Implantation Site in Normal Pregnancy

A Study With Monoclonal Antibodies

SALIM E. KABAWAT, MD,MAHPAREH MOSIOUFI-ZADEH, MD,

SHIRLEY G. DRISCOLL, MD, andATUL K. BHAN, MD

In the present study, the presence ofmajor histocompati-bility complex antigens (MHC) and the degree and na-ture of inflammatory response in the human placentawere determined by staining frozen tissue sections withmonoclonal antibodies and an immunoperoxidase tech-nique. Although class I (HLA-A, B, and C) and Class II(HLA-DR, Ia-like) MHC antigens were not demonstratedin the syncytiotrophoblast, Class I antigens were foundin trophoblast ofthe placental septum, shell, and implan-tation site and in the chorionic villous stroma. There wasno staining for Ia-like antigens in the fetal componentsof the placenta. T cells were scarce and evenly scatteredin the normal implantation site. No T cells infiltrated the

THE MECHANISM by which the mammalian fetalsemi-allograft is tolerated in the womb of its immuno-logically intact mother has been the subject of numer-ous hypotheses.1 Because a principal component of al-lograft rejection lies in the recognition by the host offoreign major histocompatibility antigens (MHC) onthe graft, attention has been focused on the degree ofexpression of these antigens in the fetal and chorionictissues. Trophoblast in direct contact with the mater-nal cells is the most important tissue in this respect.The expression of MHC antigens in the human and

mouse trophoblast has been the subject of controversy,reported results of studies differing according to themethods used.2-5 Although HLA-A, B, and C antigenshave been demonstrated in the human villous stromalcells but not in the villous trophoblast,6 8 Ia-like anti-gens (HLA-DR) have not been identified in the chori-onic villi.6 8 These findings may account for lack of im-mune rejection of the syncytiotrophoblast that is indirect contact with maternal blood in the intervillousspace. However, the demonstration of HLA antigensin the trophoblastic columns and in the trophoblast ofthe placental shell8 requires another explanation for the

From the Departments of Pathology, Massachusetts General Hospital,Brigham and Women's Hospital and Harvard Medical School,Boston, Massachusetts

chorionic villi. B cells and natural killer cells were notidentified in the human placenta. Macrophages con-stituted more than 20% ofthe decidual cells and had mor-phologic features identical to those of "small decidualcells." The lack ofT-cell infiltration ofthe fetal placentalstructures and their scarcity in the implantation site sup-port the notion that T-cell-mediated immune responseagainst placental antigens is not generated by the mater-nal host in normal pregnancy. The abundance ofmacro-phages at the implantation site may be related to theirpossible role in the suppression ofimmune response. (AmJ Pathol 1985, 118:76-84)

tolerance of these fetal structures by the maternal im-mune system. Trophoblast at both these sites is in di-rect contact with maternal tissues. That an immuno-logically privileged environment is provided, eitherthrough suppression of the maternal immune responseor by a mechanical barrier, is still of considerableinterest.9We examined the normal implantation site, infiltrat-

ing trophoblast and decidua, using monoclonal anti-bodies that recognize major histocompatibility antigensand antigens associated with the different immune-active cell subsets. For this purpose, a sensitiveavidin-biotin immunoperoxidase technique was usedon frozen tissue sections. We reexamined MHC anti-

Supported by Grants CA 29601 and HL 18646 from theNational Institutes of Health. S. E. Kabawat is a recipientof a USPHS Research Service Award T32-CA09216.

Accepted for publication July 27, 1984.Address reprint requests to Dr. Atul Bhan, Department of

Pathology, Massachusetts General Hospital, Fruit Street,Boston MA 02114.

76

IMPLANTATION SITE IN NORMAL PREGNANCY 77

gen expression on the trophoblast, confirmed publishedstudies, and examined MHC antigen expression oninfiltrating trophoblastic cells of the implantation site.We also identified the inflammatory cells infiltrating thenormal human decidua, providing evidence of a rela-tive paucity of lymphocytes in the implantation site, anddemonstrated that small decidual cells have macrophagemarkers.

Materials and Methods

Freezing and Storage of Tissue

Sections of placenta, basal plate, and implantationsite were obtained from normal pregnancies: two atterm, two delivered prematurely (at 32 and 33 weeks),and seven elective abortions (two each at 15 and 16weeks, and one each at 17, 18, and 19 weeks).

Samples of endometrium from nonpregnant womenwere obtained following hysterectomy for conditionsnot affecting the endometrium. Three proliferative and

one midsecretory endometria were frozen. All speci-mens were snap-frozen, embedded in OCT compound(Ames Co., Division of Miles Laboratories, Inc., Elk-hart, Ind), and stored at -70 C.

Monoclonal Antibodies

The reactivity and sources of the monoclonal anti-bodies used in this study are summarized in Table 1.

Heteroantiserum

One antiserum, a rabbit-derived anti-lysozyme(Dakopatts, Copenhagen, Denmark) was used.

Avidin-Biotin Immunoperoxidase Technique

Four-micron cryostat sections were air-dried andfixed in acetone.19 The immunoperoxidase techniquewas carried out with the avidin-biotin-peroxidase kit

Table 1 -Reactivity of Monoclonal Antibodies Used

Monoclonalantibody

Anti-HLA

Pattern of reactivity

Common framework determinant on HLA-A,B, and C specificities

Anti-la (HLA-DR) N

Anti-P2 microglobulin Li

Anti-Bl Al

Anti-lgM 1g

OKT3 11

Anti-Leu-4 8(

Anti-Leu-1 9E

Anti-T4, Anti-Leu-3a H

OKT8 C

OKT6 La

OKT1 0 Pi

OKT9 Tr

OKT1 1 St

Anti-Leu-7 (HNK-1) Nl

OKM1 G

Anti-Leu-M3 M

Anti-leukocyte GIantigen (T200)

* Peripheral blood lymphocytes.t Chronic lymphocytic leukemia.t Natural killer and killer cells.§ And unpublished observations.

lonpolymorphic determinant on theHLA-DR molecule

ight chain of the HLA-A, B, and C molecule

,l human B cells except plasma cells

]M (heavy chain specific) includingB-cell surface Ig00% E rosette+ PBL*

0-95% E rosette' PBL*

5% E rosette+ PBL,* lg+ CLLt cells

lelper/inducer T-lymphocytes, macrophages

:ytotoxic/suppressor T-lymphocytes

angerhans cells and cortical thymocytes

lasma cells, minority of B cells andmonocytesransferrin receptor

,heep erythrocyte receptor

IK and K cellst;ranulocytes, monocytes, and 40% NKcellstlonocytes, macrophages, Langerhans cells,and granulocytes

aIycoprotein on all hematopoeitic cellsexcept most plasma cells

Source

Bethesda Research Laboratories,Gaithersburg, Md

Ortho Diagnostics, Raritan, NJ

Becton-Dickinson, Sunnyvale, Calif

Coulter Immunology, Hialeah, Fla

Dr. Lee Nadler, Dana-FarberCancer Center

Ortho Diagnostics

Becton-Dickinson

Becton-Dickinson

Coulter Immunology (anti-T4)Becton-Dickinson (Leu-3a)

Ortho Diagnostics

Ortho DiagnosticsOrtho Diagnostics

Ortho Diagnostics

Ortho Diagnostics

Becton-Dickinson

Ortho Diagnostics

Becton-Dickinson

Hybritech, La Jolla, Calif

Reference

5, 10

11

12

13

14

15, 16

15, 16

17, 1819, 20

21

22, 23

13, 22

22, 24

22, 25

26

27

28§

29§

Vol. 118 * No. I

78 KABAWAT ET AL

(Vector Laboratories, Burlingame, Calif). Sections wereprewashed in phosphate-buffered saline (PBS) andpreincubated with normal horse or goat serum for satu-ration of nonspecific binding sites. Suitable dilutionsof the monoclonal antibodies or the rabbit anti-serumwere then left on the sections for 30 minutes and thenwashed three times in PBS, each for 5 minutes. The sec-tions were then incubated with biotin-labeled horse anti-mouse IgG antibody or goat anti-rabbit antibody, andendogenous peroxidase activity was blocked in a 0.3%0osolution of hydrogen peroxide in PBS. A final incuba-tion with biotinylated avidin-peroxidase complexpreceded color development in 3-amino, 9-ethylcar-bazole solution (Aldrich Chemical Co., Milwaukee,Wisconsin) in acetate buffer (pH 5) with hydrogenperoxide. Dilutions of all biotinylated antibodies andperoxidase complex were as recommended in the kits.Slides were subsequently counterstained in Lerner's 3hematoxylin (Lerner Laboratories, New Haven, Conn)and mounted in Elvanol (E. I. DuPont de Nemours &Co., Inc., Wilmington, Del). Negative controls for eachcase also included a slide incubated with PBS alone inthe primary antibody step. Potency of the antibodiesused was frequently monitored on sections of humantonsil, lymph node, and thymus. One cryostat sectionfrom each block was stained with hematoxylin and eo-sin (H&E) and examined for correlation of morpho-logic features with patterns of staining.

Fc and C3 Receptors

Decidual cells were separated as published else-where.30 Fragments of decidua were obtained fresh; villiand fetal fragments were discarded on the basis of theirgross appearance or as stained with toluidine blue on

frozen tissue sections. Fragments were then minced;decidual cells were separated using collagenase, tryp-sin, and mechanical teasing.30The method of preparation of sensitized (antibody

[A]- and complement [C]-coated) ox red blood cells (E)was published previously.31 E, IgGEA, IgMEA, and Ig-MEAC cells were prepared. For detection of Fc and C3receptors, suspensions of decidual cells were adjustedto 1.5 x 106 cells/ml and 0.3 ml were incubated withequal volumes of E, IgGEA, IgMEA, and IgMEAC 1Wosuspensions for 30 minutes at 37 C without centrifuga-tion (IgMEA and IgMEAC) or for 3 minutes with cen-

trifugation at low speed (IgGEA and E). The cells werethen left for at least 1 hour at 4 C; then they were stainedwith trypan blue, and the percentage of rosetted cellsof all viable nucleated cells was determined in a

hemocytometer. Finally, concentration of all cells wasadjusted to 1 x 106/ml, and H&E-stained cytocen-trifuge slides were prepared to examine the morpho-logic features of the rosetted cells.

_a,& ~~~~~~4w

Figure 1- Frozen tissue section of a proliferative endometriumstained with anti-la antibody. Numerous la-positive cells, includingsome dendritic cells (B, arrow), are scattered in the stroma. Endo-metrial epithelial glandular cells and stromal cells are la-negative.(Immunoperoxidase with hematoxylin counter stain; A, x 160; B,x 1008)

Nonspecific Esterase Technique

Four-micron cryostat sections were fixed in 6%o form-aldehyde-calcium, stained in Jaubert's solution32 for15 minutes, and counterstained with hematoxylin.

Results

Endometrium From Nonpregnant Women

Endometrial surface and glandular epithelium was

HLA- and P2M-positive and Ia-negative. In the stroma,in addition to lymphocytes, two types of cells reactedwith HLA, P2M, and Ia antibodies. One type of cellhad abundant cytoplasm, with cytoplasmic dendritessurrounding adjacent cells, and round or oval, centrallyplaced nuclei. The other type of HLA-, f32M-, and Ia-

positive cell was smaller, round to oval, with round tooval, central or eccentrically placed nuclei. These cellssometimes contained hemosiderin granules and are

probably histiocytes (Figure 1). Each of these types com-prised 5-10 cells per high-power field. They were read-ily differentiated from the stromal cells proper, whichhad fusiform to round nuclei and scant cytoplasm and

A

P' Bil

AJP * January 1985

IMPLANTATION SITE IN NORMAL PREGNANCY 79

Table 2-Major Histocompatibility Complex Antigens inthe Human Placenta and Implantation Site

HLA

Antigens

P2M la

Figure 2-Frozen tissue section of proliferative endometrium stainedwith OKT8 antibody. Rare T8-positive lymphocytes are found in closeassociation with luminal epithelium (arrows) in a manner reminiscentof intraepithelial lymphocytes.19 (Immunoperoxidase with hematoxy-lin counter stain, x 256)

were HLA-, P2M-, and Ia-negative. Stromal histiocytes,the round and oval Ia positive cells, also reacted withanti-leukocyte common antigen (T200), with OKM1,anti-T4, and anti-Leu-3a antibodies. The larger dendriticcells did not. The number of cells of the two types didnot seem to change in the secretory phase of the men-strual cycle. Unlike the stromal cells proper, they didnot display predecidual reaction.

Antibodies directed against lymphocyte surfacemarkers stained numerous cells with cytologic featuresof lymphocytes scattered throughout the endometrialstroma or aggregated in lymphoid follicles. Most wereT cells (Leu-l-, Leu-4-, T3-, and Tlla-positive. Abouthalf were cytotoxic/suppressor T8 type, and half werehelper/inducer T4-positive. T8-positive cells were alsofound adjacent to, or between glandular epithelial cellsand cells of the surface epithelium (Figure 2). Approx-imately 2-3 T cells were found per high power field. B

TrophoblastSyncytiotrophoblastLanghans layerPeripheral trophoblast*

Villous stromaConnective tissueVesselsHofbauer cells

DeciduaLarge decidual cellsSmall decidual cells

+

+

+

+

+

+

+

+_

+_

+_

+_

+_

+ +

* Cytotrophoblast of the placental shell, septa, and site.

lymphocytes were mostly restricted to lymphoid folli-cles, being absent or very rare in the stroma.

Placenta and Implantation Site

The results of immunohistochemical staining, studiesof Fc receptors and staining for nonspecific esterase aresummarized in Tables 2 and 3.

Neither HLA nor P2M antigens were demonstratedin the syncytiotrophoblast. Although trophoblastic cellcolumns were not present in the sections studied,trophoblast of the placental septa and shell and infiltrat-ing trophoblastic cells of the placental site stainedweakly for anti-HLA and anti-f32M antibodies. How-ever, many infiltrating multinucleated trophoblast cellsdid not stain for HLA or f2M. The villous core, in-cluding connective tissue, vessels, and Hofbauer cells,was strongly reactive with both antibodies.

Table 3-Lymphocytes and Macrophage Markers in the Human Placenta and Implantation Site

Antigens

T200 T11A Leu-4 Leu-1 T3 Leu-3a/T4 T8 T6 T9 Bi OKM1 LeuM-3 NSE* Lyst

TrophoblastSyncytiotrophoblastLanghans layerPeripheral trophoblasts

Villous stromaConnective tissueVesselsHofbauer

DeciduaLarge decidual cellsSmal decidual cells

+

- + + - - - +

+ - -

+, positive staining; -, no staining.* Nonspecific esterase.t Lysozyme.t Variable staining.

+

+

_ + + +

- + -_ _ + -

- + Vt + + + +

Vol. 118 * No. 1

80 KABAWAT ET AL

I

.I....*--A;1

Figure 3-Frozen tissue section of the placenta and implantation sitefrom a 17-week pregnancy stained with anti-HLA antibody. Thestroma of the chorionic villi is strongly HLA-positive, whereas syn-cytiotrophoblast (arrows and lower inset) is HLA-negative. Peripheraltrophoblastic cells of the placental shell (PT and upper inset) arefaintly stained. Maternal epithelial and stromal cells are strongly HLA-positive (arrowheads). (Immunoperoxidase with hematoxylin counterstain, x 100; insets, x640)

In the endometrium, large and small decidual cellsand the various infiltrating inflammatory cells were

strongly positive for HLA and P2M antibodies (Figure3). Ia antigens were not demonstrated in any chorionictissue, including trophoblast, villous stroma, and fetalblood vessels. Only the small decidual cells, the en-

dothelial cells of the maternal vessels, and rare lympho-cytes stained with anti-Ia antibody (Figure 4).The luminal surface of the syncytiotrophoblast

stained with OKT9 antibody that recognizes transfer-rin receptor. Cytotrophoblast in all sites was T9-negative(Figure 5). None of the other monoclonal antibodiesdirected against T- or B-lymphocytes and macrophagesreacted with any trophoblastic cells. In the villousstroma, Hofbauer cells, the fetal tissue macrophages,33reacted with anti-lysozyme but not OKM1 and anti-Iaantibodies. They also reacted with anti-T200, T3, T4,Leu3a, Tlla, and Bi antibodies. Cells recognized mor-

phologically as lymphocytes were not found in the vil-lous stroma.Two groups of decidual cells were identified on fro-

zen tissue sections stained with hematoxylin and eo-

sin. Twenty to 30% of decidual cells were small and dis-tinguishable from the larger cells by size, oval shape,and darker blue hematoxylinophilic nuclei. They hadone or occasionally two round or oval eccentric nuclei.The large decidual cells were polygonal in shape andhad more abundant cytoplasm and larger, vesicular,light-staining, centrally located nuclei with an occa-

sional small nucleolus. Both types of decidual cells wereeasily differentiated from infiltrating trophoblastic cellsthat had one or several large nuclei and dense basophiliccytoplasm. Large and small decidual cells were trans-

ferrin-receptor-positive. The small decidual cells thatwere Ia-positive also reacted with anti-T200, OKM1,anti-T4, Leu-3a, anti-Leu-M3, and anti-lysozyme anti-bodies (Figure 6).

In all cases, rare lymphocytes, in average about 3-4per high power field, infiltrated the decidua. Most ofthese were phenotypically T cells (T3-, Leu-l-, Leu-4-,and Tlla-positive); the majority stained with anti-T4and anti-Leu-3a antibodies that recognize helper/in-ducer T cells. T8-positive cells were occasionally seen.The intensity and nature of the infiltrating lymphocytesdid not seem to vary with gestational age. None of thelymphocytes reacted with anti-IgM and Bi antibodiesthat recognize B-lymphocytes. In two cases up to one-third of the small decidual cells were T1O-positive. NoHNK-1-positive cells were found at any site.

Fc and C3 Receptors

Results of receptor analysis are presented in Table4. The percentage of IgG Fc-receptor-positive cells incell suspensions from decidua varied between 24% and417o. Only 3% of cells in suspension had C3 recep-tors. On cytosmears, most of the Fc-receptor-positivecells displayed morphologic characteristics similar tothose of small decidual cells. In addition, some cellswere vacuolated, and some contained dense inclusionssuggestive of hemosiderin.

A

B

Figure 4-Frozen tissue sections of the placenta and implantationsite (A) and the implantation site (B) of a 17-week pregnancy stainedwith anti-la antibody. Only small decidual cells (macrophages) arela-positive (arrowheads). (Immunoperoxidase with hematoxylincounter stain; A, x 100; B, x 160)

AJP * January 1985

IMPLANTATION SITE IN NORMAL PREGNANCY

Nonspecific Esterase Staining

Strong staining with Jaubert's solution was found insyncytiotrophoblast, Hofbauer cells, and the smalldecidual cells. Weaker staining was noted in cytotropho-blast in all sites. Large decidual cells were negative oronly faintly positive (Figure 7).

Discussion

Since recognition ofMHC antigens constitutes a ma-jor component of allograft rejection, the expression ofthese antigens in the trophoblast has been the focus ofnumerous studies in human beings and in the murinesystem. In the mouse, the results seemed to vary accord-ing to the method of study. Whereas some investiga-tors have not been able to demonstrate H-2 antigens ona cultured murine blastocyst trophoblast by a direct im-munoperoxidase method,2 more recent work with theuse of radiolabeled antibody3 demonstrated these an-

tigens on dissociated trophoblastic cells. Furthermore,injection of anti-paternal H-2 haplotype radiolabeledantibody in the maternal blood resulted in labeling ofthe trophoblast.

Although several human studies agree that MHC an-

tigens (Class I and Class II) are not expressed on thevillous trophoblast,618 in one study HLA antigens were

demonstrated on cytotrophoblast of the early placen-tal columns.8 To test whether the apparent differencebetween types of trophoblast is due to the sensitivityof the method of study, especially in view of the con-

troversy in mice, we reexamined the subject, employ-ing a highly sensitive method.

In this study, HLA antigens were demonstrated inthe stroma of chorionic villi, but not on the covering

Figure 6-Frozen tissue section of the implantation site of a 16-weekpregnancy stained with anti-Leu-3a antibody. Cells with the morpho-logic appearance of small decidual cells (macrophages) are Leu3-a-positive. (Immunoperoxidase with hematoxylin counter stain, x 400)

trophoblast. Infiltrating trophoblastic cells of theplacental shell and invasive trophoblast of the placen-tal site were weakly HLA-positive. Whether the HLAexpression on these cells includes both maternal andpaternal haplotypes remains to be examined with anti-bodies that recognize human HLA allotypes. The pos-sibility remains that this positive reaction is due toadsorption of maternal HLA antigens from the sur-rounding decidua. Alternatively, these cells may escapeimmune recognition by masking or modulating thepaternal haplotype of their HLA antigens. MaternalHLA haplotypes were readily demonstrated on lympho-cytes from the newborn.34 In contrast, paternal anti-gens were often difficult to demonstrate until lympho-cytes were incubated overnight in tissue cultureconditions.34 Failure to demonstrate HLA antigens onthe syncytiotrophoblast seems to be due to their ab-sence, rather than masking, because digestion with mul-tiple enzymes failed to convert trophoblastic reactivityto positive.6Although Ia antigens have been described in placen-

tal vessels with no further indication as to their specificsite,35 we did not find reactivity with anti-Ia antibody

in the chorionic villi, including vascular endothelialcells, or in the cytotrophoblast. This finding is interest-ing in view of the universal expression of Ia antigens

Table 4- Fc and C3 Receptors on Decidual Cells*

Gestationalage ofdecidua %E + * % EAIgG + % EAIgM + %EAC +

Figure 5-Frozen tissue section of the placenta and implantation siteof a 17-week pregnancy stained with OKT9 antibody. Staining is pres-ent on the luminal surface of syncytiotrophoblast (arrows) and indecidual cells (D). Peripheral trophoblastic cells of the placental shellare OKT9-negative (arrowheads). (Immunoperoxidase with hematoxy-lin counter stain, x 100)

1) 16 weeks2)16 weeks3)15 weeks4)15 weeks

0

0

0

0

24284131

0

0

0

0

3213

Cell suspensions prepared from decidua were incubated with redblood cells or sensitized red blood cells (see Materials and Methods).

* Percentage of rosette-forming cells in the total number of viablecells counted.

81Vol. 118 * No. I

82 KABAWAT ET AL

A

Ip

B oj

it.. ..Pis W ...

Figure 7-Frozen tissue section of the placenta and implantation siteof a 32-week pregnancy stained for nonspecific esterase. Strongstaining is found in syncytiotrophoblast, Hofbauer cells in the villousstroma, and in small decidual cells (macrophages, arrows). Weakstaining is found in peripheral trophoblast (PT). (Hematoxylin counterstain; A, x100; B, x640)

in the endothelial cells of the human adult.36 However,Ia antigens cannot be demonstrated in the cultured um-bilical cord endothelial cells without induction withlymphokines (gamma interferon).37The lack of expression ofMHC antigens on the syn-

cytiotrophoblast, the chorionic ("fetal") tissue in directcontact with the maternal intervillous blood, may ex-

plain why this tissue is not rejected. However, other hy-potheses must be advanced to explain the tolerance ofthe peripheral trophoblast if its expression of HLA an-

tigen is verified. One explanation may be the develop-ment of blocking antibodies that can mask the HLAdeterminant. Ten percent to 30Wo of pregnant womendevelop antibodies to paternal HLA antigens, the fre-quency increasing with parity.38 Another possibility isthe development of tolerance through suppressor cellactivity in the implantation site.39Some additional finding of our study should be

noted. The presence of transferrin receptors on the sur-

face of human syncytiotrophoblast was described byimmunohistochemical means and by binding of trans-

ferrin.40 The present study with the monoclonal anti-body T9, which binds to transferrin receptor,24 confirmsthese results. The cytotrophoblastic cells were T9-negative, probably reflecting a difference in function,because transferrin receptors on the surface of chori-onic villi are involved in the transfer of iron to the fe-tus.40 Binding of transferrin to the surface of tropho-blast has also been implicated in steric inhibition of theimmune response.5Another question addressed in this study regarding

the tolerance of the fetal allograft concerns the inten-sity and nature of the inflammatory infiltrate in the im-plantation site, and how it differs from response in otherallografts, such as skin allografts.41 Tissue allograft re-jection is generally characterized by marked lympho-cytic infiltrate, in particular of T cells, in the allograftand in the graft bed.41 In rejected skin allografts, T4cells tend to be more abundant in the dermis, especiallyperivascularly, while T8 cells infiltrate the epidermis.41In sharp contrast, although present in small numbersin the implantation site, T-lymphocytes were not in-creased over the number present in normal nongesta-tional endometrium, nor did they infiltrate any chori-onic structures.An interesting observation made in the present study

is the abundance of macrophagelike cells in the decidualstroma. These cells probably represent the same cellsthat were previously labeled small decidual cells,42 whichhave morphologic,42 histochemical,43 and ultrastruc-tural44 features distinct from those of the larger decidualcells. Morphologically, the large decidual cells areround, with delicate eosinophilic homogenous cyto-plasm. In contrast, the small decidual cells are spindly,with dense nuclear chromatin and a cytoplasm that con-tains diastase-resistant, periodic acid-Schiff (PAS)-positive granules.42 These small decidual cells have longbeen known to be esterase-positive.43 The decidua alsocontains the placental site trophoblastic cells, readilyrecognized by their large nuclei and their intenselybasophilic cytoplasm.42 One ultrastructural study de-scribed two types of decidual cells,44 large and small.The smaller cells were noted to have cytoplasmic pro-trusions and were closely associated with lymphocytes.A more recent study with electron microscopy andhistochemistry45 describes a single, distinct type ofdecidual cells, held together only by connective tissue.No mention is made in this study of small decidual cells;instead, large numbers of macrophages with large veil-like cytoplasmic extensions were noted. We suggest thatthe small decidual cells are tissue macrophages. Theyconstitute 20-30 We of the total decidual cells and pos-sess many characteristics of macrophages: ie, Ia-like an-tigens, Leu-M3, OKM1, T4 and Leu-3a antigens, andFc receptors. T4 and Leu-3a markers that are usually

AJP * January 1985

Vol. 118 * No. 1 IMPLANTATION SITE IN NORMAL PREGNANCY 83

associated with helper T cells were also described onmacrophages. 19.20 Small decidual cells also containnonspecific esterase and lysozyme and were previouslyfound to contain acid phosphatase and residualbodies.45 In the mouse, most of the decidual cells bearFc receptors46 and Thy-I antigens30 and are of bone mar-row origin.47 We suggest that the small decidual cells,ie, macrophages, are the human equivalent of bone-marrow-derived cells in the mouse. However, the per-centage of mouse decidual cells traced to the bone mar-row was higher. This difference may be species-relatedor may be attributable to the decidualization in themouse being induced by oil, rather than by implanta-tion of a blastocyst.47Although in humans it has been suggested that de-

cidual macrophages may represent Hofbauer cells thathave migrated from the chorionic villi,45 in the presentstudy differences in surface markers between the twomacrophage populations were demonstrated. Theirmaternal origin is thus more plausible, probably migrat-ing from the bone marrow. Furthermore, in a previousstudy "small developing decidual cells" were foundaround the maternal capillaries at the beginning of preg-nancy, closely associated with lymphocytes.44 The sametype of macrophages were demonstrated in the en-dometrium from women who were not pregnant. Somecontained hemosiderin granules, suggesting a scaven-ger function. These findings are consistent with thoseof a recent report of abundant macrophages isolatedfrom the endometrium of women of the reproductiveage, but not from that of postmenopausal women.48We also found larger, dendritic, Ia-positive cells in theendometrial stroma of nonpregnant women. These cellsdid not exhibit other macrophage markers, thus differ-ing from the macrophages described above. They werenot found in sections from gestational endometrium.

It has been postulated that the large number ofdecidual macrophages may protect the mother from in-vading trophoblastic cells.45 It is also likely that mac-rophages in the implantation site may have a role insuppression of the immune response, and in turn mayrelate to their capacity to synthesize prostaglandins.49In support of this, suppressor-cell activity associatedwith non-T small decidual cells obtained from pregnantrat uterus has been demonstrated.39

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2. Billington WD, Jenkinson EJ, Searle RF, Sellens MH:Alloantigen expression during early embryogenesis andplacental ontogeny in the mouse: Immunoperoxidase andmixed hemadsorption study. Transplant Proc 1977,9:1371-1377

3. Chatterjee-Hasrouni S, Lala PK: Localization of H-2 an-tigens on mouse trophoblast cells. J Exp Med 1979,149:1238-1253

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8. Sunderland CA, Redman CWG, Stirrat GM: HLA A,B, C antigens are expressed on non-villous trophoblastof the early human placenta. J Immunol 1981, 127:2614-2615

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10. Ugolini V, Nunez GR, Smith RG, Statney P, Capra JD:Initial characterization of monoclonal antibodies againsthuman monocytes. Proc Natl Acad Sci 1981, 77:6764-6768

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13. Bhan AK, Nadler LM, Stashenko P, McCluskey RT,Schlossman SF: Stages of B cell differentiation in humanlymphoid tissue. J Exp Med 1981, 154:737-749

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AcknowledgmentsThe authors thank Mr. William Nelson for excellent

secretarial assistance.