterine neutrali zati on of the a-l integrin results in implantation fa ilure in thi s spec ies ' . The present study was aimed to eva luate the express ion and hormonal modulation of f ibronec tin. vi tronectin and laminin integrin

receptors by estrad iol and IL-I a in human trophoblast ce ll s. The study would help gather insights into the molecular events occurring during embryo implantation in humans and un veil strategies or rati onale contracepti ve des ign or management o f infertilit y trea tments.

Materials and Methods

/so/cil iOIl of rrop/lOb/asr cells Isolat ion and purifi ca ti on of trophob las t ce ll s was

carried out follow ing the protoco l publi shcd by us prev iously'l . Approx imately, 20-30 g o f washcd vi 1I0us ti ssue from first tri mester placentae was finel y minced, transfcrred to a digesti on flask containing calc ium and magnesium-free Hank's ba lanced salt so lution (CMF-HBSS) w ith 25 mM HEPES, 0. 1 % trypsin I :250, and 0.2 mg/ml DNase I ( 1500 Kunitz units/mg). pH 7.4, and placed in an orbital shaking water bath at 37°C for 20 min. A t the end of the incubation, the supernatant was co llected and layered over new born bov ine serum (N BBS). The remaining placenta l ti ssue was subjccted to the same digesti on procedure again ror 10 min and las tl y fo r 5 min w ith the add iti on of 100 ml trypsin-DNase (S igma, USA) solu tion in each digesti on. The supern atants co llected from all three digestions were centri fuged at 1000 g for 10 min at room temperature. T he resultant pellets were pooled and resuspended in 5 ml Dulbecco' s Modified Eag le Medium-High G lucose (DM EM-HG) and carefull y layered upon a pre-formed discon tinuous Perco ll grad ient (Gibco BRL, USA), made in CMF-HBSS. The grad ien t was centrifuged fo r 30 min at 1200 X g at 25°C. The middle layer correspondi ng to the dens ity of 1.04H- I .062 g/ml containing approximately 9Y'/0 pure popu lati on o f cy totrophoblast ce ll s was ca refull y aspirated out. The viabi lity of ce ll s after iso lati on and purifi ca ti on was checked by Trypan blue dye exc lusion and was found

to he> 90%. Purity of trophoblast ce ll s was eva luated by cy tokeratin staining for epitheli al ce ll s, which indi ca ted > 951'10 trophob last ce ll s in any single preparation.

CII/Illre of I rop/lOb/asl cells Percoll puri fied trophoblast ce ll s were plated

according to the need of the experiment with DM EM­HG containing 12 % FCS and an ti bioti cs in cul ture tlasks or in petri di shes at a concentrati on of 5 x l Oll or I x IO:i ce ll s/well respecti ve ly. For immunocytochemica l studi es, ce ll s were plated on 12 mm coversli ps. The flasks and coverslips were maintained at 37°C in steri Ie humidi fied atmosphere. After 24 hr of plati ng. all the plates were washed with medium to remove non-adherent ce ll s, replaced w ith fresh medium and maintained in culture for the required experimental peri od with daily change of medium 1o

.

Hor/l/one and cylokinc challenges To study the effect of estrogen and cy tok ines on

the ex press ion of integrin receptors by trophob last ce ll s in vil ro. the cells were washed tw ice w ith sa line after 24 hr of plating and supp lemented with fresh serum- free media containing either estradiol 1 7~ at a concentration of 750 pg/m l (Sigma, USA) or IL-I a at a concentration o f 200 pg/ ml (Genzy me. USA). A fter 12 or 2-+ hours of challenge. the cell s we re·collccted for total RNA iso lati on. The concentrations of the hormone and the cy tokine used were \V ithin the cy totox ic limits as measured earli er by MTT assay' I .

Toto/ RNA iso/arion Total R A was iso lated using TR lzo l reagent (Li fe

Technolog ies, USA) as per the manufacturer's instructi on. Essenti all y, after remov ing the media. trophob last ce ll s were collected in TRl zo l reagen t

(5 x l Oll ce lls/ml ). The samples \Vere kept fo r 5 mi n at 1'00 111 temperature to pe rmit complete dissoc iation of nucleoprotein complexes and stored at -70°C pri or to the iso lati on of total RN A. The samples \-vere thawed at 4"C and then brought to room temperature followcd

by addition of 200 ~tl chloro form per ml TRl zo l reagent. Tubes were vo rtexed for 15 sec. incubated at room temperature for 15 min and then centrifuged at

12000 g for 15 min at 4°C. T he aqueous phase was aspirated out to another Illi croccntrifuge tube to which

500 ~tl of 2-propanol was added per ml of TR lzo l used. After mi x ing by in version . the tu bes were kept at room temperat ure for 10 min and th en cen tri fuged

750 INDI AN J EXP BIOL. JULY 2003

at 12000 g fo r 10 min at 4°C. Supernatant was discarded and the RN A pellet was washed with 70% ethanol ( I mill ml of TRl zo l used) by vortex ing and spinning at 7500 g for 5 min at 4°C. The pellet was air

dried and then dissolved in 20 IJ.I of OEPC-treated water. RN A was quantitated by measuring absorbance at 260 nm using an UY spectrophotometer. The A~6c/A2x() rati o observed in different samples of isolated RNA vari ed from 1.6-2.0. They were further processed for RT-PC R analy. is as described subseq uentl y.

First strolld eDNA sYllthesis For first strand cONA synthesis, 5IJ.g of total RN A

was taken in a microcentrifuge tube to which 1.5 ~tl of oligo (dT)I~.IR (500 pg/IJ. I) was added and the vo lume made up to 12 IJ.I. The mi xture was heat denatured at 70°C for 10 min and quick chilled on ice. The following solut ions were subsequentl y added: 4 IJ.I of 5X first strand buffer (50 mM Tri s-HCI pH8 .3, 375 mM KCI, 15 mM MgCl ~ ) , 2 IJ.I of 0. 1 M OTT, I ~t1

of d TP mix ( 10 mM each of dA TP, dGTP, dCTP and dTTP) and I IJ.I of reverse transcriptase enzyme (200 U/IJ. I). Reacti on mi xture was incubated at 42°C for :2 hI' fo llow ing wh ich the en7.yme was heat denatured at 70°C for 15 min .

PolYlllerase elw ill react ioll cD As were ampli fied with spec ific primers

(ATCC, USA) for GAPOH, integrin subunits and cy tok i nes US I ng Eppendorf thermal cyc ler. Polymerase chain reacti on was performed w ith template strand , Taq 0 A po lymerase (0.5 uno III rcac tion vo lume) (MBI Fcrmentas, USA), I X reaction buffer (con taining 750 mM Tri s- HCI pH 8.8, 200 mM (N H .j) ~S04 , 0.1 % Tween 20), 1.5 mM MgCI ~, 200ilM dNTPs and 0.5 IlM of each primer. A mplifi cat ion was per formed w ith an initial denaturat ion at 94°C for 10 min fo llowed by 35 cyc les each of 94"C for I min. , annea ling tcmperature fo r I min and 72nC for I min . Final ex tension was carried out at 72"C for 10 minutes.

/lgarose gel electroplioresis The alllp i i fi ed products were resol ved eithcr on

1.5% or 2% agarose ge l dcpendi ng on the expec ted size of the amplicon and fina l ly densitometric ana lys is was performed usi ng A I pha Immunotech ge l docu­mentati on system with Chemiimagc A lphaEase™ software. In all the experiments, GAPOH was taken as internal control and intensity of GAPDH band was used to norma l ize that of the respect ive genes.

/111 III unoey! oehelll ist ry

Cell s grown in monolayer on coverslips were fixed in 4% phos hate-buffered para formaldehyde for 10 min at room temperature. Endogeneous perox idase was quenched by treat ing the ce ll s with 0.3 % H20 2 in methanol for 30 min followed by rinsing in deionized water. The coverslips were then placed in 10 mM PBS containing 2% BSA for 5 min twice. Non-spec ific binding was blocked using non-immune serum provided in the k it fo r 30 min at room temperature in a humidi fied chamber. T his was followed by overni ght incubation w ith spec i fic primary antibod ies, a .j (Santra Cruz Biotechnology, USA). a s, a o and BI (Pharmingen, USA) at I :300, I :300, I :400 and 1:750 dilutions respect i vely. A fter washing, the ce ll s were incubated for one hour at room temperature with biotinylated secondary antibody diluted accordi ng to the manufacturer 's instructi on. The cells were then incubatcd in av idin-b iotin perox idase complex for 30 min at room temperature, washed thri ce with PBS for 5 min each and treated w ith DAB substrate solution for 3-5 min for visuali sati on of antigen­ant ibody comp lex. Fina lly, the covers li ps were rinsed in water, dehydrated in graded ethy l alcohol, cleared and mounted on slides using DPX mountant.

Stat istica l analysis RT-PCR experiments were performed from three

dilTerent cu ltures, each PCR being repeated twice. Stat ist ica l analys is was performed using one-way ANOYA and SPSS 7.5 software, USA. P value less than 0.05 was cons idered to be ·tati sti ca ll y significant.

Results

Immunocy tochem istry for ER-a demonstra ted presence of e tradio l receptor in the cytot rophoblast ce ll s (Fig. I A). Trea tment w ith the hormone further upregulated its own receptor expression in these cell s (Fig. I B).

Challenge with either es tradiol or IL-I a upregu lated

the expression of a subunits of integrin s i.e. a .j , a s, aC, and a " (Fig. 2A-D) in the trophoblast ce ll s almost to a similar ex tent except a .j wh ich showed higher ex press ion in response to I L-I a as compared to

es trad iol (Fig. 2A). Expression of B, integrin was however, not modu lated either by estradi ol or I L-I a neither at the mR A level (Fig. 2E) nor at the protein leve l (Fig. 6) . The urregul ation of a subunits of

DAS &BASA K : I TEGR IN EX PR ESSION IN TROPHOBLAST CELLS 75 1

integrins by estrad iol at the mRNA level was al so refl ec ted in their protein express ion as observed by immunocy tochemistry (Figs 3-6).

To study any possible effect of es trad io l in regulating the express ion of the proinriammatory

cy tok ine, I L-I a, the cytotrophoblas t ce ll s were challenged w ith estradi ol fo r 12 hr as ex plained prev iously under Materi als and M ethods. The results as indi cated in Fig. 7 showed that besides upregulating the express ion of integrin s, es tradi o l also

signi ficantl y stimu lated the ex press ion of IL - I a in the trophoblast ce ll s.

Discussion Impl antation and successful maintenance of

pregnancy in human require the presence of hormones in the utero-placental microenvironment , predominant being heG and the steroid hormones, estrogen and progesterone. Many of the ac ti ons o f steroid hormones in controlling reproduction are strongly suggested to be mediated by loca ll y act ing factors such as growth factors and cy tokines which act in an autocrine/paracrine manner to regulate peri ­implan tati on embryo development and migration

•

necessary for placental deve lopment 12 These hormones are thought to exert their effect by modulating the ce ll adhesion molecules and cytokines present on the trophob lasts as well as the uterine endometrial ce ll s. Several integrin subunits have been observed to be ex pressed by the endometrial cell s in a cyc le-dependant manner, suggest ing the in vo lvement of stero id hormones, their modulation In the trophoblast ce ll s by these steroids however. sti II remains to be unexplored.

Integrins have been considered as markers o f endometrial receptiv ity and implantati on in humanl:1 . Ex tensive studies by Lessey and co-workersl4 have shown the involvement of in tegrin s during the implantation w indow in women. Three integrin

subunits, ai, a4 and ~:1 have been shown to be spec i fi ca ll y ex pressed in glandular epithel i Ulll on cyc le days 20 to 24 corresponding to the putati ve window of implantat ionI4.lx. The vitronectin receptor.

av~:1' seems to appear abruptl y on cycl e day 20. des ignat ing "opening" of the window, whi le a4 ~1. a fibroneetin receptor is present from ovulation to cyc le day 24 when it s di sappearance "c loses" the window.

Though. abnorma l expression of a.I ~ 1 integ rin has

I A

I B

Fig. I - Imillunocytochcmica l locali zati on of estrogen receptor-a in trophoolas t ce ll s (A) contro l cytotrophoo last ce ll s. 48 hr. (B) estrogen treated cytotrophoblast cell s. 41\ hI' showing upn:gulation of ER-a ill response to cstradi ol. X -100

752

I,

~ 1 (,

~ 1 I

1 --

. ~ II .'

" II (1

" II. 11 .'

~ .x "

.\

1 ,

I" 1 •

~. ::t I ,'

c

I l'llrtll·, l I " I II' ~l n

Control E2

I (', . n[I •• 1

- I '

1(,

I I

C I:

-'" -

.-" I

.- n .: -"

E

INDIAN J EX P BIOL. JUL Y 20m

II 1.111')\.1

IL- l a

U ,'

OM (; ,\I'IJII

II 1.dph.,

T

I ru "'q

~~J!

1,.1\11, • . I ':I!' ~"I'

1 , 1 " ~

'S. 1 ,

::! I --·Z ( I ~

.~ II!.

...;

" I, .~ -"

B

1 ,

~ III

~ I I

I.:

J)

0 " 1.1 11'11.1

I ( ','"' 11.·1 I ,II. ~ ~ l. ":11

a\"

II . I .llph.1

Fig. 2 - RT-PCR amplification and densitomctric analys is of integrin receptor mRN A in estrogen and IL- i a treated ce ll s YS cont ro l cells (A) RT-PCR and densitomet ri c analysis of aJ mR A ex pression in cultured trophoblast cell s showing an upregul ati on in its ex press ion in estrogen and IL- I a treated cell s as compared to the untreated co nl ro l cell s (8 ) RT-PCR and densitomet ric analysis of a s mRNA expression in trophoblast cell s showin g a significant increase in the expression in

es trogen and IL- Ia treated ce ll s (C) RT-PC R and densitometri c anal ys is of ab mRNA ex press ion in trophoblast ce ll s demon strat ing a sign ificant increase in th,;

expression in es trogen and IL- I a treated cell s (D) RT-PCR and densitometric anal ys is of ay mR A ex press ion in trophob last ce ll s showing a signifi ca nt inc.ease in the ex press ion in

estrogen and IL- I a treated cell s (E) RT- PCR and densitomet ric anal ys is of ~I mRNA ex press ion in cultured trophoblast ce ll s show ing simil ar express ion in es trogen and IL- I a treated ce ll s to the Cont ro l ce ll s

DAS &BASA K : INTEGR IN EXPRESSION IN TROPHOBLAST CELLS 753

3A

4A

< (

.,

5A 5B

6A 6B

Fig. 3 - Modulati o n of u J protein in human first tc rm cy to trophohbst ce ll s by estrogcn (A ) cont ro l cytotrophob last ce lls . 48 hr (8 ) Estrogcn treated

cytotrophob last cc ll s . 48 hr showing its upregul a ti on in respo nse to estrad io l. X 200 Fig . 4 - Modul ,tli un o f u, protein in human first tc r!ll cy to trophoblast cc ll s by estrogcn (A ) c·.lIllro l cyto troph obla, t cel". 48 hr ( 8 ) Estroge n trea ted

cytotrophublast ce ll s. 48 hI' showing its upregulalion in rcspo nsc to cstradi o l. X 21)0 Fig. 5 - Modul ation o f u. protei n in human first tCrill cy to trophob last ce ll s by estrogen ( 1\ ) c011lrol cyto trophobla st cells. 4 hI'. ( B ) ESlrogcn treatcd

cytolrophob iasl cells. 48 hr showing il s upregulali,)n in rcspo nsc 10 cSlradiol. X 200 Fig. 6 - lmll1un,1cylochc ll1i ca l loca lif.ali o n o f P, prole in in human firsl I CI'll) cylOlrophoblasl ccll s. ( A ) conl rol cytOlroph oblast cc ll s . 48 hr. (8 ) Estrogcn

treated cytotrophob last cell s. 4 hI' , howing ab,cnce o f ils reg ulati on in rcsponsc to est rad io l. X 200

754 INDI AN J EXP BIOL, JUL Y 2003

Con tro l

I S

- I (, ~

'§ I .j

- , I --

-n s .-

- (I (. J ' !'«UI." 1J.j

~ , 0 --

~ I I

Cl) lllJPI i· .... l fUt:CII

Fig. 7 - RT-PCI< and dcnsitomctri c analys is of IL- I a mR NA ex pression in trophoblast ccll s show ing a significant incrcasc in thc cxpression in cstrogcn trcatcd ccll s

been assoc iated w ith certain unex plained cases of infeni l itl ·'9 , the factors in vo lved in the regulati on of integrin ex pression during bl as tocyst ac ti vati on and implantat ion is not known.

Limited informati on exists on Ihe integrin express ion by human trophoblast ce ll s at the time o /" implantati on. The surface o f the hu man oocy te and the blas tomeres of the earl y embryo have been shown

to ex pre. sa] , a v , ~ I . ~3 , ~ .j and ~5 from the oocy te to the bl astocyst stage::!o suggesting the formati on o /"

/"uncti onal integrin dimers, a]~" a\ ~ " a\'~3 and av~5 at the time of implantati on. First-trimester human trophob last ce ll s have been shown to express both laminin and fibronectin receptors, specifi ca l ly the

al ~" a5~1 ' a6~ 1 and a6~4 integrin heterodimers21

. It has further been shown that first trimester human trophob lasts exhi bit a distinct set of integrins

including ai, as, a v and ~I subunits and av~3 / ~S vitronectin receptor and to be negati ve for a i, and ~4 integ rin subunitsn .

The presence of es trogen receptor (ER) in the human placenta has been controversial with a few in vesti gators demonstrating its presence in the trophobl ast ce l ls while others claiming its absence. Patri ci023 first reported the presence of ER in human placenta obtained from normal deli ver ies as well as cesari an cases. Bi Iliar ef al. 2

.j usi ng i mmunocyto­chemistry further demonstrated the presence of ER protein in synty tiotrophoblast cell s suggesting that these are poss ibl y the es trogen recepti ve cell s. Rossmani th25 on the other hand, fa iled to detect the ER in human trophobl ast ce lls and suggested that the

acti on o f estrogen on the placenta may be mediated by the non- class ica l membrane bound receptor. The present study not only clearl y demonstrates the presence o f es tradi ol receptor in the human first trimester cy totrophoblast ce ll s, but also shows for the first time that es tradi ol stimul ates the ex pression of

ER-a in these cells (Fig. I ). The findi ngs suggest the possibi l it y of an autocrine regulati on by es trogen in inducing it s own receptor in the trophoblast ce ll s and further mediating the signalling effec t of estrogen in these ce ll s.

One of the act ions o f es tradio l in the trophob last ce ll s seem to be induction of integrins indi ca ting another mechanism by whi ch es trogen helps the process of implantati on apart from making the uteru s recepti ve for impl antati on. The upregulation of a .) , as. a 6, and a v integrin subun its by estradio l as observed in our studi es confirm the hypothesis.

A secondary surge o f es trogen is kn own to be necessary for implantation in mice26. A lthough, the presence or need o f thi s secondary surge of estrogen is debatable in hu m<lns27

, i ll \' if ro fertil ized hu man embryos seem to secrete estradiol just around 5-8 days post- fert ili zati on in culture, which is normall y the peri od of implantation ill vivo::!x -2Y . A lso, estrad iol levels in materna l serum appear to renec t the

folli cular IL- I ~ leve l and correlates with the outcome o f embryo transfer aner ill vif ro fertili zation31J

-31. Thi

correlation between estrogen, IL-I a level and successful implantation after IV F may be due to the positi ve effects o f these li gands on the cell adhes ion molecules and integrin subunit ex pression on trophoblast ce ll s.

Indeed the present study clearl y demonstrates the

poss ibility of such a correlati on as IL- I a induced the

ex pression of a .), a s, a 6, a v , integrin s in the tro­phoblast ce ll s Furthermore, es tradio l stimulated the

express ion o f IL- I a in these cell s, suggest ing thereby the poss ibility th at estrogen mediated regulati on of the integrin express ion may be through the proinfl alllma­tory cy tok i ne duri ng i mp lantation_

Acknowledgement Thi s study has been supported by the Indo-French

Center for the Promotion o f A dvanced Reearch, ew Delhi . Fe llowshi p prov ided to SB by ICMR, ew Delhi , is acknowledged.

Refernces I Basak S. Dhar R & Das C. Stcroids Modui alc thc Express ion

of <14 In tcgri n in Mousc blaslOcysts alld Utcrus During Impl anta tion. lJiol RC'prod, 66 (2002) 1784.

DAS & BASAK : INTEGRIN EX PR ESSION IN TROPHOBLAST CELLS 755

2 Basak S, Dubanchct S. Zourbas S. Chaouat G & Das C, Ex prcssion of pro-inflammatory cy tokincs in mousc blastocysts during implantation: Modulation by stcroid hormoncs. Alii J Repmd 1111111111/01. 47 (2002) II.

3 Cross J C. Werb Z & Fisher S J. Implan tati on and thc placcnta : kcy pieccs of the devc lopmcnt puzzlc. Sciel/ce. 266 ( 1994) 150R.

4 Duc-Goiran P, Mignot T M . Bourgcois C & Fcrrc F. Embryo-matcrnal intcracti ons at the implantati on site: A dc lica tc cquili brium. Ellr J Db.Her G\'Ilecol Reprod BioI. R3 ( 1999) 85.

5 Bowen J A & Hunt J S. Thc rolc of intcgrins in rcproducti on. Proc Soc E.rp Bioi M ed. 223 (2000) 33 1.

6 Ezra Y & Schenkcr J G, Abort ion rate III ass istcd reproducti on-true increasc? Early Preg Bioi Med. I ( 1995) 17 1.

7 Wil cox A J. Wcinberg C R. O'Connor J F. Baird D D, Schlattcrcr J P. Canfield R E. Armstrong E G & Nisula B C. Incidcncc of earl y loss of pregnancy. N EI/gi J M ed. 3 19 ( 1988) 189.

8 Gonzalcz R R, Palomino A. Boric A . Vega M & Devoto L. A quantitati vc eva luation of alpha I , alpha 4. alpha v and beta 3 cndomctrial integrins of fc rtile and unexplaincd infertile women th roughout thc menstru al cyc lc. A flow ey tomctrie appaisa l, 1-/11111 Reprod. 14 ( 1999) 2485.

9 Sanyal M. ag T C & Das C. Loca li za ti on of nitri c ox idc synthase in human trophoblas t cclls: ro lc of nitri c ox idc in trophoblast prol iferation and di ffc rentiati on, Alii J Reprod 1111111111/01. 43 (2000) 70.

10 Bhattacharya S. Chaudhary J & Das C. Antibodics to hCG inhibit progcsteronc producti on from human syncyti otrophoblas t cell s, Placel/ f(J . 13 ( 1992) 135.

II Das C & Sanya l M . Immunomodulators III human trophoblas t-uterus cross ta lk : cy tokines. growth factors :lIld nitric oxide. In: Rcproducti vc Immunology, Ed. Gupta SK, Narosa Publi shing Housc, New Dclhi , India. ( 1999) 99.

12 Smith S K. Charnock-Joncs D S & Shark Icy A M , The ro le of leukemia inhib itory factor and interleukin -6 In human n.:p roductior~ 1-/11111 Repmd. 3 ( 1998) 237.

13 A plin 1. Adh.:,ion molecules in implantat ion. Rev Reprorl . 2 ( 1997) 84.

14 Lesscy B A . The us.: of integ rins for the assessmcnt of uterinc recepti vity. Fenif Sreril , 6 1 ( 1994) 8 12.

15 Tabibzadeh S, Immunoreactiv ity of human endometrium: Correlation with endometri al dating. Fenl Steril . 54 ( 1990) 624.

16 Lessey B A, Damjanov ich L , Couti faris C. Castelbaum A. A lbelda S M & Buck C A. Integrin adhesion molecu les in the human endomctrium, J C/il/ 111I'esr, 90 ( 1992) 188.

17 Lessey B A. lIesanmi A O. Lcssey M A. Ri bcn M . Harris J E & Chwa li sz K, Luminal and glandular endomct ri al epithelium express intcgrins differentiall y th roughout the menstrual cyc le: Implications for implantati on. contraception. and in fert i l ity. Alii .I Reprod 1111111111101. 35 ( 1996) 195 .

18 Lessey B A . Implantati on defects in infertile womcn \V ith endometri osis . AIII/ N )' A cad Sci. 955 (2002) 265.

19 Rcddy K V & Mchalji P K . Integrin cell adhesion molecules in endometi rum of fert il e and in fert i le womcn throughou t mcnstrual eyc lc. 'I/ dio J Exp Bioi, 37 ( 1999) 323.

20 Campbell S. Swann H R. Seif M W, Kimber S J & Aplin J D. Cc ll adhesion molccules on the oocy te and preimplantati on human embryo. 1-/11111 Neprod. 10 ( 1995) 157 1.

2 1 Burrows T D. King A . Smith S K & Loke Y W. Human trophoblast adhesion to malrix proteins: Inhibition and signal transducti on. 1-/11111 Reprod. 10 ( 1995) 2489.

22 Irvi ng J A. Lysiak J J. Graham C H. Hearn S. Han V K & Lala P K . Charaet.: ri sti cs of trophob last ce ll s migrati ng from first trimester chorionic villus expl ants and propagaled in culture. Placel/ta . 16 ( 1995) 4 13.

23 Patri cio B. Demonstrat ion of ER and PR in human placenta in normal dcli vc ries and ti ssues obta ined from cc. arian deli ve ri es. Rev Fr Cyl/ecol Dbsrer. 89 ( 1994) 145.

24 Billi ar R B, Pepe G J & A lbrecht E D, Immunocy tochcmieal identifi cation of the oestrogen receptor in the nuclei of culturcd human placental syneyti otrophoblasts. Placel//([. 18 ( 1997) 365.

25 Rossmanith W G. Wolfahrt S. Ecker A & Eberhardt E, Thc demonstration of progesterone. but not of cstrogen receptor in the developing human placenta. 1-/01' Me/([/J Rt's. 2Y ( 1997) 604.

26 McCormack J T & Greenwa ld G S. Progesteronc and es tradiol- 1713 concentrat ions in the peripheral plasma du ring prcgnancy in the mouse. 1. EI/docrillol. 62 ( 1974). 10 1.

27 Dc-Ziegler D. Fanchin R. De-Moust ier B & Bulletti C. The hormonal control of endometrial recepti vit y: Estrogcn (E2) and progesterone . .I Reprod 11I11I1I1I1U1. 39 ( 1998) 149.

28 Shutt DA & Lopala A , The sec retion of hormones during the cullure of human preimplantation embryos with corona cc ll s. FerrilSteril . 35 ( 198 1) 4 13.

29 Edgar D H. Jamcs G B & Mills J A. Stero id secreti on by human early cmbryos in cul ture. I-/U/II Rep rod. 8 ( 1993) 277 .

30 Zo lt i M , Ben-Rafael Z, Meirom R. Shemcsh M . Bida D, Mashi ach S & Apte R. Cy tokine in volvement in oocytcs and earl y embryos, Ferril Steril. 56( 199 1) 265 .

31 Su nder S & Lenton E A , Endocrinology of the peri­implantation pe riod. Bailfieres Best Pract Res elill Obstet Cwwecol, 14 (2000) 789.