Placenta (2002), 23, 672–684doi:10.1053/plac.2002.0841, available online at http://www.idealibrary.com on

Structural Differentiation of Human Uterine Luminal and Glandular

Epithelium during Early Pregnancy: An Ultrastructural and

Immunohistochemical Study

R. Demira,e, U. A. Kayislia,b, C. Celik-Ozencia,b, E. T. Korguna,c, A. Y. Demir-Weustend and A. Aricib

Departments of a Histology and Embryology, Faculty of Medicine, Akdeniz University, Antalya, Turkey; b Obstetrics andGynecology, Yale University, School of Medicine, New Haven, CT, USA; c Histology and Embryology, Karl-FranzensUniversity, Graz, Austria, and d Obstetrics and Gynecology, AZTM, Maastricth University, The Netherlands

Paper accepted 6 May 2002

The differentiation of human endometrial epithelium is a dynamic event that occurs throughout the menstrual cycle and earlypregnancy. The structural transformation and differentiation of human uterine luminal and glandular epithelium of early humanpregnancy (n=14) was investigated ultrastructurally and immunohistochemically using antibodies against cytokeratin (CT),endothelial marker CD31, Fas, and proliferating cell nuclear antigen (PCNA). Ultrastructurally, luminal epithelial cells showeddistinctive euchromatic nuclei with prominent nucleoli and relatively loose cell membranes in all poles (apical to basal). Subcellularcomponents were easily recognized in luminal epithelium except in degenerating cells. Mainly two cell types, dark and clear cells,formed the glandular epithelium. In the early gestation period, microvilli were abundant on the apical and apico-lateral poles ofthese cells. Only a few cytoplasmic projections were observed in dark cells. Numerous cilia were observed on the apical pole ofsome clear cells, located at the adluminal segment. In contrast, dark cells lacked cilia, nuclear channels, or giant mitochondrialprofiles. Glycogen synthesis and apocrine secretion were recognizable for several days during early gestation. The apocrinesecretory activity differed among dark cells of the glandular epithelium. The immunoreactivity of PCNA and Fas, andultrastructural observations in the glandular epithelium suggest that, even in different segments of the same gland, epithelial cellsdo not regress during early gestation, but proliferate, perhaps representing a resistance against trophoblastic invasion. Thesemorphological and molecular changes suggest that both luminal and glandular epithelium may play an important role in cellulardefense and limitation for trophoblastic invasion during early pregnancy since plasma membrane alterations of the surfaceepithelium take place at the apical, basal and lateral poles compared to early secretory phase endometrial cells. Besides glandularepithelium may be consequently responsible for uterine secretions, which may be critical for early embryo development.

� 2002 Elsevier Science Ltd. All rights reserved.Placenta (2002), 23, 672–684

e To whom correspondence should be addressed at: Department ofHistology and Embryology, Faculty of Medicine, Akdeniz University,Campus, Antalya 07070, Turkey. Tel./Fax: +90 (242)-2274486;E-mail: demir@hipokrat.med.akdeniz.edu.tr

INTRODUCTION

Mutual recognition of the implanting embryo and theendometrium is of primary importance during the first stagesof implantation. It requires the establishment of a tight contactbetween the apical poles of the cells, the so-called ‘cellparadoxical receptivity’ (Denker, 1993), which is necessary fortemporary pseudo-symbiosis between these two tissues(Demir, 1997, 2000). Prior to contact with the uterine epi-thelium, the blastocyst undergoes a series of physiologicaland developmental changes, and shows an exquisite dialogueduring the implantation window (Simon et al., 1996).Adhesion and implantation of the blastocyst to the decidua are

0143–4004/02/$-see front matter

controlled by proteolytic processes, and adhesion moleculessuch as integrins and integrin-related proteins that allow themural trophoblast to penetrate into the decidua and invadethe maternal vasculature (Blankenship et al., 1993; Kayisliet al., 2002).

The human feto-maternal interface is a dynamic site whereextensive and rapid cellular and extracellular matrix (ECM)turnover takes place both in the endometrial epithelium andthe decidual stroma. During early gestation, different cell typessuch as typical stromal-type decidual cells, maternal leukocytesand various trophoblastic cells can be distinguished in theendometrial stroma (Benirscke and Kaufmann, 1995; Aplin,1996; Kaufman and Castellucci, 1997).

Structural changes in the endometrial epithelium through-out the menstrual cycle are regulated primarily by steroidhormones (Verma, 1983; Dockery et al., 1993). Additionally,ultrastructural transformation of the glandular epithelium

� 2002 Elsevier Science Ltd. All rights reserved.

Demir et al.: Differentiation of Uterine Epithelium during Early Pregnancy 673

depends on different distribution patterns of cytoplasmic andnuclear receptors for progesterone and oestrogen in theendometrium (Dockery et al., 1988; Li et al., 1991).

A number of studies which focused on structural differencesin the glandular epithelium have described the ‘post-ovulatorytriad’, namely, the presence of giant mitochondria, subnuclearglycogen, and the nuclear channel system during the secretoryphase (Spornitz, 1992). It has been reported that formation ofthe nuclear channel system is initiated during the mid- tolate-proliferative phases of the cycle and it remains until thelate secretory phase (Dockery et al., 1988, 1993).

In spite of the aforementioned studies and the alternativemodels, which suggest that progesterone, oestrogen (Spornitz,1987), and LH (Dockery et al., 1988, 1993) are responsible forthe structural transformation of glandular epithelium, theregulation of the ‘post-ovulatory triad’ during pregnancyremains unclear (Johnson and Chatterjee, 1993).

In the present study, we aimed to clarify the ultrastructuraltransformation and differentiation of human endometrialluminal and glandular epithelium, and the roles of possiblemolecular markers that might be responsible for proliferationand apoptosis of glandular epithelium during early pregnancy.

MATERIALS AND METHODS

Human decidual tissue samples were obtained after voluntarylegal termination of normal pregnancies by curettage. Tissueswere supplied from the Department of Obstetrics andGynecology, Medical Faculty, Akdeniz University, and theClinic of Obstetrics and Gynecology, Government Hospital,Antalya. Written consent was obtained from patients beforethe procedure. Consent forms and protocols to use the tissuewere approved by the Ethical Committee of Medical Faculty ofAkdeniz University.

Tissue collection and storage

Decidual tissues were derived from 14 human implantationsites from day 18 through day 41 post-conception (p.c.). Fourtissues from the early secretory phase of non-pregnantendometrium were used as control group. Six early deciduaspecimens [aged 18 to 25 days (p.c.)], and four late deciduaspecimens [aged 28 to 33 days (p.c.)], were dated as describedin our previous study (Demir et al., 1989) and by studyingembryonic developmental details following the Carnegieclassification (O’Rahilly, 1973). An additional four specimens(three, aged 38 days p.c.; one, aged 41 days p.c.) were dated byhistory. Age of pregnancy was determined from women’shistories, according to the first day of the last menstruationperiod. Human decidual currettings were either snap-frozen inliquid nitrogen, or fixed immediately in 4% paraformaldehydefor 12 h. After routine processing, samples were embedded inparaffin. Six �m paraffin sections were obtained by microtomeand were used for immunohistochemical localization of cyto-

keratin (CT), endothelial cell marker (CD31), proliferating cellnuclear antigen (PCNA), and Fas (CD95).

Immunohistochemistry

Immunohistochemical detection procedures have beendescribed previously (Arici et al., 1998, Kayisli et al., 2002),and were slightly modified for our antibodies. Briefly, thinsections were deparaffinized in xylene, and immunohisto-chemistry was performed as follows: samples were dehydratedwith 70% alcohol and distilled water. To reveal antigenmasking, sections were exposed to microwave in citric bufferedsolution (pH 6) and were incubated with proteinase K(DAKO, Carpinteria, CA, USA) for 10 min. Thereafter slideswere immersed in absolute methanol containing 3% H2O2 toblock endogenous peroxidase activity (37�C, 15 min). Afterseveral rinsing in phosphate buffered saline (PBS, pH 7.2) atroom temperature for 15 min non-specific binding sites weredecreased by normal multi-species serum (Signet, Dedham,MA, USA) at 37�C for 20 min. Sections were incubated withthe primary antibodies against cytokeratin7, endothelial cellmarker, CD31, proliferating cell nuclear antigen, PCNA andFas antigen for 1 h at room temperature. For negative controls,primary antibodies were replaced by their non-immune iso-types. After several rinsing in PBS, sections were incubatedwith biotin conjugated anti-mouse and anti-rabbit IgG (sec-ondary antibodies; DAKO) at room temperature for 15 min.Thereafter, sections were rinsed in PBS and incubated withstreptavidin-peroxidase complex (DAKO) at room tempera-ture for 15 min. Following several washing steps with PBS, theimmunostaining was developed with 3,3-diaminobenzidine(DAB) or 3-amino 9-ethylcarbizol (AEC) (DAKO) at roomtemperature for 10 min. Sections were counterstained slightlywith hematoxylene, mounted with oversleeps (Sigma,St Louis, MO, USA) and examined under light microscope.The panel of antibodies used in the study is provided inTable 1.

Ultrastructural analysis

Human decidua tissue samples were fixed by immersion in2.5% glutaraldehyde in 0.1 cacodylate buffer (pH 7.4) atroom temperature for 4 h, and post-fixed in 1% phosphate-buffered osmium tetroxide for 2 h (Demir, 1980). Specimenswere dehydrated in ascending concentrations of ethanol andwere embedded in Araldyte-epoxy resin. Semi- and ultra-thinsections were prepared by ultratome. Semi-thin sections werestained with toluidine blue. Thin sections were double stainedwith uranyl acetate and lead citrate and examined with a ZeissEM10 electron microscope.

Quantitative analysis

To compare the microvilli number on the free surfaces of clearand dark cells of glandular epithelium, 23 electronmicrographs

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were randomly selected (between 23 and 38 days p.c. ofearly pregnancy) and all of the micrographs were magnifiedidentically (�5000) to show glandular and luminal structures.

For evaluation of the microvilli and cytoplasmic hills ofluminal epithelium, transmission electron microscopy ()micrographs were standardized under the same magnification(�2500) with a Zeiss EM10. In the randomly chosen 64electronmicrographs (eight from each group) between 18 and30 days p.c., the microvilli and cytoplasmic hills of theepithelial cells were counted and described as, total numbers ofuterinal cells and their microvilli and cytoplasmic hills onapical and lateral surfaces separately. Following the quantifi-cation, the electronmicrographs were arranged according togestational days of early pregnancy respectively.

Statistical analyses

The numbers of microvilli and cytoplasmic hills of the cells ofthe luminal epithelium were not normally distributed, andthen were analysed with non-parametric analysis of variance byranks (Kruskal–Wallis). Statistical calculations were performedusing Statistical Package for Social Sciences (SPSS) forWindows, version 6.0 (SPSS, Chicago, IL, USA) and SigmaStat for Windows, version 2.0 (Jandel Scientific, SPSS,Chicago, IL, USA). Results were considered statisticallysignificant when P<0.05.

RESULTS

Immunohistochemical observations

Tissue sections of human decidua from days 18–41 p.c. ofearly pregnancy were immunostained to evaluate the distri-bution of CT, CD31, PCNA and Fas proteins. CT antibodywas used to determine luminal and glandular epithelium andinterstitial extravillous trophoblast cells (IEVT); CD31 anti-body was used to determine blood vessels; PCNA antibody wasused to determine proliferating cells, and Fas antibody wasused to determine cells that have a capability marker forapoptosis. CT was found in the glandular epithelium and wasdetected in cytoplasm of invading IEVT cells distributed inthe decidual stroma, but not within the glandular epitheliumcells, except for very rare cells situated around the sub-basalpart of the uterine glandular epithelium (Figures 1a, c). Someof intramural giant multinucleated trophoblast cells, weakly

positive with CT, were found in the vascular lumen(Figure 1a, insert). In the decidual stroma, although conven-tional stromal and decidual cells were not immunoreactive forCT, invading IEVT cells were strongly positive in all samplesand the CT immunoreactivity gradually increased as preg-nancy progressed. The vascular endothelium was definitelystained by CD31 antibody in the decidua (Figures 1b, d). Inthe decidual areas in where high trophoblastic invasion takesplace, advanced differentiation and transformation was seen inthe media layer of the vascular wall, detected by CD31antibody (Figure 1b, insert).

PCNA immunoreactivity was observed in both stromal andglandular cells (Figure 1e) but not in the luminal epithelium orin the blood vessels’ walls during early days of pregnancy.Interestingly, while some cells were positive in differentsegments of the same gland, the remnant cells were negativefor PCNA (Figure 1e). In contrast, the immunoreactivity ofPCNA in the decidual stroma decreased in late deciduasamples (38–41 days p.c.), to levels similar to those of earlydays’ glandular cells.

Fas immunoreactivity was strongly detected in the glandularepithelium, generally, but some uterine glands were immuno-negative for Fas expression (Figure 1g). Similar to the PCNAimmunostaining, we also observed immunopositive andimmunonegative stained regions in the different segments ofthe same gland (Figure 1g and lower insert). Some of the Faspositive glandular cells located adluminally showed somevacuoles and picnotic nuclei and irregular cytoplasm, while theFas negative cells had a more regular nucleus and cytoplasmenclosed within cell membranes (Figure 1g upper and lowerinserts).

Ultrastructural observations

Table 1. Panel of antibodies used for immunostaining

Antibodies Source Catalog No. Reactivity Host Isotype Dilution

Cytokeratin 7 DAKO M7018 Human Mouse Monocl-IgG1 1:300–1:500Endothelial cell marker, CD31 DAKO M0823 Human Mouse Monocl-IgG1 1:30–1:50PCNA DAKO N1529 Human Mouse Monocl-IgG2A 1:30–1:50Fas Transduction Lab. F22120 Human, rat Mouse Monocl-IgG1 1:200–1:500

Luminal epithelium. The ultrastructural appearance ofuterine epithelial cells during human early pregnancy is shownin Figure 2–4. During the early days of pregnancy, the luminalepithelial cells possess relatively loose cell membranes atthe apical and lateral walls as well as at the basal region(Figures 2a, b). Two epithelial cell types were recognizedon the luminal surface. The first type had straight cellborders (Figure 2a, b) and the second type had dark cytoplasmwith picnotic nuclei and degenerated cellular fragments(Figure 4a, b). The number of microvilli on the apical cellmembrane was decreased and shortened cytoplasmic hills were

Demir et al.: Differentiation of Uterine Epithelium during Early Pregnancy 675

Figure 1. Immunostaining of cytokeratin positive glandular epithelium, interstitial and intramural trophoblast cells (a, c) and CD31 positive vascular areas (b,d) in serial sections are seen. A weakly stained multinucleated trophoblastic giant cell is seen in the vascular lumen (insert a). Abundant cytokeratin positive cellsare located around the vessel. CD31 positive platelet cell accumulation against trophoblastic invasion is seen in vessel lumen (insert b). Many mononuclear cells,but few cytokeratin positive cells localized between the uterine glandular epithelium compared to vessel wall are seen (c and d). Strong immunopositive reactionfor PCNA is seen in the basal cells of glandular epithelium, but interestingly, some of the cells do not have immunoreactivity for PCNA (e). Negative controlstaining (f). Fas positive and negative cells of glandular epithelium (g) are seen in human decidua on day 18 p.c. of early pregnancy. Fas positive (upper insert)and negative glandular cells (lower insert) are comparable. Strong immunoreactivity for Fas was observed in the cells of glands (upper insert), reflecting apredisposition to apoptotic processes. Fas and PCNA positivity or negativity on same gestation day is detectable with opposite accordance, suggesting a balancebetween proliferative and apoptotic processes. Scale bars=100 �m.

676 Placenta (2002), Vol. 23

diminished as pregnancy progressed. Shortened microvilli andcytoplasmic hills diminished the total size of apical and lateralplasma membrane (Figures 2a, b).

From day 18 of early pregnancy on, microvilli weregradually replaced by shorter, irregular cytoplasmic hills, andthey completely disappeared by day 26 of gestation. By then,the lateral pole of the cell consists of different forms of highlyirregular microvilli (lateral protrusions), and cytoplasmic hills(Figure 3). Spaces between cells were extended by manyvacuoles of various sizes and shapes (Figures 2–4). When thenumber of microvilli and cytoplasmic hills on the luminalepithelium were compared throughout the early days ofpregnancy, we observed that the number of microvilli in theapical and lateral poles showed a significant decrease inpregnancy days compared to early secretory phase (P<0.05).The number of cytoplasmic hills in the apical pole showed asignificant increase starting from pregnancy day 23 (P<0.05),but the increase for the lateral pole started to become signifi-cant at the pregnancy day 26 when compared to early secretoryphase (P<0.05, Figures 5a, b).

Desmosome-like complexes between two adjacent cells wererecognized at the extended intercellular spaces (Figures 2, 3).The basal pole of the cells was lined by distinctive hom-ogeneous basal lamina, which was augmented by inter-digitations and showed a marked undulation (Figures 2b, 3).The euchromatic and round nuclei with distinctive nucleoliand double membranes were situated at the basal part of thecells (Figures 2, 3). In active cells, sub-cellular organelles, i.e.,mitochondria, endoplasmic reticulum, and polysomes wererecognized, but most were damaged (Figures 1, 4).

With further degeneration of luminal epithelial cells, theircytoplasm was darkened and contained many large vacuolescontaining different soluble substances (Figures 4a, b). Sub-cellular components were not recognized in these cells, whereoccasional glycogen particles were present. In some areas ofluminal degradation, the basal lamina was mixed with otherstromal elements but was still recognizable (Figure 4b).Degeneration of the basal lamina may be related to the stromaland epithelial architectural destruction due to the extensiveremodelling of decidua.

Figure 2. The luminal epithelial cells are observed to possess a relativelythinner cell membrane in lateral as well as apical and basal parts (a and b).Abundant microvilli and cytoplasmic hills (with arrowheads) on the free apicalsurface (a) and long microvilli (lateral projections) in the lateral spaces betweenneighbour cells are seen. Intercellular junctions between two luminal epithelialcells are clearly seen as desmosome-like structures (with arrows) and zonulaoccludes. Pinocytotic vesicles on the surface of the cells near the microvilli base(a), numerous vacuoles (V) with soluble contents were distributed throughoutthe cytoplasm, and rough endoplasmic reticulum cisternae (rER), and electrondense lipid droplets (L) of various sizes were dispersed at the basal parts of thecells (a and b). Distinctive euchromatic nuclei (N) and nucleoli (Nol) were alsoseen. All of the cellular components are recognized in metabolic activity.Definite homogenous basal lamina (BL) showing an undulation between thestroma (ST) and luminal epithelium were seen. On days 18 and 21 p.c. of earlypregnancy. Scale bars=4 �m.

Glandular epithelium

In the early stages of gestation, glandular epithelium consistedof columnar and pseudostratified epithelia. Cells were restingon a basal lamina and were connected to each other byintercellular interdigitations and desmosomes (Figure 6).Their height varied depending on gestational age. Two maincell types with structural diversity were seen in the glandularwall, reflecting their stages of transformation: glycogen richcells (dark cells, Figures 6, 9) and glycogen poor cells (clearcells, Figures 7, 8).

Uterine glands always had a continuous basal lamina, whichshowed a fine homogenous undulated line (Figures 6, 7, 8)related to membrane digitations at the basal pole of the cell.

Demir et al.: Differentiation of Uterine Epithelium during Early Pregnancy 677

Figure 3. High magnification of middle and basal parts of the cells of luminal epithelium are seen. Extended intercellular spaces (IS) including many longmicrovilli (lateral projections) and cytoplasmic hills of different sizes, in floating or attached forms (arrowheads), connect cells with each other. Large vacuoles linedside by side with small ones (asterisk) between cells are recognized. Distinctive nucleus (N), rough endoplasmic reticulum cisternae (rER), mitochondrion (M) andother sub-cellular components are recognized. Basal lamina (BL) showing an undulation by cellular interdigitations related directly to stromal cells (ST) was seen.On day 23 p.c. of early pregnancy. Scale bar=4 �m.

These digitations increased with the progression of gestationalage. All basal lamina undulations (i.e., invaginations anddigitations) showed connections with anchoring fibrils thatwere related to stromal elements (Figures 7, 8). Basal parts ofglandular cells appeared to be connected to the surface ofbasal lamina with some hemidesmosome-like structures thatwere adhering to an amorphous substance and forminglabyrinth-like, fine inter-digitations complexes (Figures 6–8).

Clear cells were in close proximity to the basal lamina andshowed more metabolic activity with a well-developed endo-plasmic reticulum, Golgi apparatus, mitochondria and abun-dant cytoplasm. Due to their active components and organellestructures, clear cells were presumed to be main (stem) cellsfor further proliferation, differentiation and transformation ofglandular epithelium (Figures 6–8). Some of the clear cellshave few cilia structures (Figure 8). The number of cyto-plasmic cilia decreased through the gestational progression.Other clear cells with some signs of early glycogen synthesiswere also seen and could be interpreted as ‘intermediate’between clear and dark cells (Figures 6, 8, 9). They havemoderate amounts of glycogen particles and active roughendoplasmic reticulum (rER) cisternae, but have fewer poly-some accumulations and do not have a dense dark cytoplasm.

Details of the organelles of the dark epithelium cells weredifficult to identify in gland units. Myelin figures, withdifferent configurations and contents, were seen both in darkcells and in intermediate cells (Figures 9, 10). Dark cellscontained a small number of microvilli on their luminalsurface. Irregular, hill-shaped short projections of cytoplasmwere also seen, representing glycogen discharge (Figure 10).

All the glandular cells exhibited abundant microvilli onsurfaces, which were adjacent to lateral spaces near the glandlumen. Clear cells and some dark cells that did not exhibitglycogen accumulation had regular shapes and diameters, andresembled cells of absorptive tissues such as jejunum andkidney tubules (Figure 8 and insert). In dark cells, mostmicrovilli were short and swollen or twisted, and their numberdecreased (Figures 9, 10). The microvilli number in clear anddark cells decreased as days of pregnancy advanced (Figure5a). The number of microvilli in clear cells was higher than thenumber in dark cells (Table 2, P<0.05). However, the numberof microvilli was not statistically different for given typesamong pregnancy days studied.

The nuclear structure of glandular cells was often elliptical,regular, euchromatic, and finely granular containing electrondense nucleoli. Nuclear membranes with a double layer

678 Placenta (2002), Vol. 23

Figure 4. Intercellular spaces formed by large vacuole (V) lined side by side(1, 2, 3, 4 . . .) between two cells. Intercellular junctions (arrows) are clearlyseen between cells (a). In a parallel section of the basal part, degeneratingluminal epithelial cells have large vacuoles (V) formed near basal lamina (BL),which shows a thickness and undulation, and in some areas the basal lamina isfragmented and mixed with stromal (ST) elements. Many microvilli andcytoplasmic hills (arrowheads) are seen in intercellular spaces. Picnotic nuclei(PN) and dark condensed cytoplasm with unrecognized cell organelles areobserved (b). On days 26 and 30 p.c. of early pregnancy. Scale bars=4 �m.

Figure 5. The number of microvilli in apical and lateral poles of endometrialluminal epithelium in early secretory phase (ESP) and pregnancy days between18–30 p.c. The number of microvilli in the apical and lateral poles showed asignificant decrease throughout the pregnancy days compared to early secre-tory phase (P<0.05) (a). The number of cytoplasmic hill in apical and lateralpoles of endometrial luminal epithelium in early secretory phase (ESP) andpregnancy days 18–30 p.c. The number of cytoplasmic hills in the apical polesshowed a significant increase starting at pregnancy day 23 (D23) (P<0.05), butthe increase for the lateral wall did not reach significance until pregnancy day26 (D26) when compared to early secretory phase (P<0.05) (b). Median, 25thand 75th percentiles are shown for each group. Error bars represent 10th and90th percentiles.

structure were evident in some pores, while the nuclearchannel system was not detectable. Some cell nuclei showed asmall number of deep gaps (Figures 6–8 and insert).

In early stages of pregnancy, the rER of glandular epi-thelium was mostly cisternae and was distributed rather evenly(Figure 9). The members of free and attached ribosomes,polysomes (to rER cisternae) were increased, particularly inthe supranuclear regions of dark cells as compared to those ofclear cells. Ribosomes were distributed to incorporate intopolysomes, which are important in synthesizing glycogen priorto its accumulation in the cytoplasm (Figures 6, 8). Mitochon-dria were numerous, usually oval or rod-like shape with lamella

Demir et al.: Differentiation of Uterine Epithelium during Early Pregnancy 679

cristae that occasionally did not appear. Large mitochondriaswollen to giant sizes were not seen during these early periodsof gestation (Figures 6, 7). The Golgi system was found inclear and intermediate cells, and appeared almost exclusivelyin the basolateral and perinuclear area of the cells in a moreenlarged form (Figures 6, 8). Lysosomal elements weredistributed in the cytoplasm of dark cells as a result ofautophagosis.

Glycogen formation and discharge by dark cells

The electron density of the glandular cells varied according toamount of glycogen accumulation. Glycogen-rich cells showedan advanced progression of glycogen formation and their apicalregions were filled almost entirely with glycogen accumulations(Figures 9, 10). Large quantities of glycogen discharge fromthe apical pole of epithelium into the luminal area wereobserved during early pregnancy. The glandular lumen wasfilled with a mixed substance that consisted of large lipiddroplets, glycogen particles, and glandular mucus. The firststep was an accumulation of apical glycogen, which causesthe apical cell membrane to bulge into the glandular lumen(Figure 10). These bulging areas did not contain any

sub-cellular elements. In these areas, microvilli were frag-mented and were broken off, so these cytoplasmic elementswere released from these potentially fragile membranesinto the gland lumen by an apocrine secretion for glycogendischarge.

Figure 6. A topographic view of the glandular epithelium containing the main two cell types (clear cell=CC and dark cell=DC) is seen in the glandular wallwith structural diversity reflecting the stages of transformation. Many microvilli (Mv) are seen on the apical surface. Euchromatic nuclei (N) with distinctivenucleoli, subcellular components and glycogen particles (gp) are seen. Human decidua, on day 21 p.c. of early pregnancy. Scale bar=4 �m.

DISCUSSION

The human uterine luminal and glandular epithelium have anextensive dynamism around the implantation process. Luminalepithelial cells show some atypical transformation processes viaregression (Murphy, 1995). The structural alterations ofplasma membrane are extensively described in endometrialluminal epithelium of different animals (Niklaus et al., 2001;Png and Murphy, 1997). Luminal and glandular epithelial cellslose their apical microvilli. Also a move in the lateral directionof the cell membrane and alterations in the luminal epithelialplasma membrane could occur as ‘plasma membrane transfor-mation’ is suggested to be the main factor for receptivity(Murphy, 1995, 1998). At the apical surface, the number ofmicrovilli decreases and blunt cytoplasmic prominencesincrease. On the contrary, the number of microvilli at thelateral surface of the plasma membrane increases, intracellular

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Figure 7. Typical clear cells (CC) of the glandular epithelium are seen in the glandular wall with structural diversities. Cells are in close proximity with eachother and with the basal lamina (BL), connecting by fine interdigitations (asterisk). Elliptical euchromatic nucleus (N) with distinctive fine chromatin and cellorganelles is seen. Basal lamina (BL) shows an undulation condition against to stromal elements (ST). In some areas the basal lamina is mixed with the stromalmatrix. E: Capillary lumen with erythrocyte. Human decidua, on day 23 p.c. of early pregnancy. Scale bar=4 �m.

distance expands due to vacuolization, connection complexesdecrease, and tight connections between two neighbour cellsdisappear. The lateral side of the plasma membrane compen-sates this decrease by leading to a formation of loose architec-ture. This dynamism of the plasma membrane corresponds tothe expression of ‘plasma membrane transformation’, which isindicated in some rodents during pre-implantation phase (Pngand Murphy 1997; Murphy, 2000). Results of our study are inaccordance with these findings. On the other hand, ourfindings suggest that the cytoplasmic projection on the freesurface of the cell might be called ‘cytoplasmic scallops’, whichare related to luminal surface interactions with apocrinesecretions of these cells. Results of this study suggest thatcellular protection and resistance against destruction ordegeneration occur in two different epithelial cell types in theluminal surface of the endometrium. Ultrastructural findingsof the luminal epithelium of the pregnant uteri suggest alimited resistance to trophoblastic invasion. Our study hassome limitations due to the lack of stereological methodsdescribed by Mayhew (1991; 1996) and should be used insubsequent studies.

The present study indicates that the basal plasma mem-brane, together with the basal lamina, is highly folded. Thebasal lamina of early pregnancy day samples (18 and 30 daysp.c.) had increased undulation and thickness, running parallelto the basal plasma membrane of the luminal epithelial cells.The undulations and thickness of the basal lamina increasedduring the progressing pregnancy days. Structural changesoccurring in the basal lamina under the influence of cyclichormones might affect the expression phenotype of luminalepithelium (Dockery et al., 1998). It has been suggested thatoestrogen alone could induce the undulation of basal plasma

membrane and could affect the basal lamina thickness in rat(Shion and Murphy, 1995). On the other hand, progesterone isresponsible for most of the increase in basal lamina folding andthickness, as well as the loss of connection complexes betweenthe cells (Pakarinen et al., 1998). The increased undulation ofthe basal plasma membrane, together with the thickness ofbasal lamina, is probably an indicator for luminal epithelial celltransformation, in response to increased gestational hormonesand tissue remodeling, which occurs during early pregnancy.The molecular function of the basal lamina is not only a simplefiltering function between the stroma and luminal epithelium,but it involves other important roles during implantation(Dockery et al., 1998). According to our findings, increasedthickness and undulation of basal lamina should influence thecell metabolism responsible for the molecular transport tothe stroma, and should play an important role during thepenetration of the blastocyst.

Adashi and Akashi (1981) have reported that ciliogenesiscould not be recognized in the glandular epithelium. Ourobservations on ciliated cells around the glandular openings arein accordance with previous reports (Spornitz, 1992), but arenot in accordance with opinions of Adashi and Akashi (1981).No clear concept exists regarding their function, but wepostulate that cilia may play a role in removing productsdischarged by the cells.

Recently, immunoreactivity for PCNA in endometrialglandular and stromal cells was described throughout themenstrual cycle (Yamanaka et al., 1996; Takai et al., 2000).Our immunohistochemical findings suggest that during earlypregnancy while the glandular epithelium proliferate anddifferentiate, there is also some evidence for apoptosis indifferent parts of the same glandular segments, which is likely

Demir et al.: Differentiation of Uterine Epithelium during Early Pregnancy 681

Figure 8. Ciliated clear cells (CC) attached to each other by connected complexes, and to the basal lamina (BL) by basal interdigitations (asterisk) are seen. Ciliarystructures (CS, upper insert) are clearly evident on the free surface of the luminal epithelium. Many microvilli (Mv) on the luminal apical surface of the cells (lowerinsert) are observed, resembling kidney tubules or jejunum. Rough endoplasmic reticulum cisternae (rER), Golgi complex (G), mitochondria (M) and glycogenparticles (gp), euchromatic nucleus (N) and stromal elements (ST) are observed. Human decidua, on day 25 p.c. of early pregnancy. Scale bars=4 �m.

to be related to the expression pre-apoptotic molecules such asFas. This is interesting since the glandular dynamism requiresa resistance to apoptosis to protect its structure and continueits function. Alterations in cell content, and disappearance ofsome ultrastructural findings of sub-cellular components, asevident by ultrastructural observations, support this prolifer-ation and transformation hypothesis. Our results suggest thatglandular epithelium may continue its presence by a balancebetween proliferation and apoptosis affected by PCNA and Fasproteins.

We do not share the opinion that the nuclei leave their basalposition and move towards the centre of the cell, and that thenuclear membrane begins to show undulations and mitoticfigures (Verma, 1983; Spornitz, 1992). Following our obser-vations by , we conclude that nuclei of dark cells are denserthan these of clear cells, and dark cells have a distinctnucleolus. The nuclear channel system (Dallenbach-Hellweg,1987), which is embedded in matrix, if present, may play a role

in the exchange of proteins between nucleoli and cytoplasm forenzyme synthesis.

Coaker et al. (1982) have reported that the giant mito-chondria can be exceptionally long and branched swellings ofnormal mitochondria. When giant mitochondria appear, thetotal number of mitochondria decreases. Spornitz (1992) hasnot accepted this opinion and has indicated that the giantmitochondria are an important postovulatory characteristic ofthe glandular epithelium. Many complex mitochondria can befound during the secretory phase of the cycle, and in womenwith unexplained infertility treated with LH hormone(Dockery et al., 1993). According to our observations, mito-chondria are numerous in clear glandular cells but theirnumber decreased in dark cells. In dark cells we have neverobserved giant mitochondria during the period between 18–41days of early pregnancy. These findings confirm other reportssuggesting that transformation of glandular epithelium dimin-ishes as pregnancy progress (Verma, 1983; Gordon, 1985). In

682 Placenta (2002), Vol. 23

Figure 9. A typical dark cell (DC) with glycogen particle accumulations and myelin figures (MF) is seen between intermediate cells (IM). The dark cell isconnected to the intermediate cell by desmosomes and cytoplasmic interdigitations (arrows). The dark cell has a condensed cytoplasm and details of sub-cellularcomponents are not recognized. Intermediate cells (IM) reflecting glycogen synthesis with abundant microvilli (Mv) on the free surface, and with abundantmitochondria (M), are attached to each other by desmosomes and interdigitations on their lateral sides. Euchromatic nuclei (N) and nucleoli (Nol). Human decidua,day 28 p.c. of pregnancy. Scale bar=4 �m.

regard to the potential role of giant mitochondria in glandularepithelium transformation, it is conceivable that it may beinvolved in the production of uterine milk and other sub-stances (Demir and Akkoyunlu, 1999). One can speculate thatthe disappearing giant mitochondria transform and fuse intoautophagic vacuoles or are divided into smaller mitochondria.This is based on the fact that numerous small mitochondria areobserved in clear cells, and most autophagic lysosomes appearas myelin figures in glycogen-rich glandular cells.

Many small round granules are seen on rER during thepreovulatory phase of the cycle, but they become larger, withparallel cisternae during the course of the cycle towards thelate secretory phase (Verma, 1983). Our findings have indi-cated that ribosomes are incorporated into newly formedglycogen, which subsequently detaches from the rER, thusforming smooth endoplasmic reticulum (sER). We found thatrER of the glandular epithelium does not exhibit vesicles ineither cell type, and becomes cisternae as the cell maturationadvances. We have also recently observed (Demir andAkkoyunlu, 1999) the relationship between rER cisternae andglycogen synthesis and accumulation. These processes also in-volve the secretion of a variety of degradative glycogen particlesand the discharge of these particles into lumen. In contrast to

some investigators (Verma, 1983; Cornillie et al., 1985), webelieve that this synthetic processes does not occur in thesubnuclear region, but in the supra and supra-lateral regions ofthe nuclei. The amount of glycogen accumulation in the apicalcytoplasm is different due to the cellular transformation andglycogen discharge process. In our findings, an apocrine type ofsecretion is recognized for several days during early pregnancy, asecretion that appears to occur at irregular intervals.

Verma (1983) has stated that the Golgi system is more elab-orate in the late proliferate phase and is found almost exclusivelyat the apical supranuclear part of the glandular epithelial cells. Inour study, the Golgi system was readily seen in epithelial cells ofearly pregnancy. There were very few Golgi apparati in clearcells of the glandular epithelium. As cell transformationadvanced, it was strictly distinguishable in both types of cells.

The existence of large numbers of lipid droplets in theglycogen rich cells in both postovulatory phases of the cycle(Cornillie et al., 1985) and in early pregnancy confirms thehypothesis that these are related to steroid hormone produc-tion in the human placenta (Demir, 1980). The observationthat the lipid droplets and glycogen particles are in closerelation with rER cisternae suggests a transitory connection orcorrelation with the steroid biosynthesis.

Demir et al.: Differentiation of Uterine Epithelium during Early Pregnancy 683

Figure 10. Dark cells of the glandular epithelium show an advanced progression of glycogen accumulation and bulged apical region, quenched almost entirelywith glycogen masses and with contents of luminal substances. A relationship between glycogen formation and rough endoplasmic reticulum cisternae (rER) isobserved. Glandular lumen (Lu) is filled with mixed substances. Many microvilli (Mv) in the lateral lumen are mixed with dense substances. Glycogen isdischarged from the glandular epithelial cell by budding-off (double arrows) and by rupture of apical glycogen filled areas of the membrane. Mitochondria (M),myelin figures (MF). Human decidua, day 38 p.c. of pregnancy. Scale bar=4 �m.

Table 2. Distribution and numbers of microvilli on clear and dark cells of glandular epithelium according to days(23–38) p.c. of early pregnancy. Differences between the number of microvilli on clear and dark cells were statisticallysignificant for all days studied (P<0.05). Number of microvilli was not statistically different for given cell type among

pregnancy days studied

Pregnancy day Cell type Number of cells Number of microvilli Mean number of microvilli

Day 23 Clear cells 31 2463 79.45Dark cells 28 1045 37.32

Day 25 Clear cells 8 608 76Dark cells 9 324 36

Day 28 Clear cells 14 1090 77.81Dark cells 12 441 36.75

Day 38 Clear cells 6 432 72Dark cells 4 136 34

6 electromicrographs were evaluated for each pregnancy day (total 24).Clear cells; total cell number=59; total microbvilli number=4593.Dark cells, total cell number=53; total microvilli number=1946.Microvilli; general total number=6539.

The basal lamina (BL) of the glandular epithelium iscomposed of an amorphous layer. In some cases, whereglandular cells are located close to the surface at the basal pole,

the BL is continuous with the BL of the stromal cells,especially decidual cells. The thickness of the BL underliningthe glandular cells is variable according to the gestational age

684 Placenta (2002), Vol. 23

and the presence or absence of extracellular matrix com-ponents (Iwahashi et al., 1996). We speculate that glandularepithelium and its BL with the high degree of undulationrepresent a cellular defence against the trophoblastic invasion.The permeability function between the stroma and glandularepithelium disappears in the endometrium during placenta-tion. The increase in the thickness of BL in glandularepithelium with advancing gestational age is a functionalmodification of endometrial resistance in both normal andpseudo pregnancies.

In conclusion, according to the ultrastructural andimmunohistochemical features, the luminal epithelium andglandular epithelium extended the protection processes. Theendometrial glandular epithelium can be described with severalfunctions; (i) limiting the trophoblastic invasion by structuraldifferentiation and by a balance between proliferation andapoptosis, (ii) glycogen synthesis, deposition and apocrinesecretion, (iii) to participate in the production and formation ofuterine milk contents, which are important for the embryonicdevelopment during early days of gestation, and (iv) transformeach other and establish contact between the implantingtrophoblast and decidua via basal interdigitations.

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