cytokeratin 8, 18 and 19 in endometrial epithelium of norplant® and

Cytokeratin 8, 18 and 19 in endometrial epithelium ofNorplant® and norethisterone enanthate injectable

progestogen contraceptive users

Sugito Wonodirekso1, Wachju Hadisaputra2, Biran Affandi2, BudiningsihSiregar3 and Peter A.W.Rogers4'5

Departments of 'Histology, Obstetrics and Gynaecology and 3Anatomic Pathology, Faculty of Medicine,University of Indonesia, Jakarta, Indonesia and 4Department of Obstetrics and Gynaecology, Monash University,

Melbourne, Australia5To whom correspondence should be addressed

Cytokeratins 8, 18 and 19 are members of thecytoskeletal intermediate filament family foundin all simple epithelia. Intermediate filamentsare dynamic intracytoplasmic structures thatcan be influenced by a number of externalfactors. Norethisterone enanthate (NET-EN) isa long-acting progestogen contraceptive that hasbeen found to arrest endometrial growth in therat Both Norplant® and NET-EN cause bleedingproblems among users which are responsiblefor >50% of withdrawals with these methods.The aim of this study was to explore changesin the expression and distribution of cytoker-atins 8, 18 and 19 in NET-EN- and Norplant-exposed endometrial epithelium which could berelated to bleeding disturbances. Seven NET-EN and 37 Norplant endometrial biopsies wereparaffin-embedded and stained immunohisto-chemically to evaluate cytokeratin expressionand distribution. The results showed thatwomen who had received NET-EN for 3-4months had a cytokeratin distribution similarto that seen in the normal menstrual cycle. Thisis in contrast to endometrium from Norplantusers in which cytokeratin expression wasreduced and the epithelial cells were more roun-ded. No relationship between cytokeratinexpression and breakthrough bleeding patternwas found. NET-EN and Norplant may actdifferently on endometrial epithelial cyto-keratin.

Key words: cytokeratin/endometrium/epithelium/norethisterone enanthate/Norplant

Introduction

Endometrial epithelium contains cytokeratins 8,18 and 19 which are cytoskeletal intermediatefilaments of ~10 nm in diameter (Quinlan et al.,1985; Nagle, 1989). Although the function ofintermediate filaments is not yet fully understood,a number of roles have been postulated. In normalepithelial cells, intermediate filaments are majorconstituents of the cytoskeletal network and aremeshed around the nucleus, with some bundles ofintermediate filaments attached to desmosomesand hemidesmosomes via a panoply of putativeintermediate filament-associated proteins (Greenand Jones, 1990). Their interactions, direct orindirect, with the plasma membrane establish astructural link between the nucleus and the cellsurface which has important implications for theorganization of the cytoplasm and cellular com-munication (Goldman et al, 1985, 1990). Cyto-skeletal elements are dynamic intracytoplasmicstructures that can be affected by external factors(Ouhayoun et al, 1990; Gijbels et al, 1992). Ithas also been hypothesized that the intermediatefilament reorganization observed in cells duringpathological situations may be linked, at least inpart, to the control of growth, either by affectingit or as a consequence of growth alteration (Skalliand Gabbiani, 1990). Other investigators havesuggested that loss of cytokeratin reduces mechan-ical rigidity, thus decreasing mechanical strengthwhich may result in reduced basal and lateral celladhesion (Green and Jones, 1990; Tropey et al,1992). To date, little information has been pub-lished about intermediate filaments in human endo-

144 O European Society for Human Reproduction & Embryology Human Reproduction Volume 11 Supplement 2 1996

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Cytokeratin 8, 18 and 19 in endometrial epithelium

metrial tissue in Norplant® and norethisteroneenanthate (NET-EN) acceptors.

It has been estimated that for the injectableprogestogen-only contraceptive NET-EN, after 1year bleeding problems are the basis for ~50% ormore of all discontinuations of this method (Odlinand Fraser, 1990). Despite recent increased interestin this problem, there is still no basic explanationfor the bleeding mechanisms or consensus on atherapeutic approach for treatment (Odlin andFraser, 1990).

NET-EN is widely used throughout the worldand is administered i.m. (Howard et al, 1982,1986; Joshi et al., 1988). It has minimal effect onthe blood coagulation system (Howard et al., 1982).Chemically, NET-EN is a C-18 steroid belongingto the 19-nortestosterone group (17-ethinyl-17P-heptanoyloxy-4-estrene-one) and is dispensed inan oily solvent mixture of benzyl benzoate andcastor oil (Topzada, 1983). NET-EN has beenshown to arrest endometrial growth in the rat(Bhowmik and Mukherjea, 1988).

Several histological changes have been observedin endometrium of women receiving long-actingprogestogens. It has been reported that womenreceiving levonorgestrel subdermal implants(Norplant) show unexpected and significantlyincreased immunostaining for nuclear progesteronereceptors (Critchley et al., 1993). Other investig-ators have observed an increase in vascular density,changes in Factor VlH-related antigen (Rogerset al., 1993; Au et al, 1994) and decreasedangiogenic activity (Subakir et al, 1995). Theaims of this study were to observe changes inthe expression of cytokeratins 8, 18 and 19 inendometrial epithelial cells from women receivingNET-EN and to compare them with the patternsof expression seen in Norplant users, as reportedpreviously (Wonodirekso et al., 1993).

Materials and methods

Endometrial biopsy collection

Ethical approval for this study was given by theEthical Commission on Research in Humans ofthe Faculty of Medicine, University of Indonesia,Jakarta, Indonesia.

The inclusion criteria were: (i) the absence of

an identifiable endometrial or uterine pathology;(ii) no haemostatic or coagulatory disordersreported; (iii) no hormonal or other therapyreceived for bleeding problems in the 3 monthsprior to or during biopsy; (iv) haemoglobin concen-trations >10 g/100 ml; and (v) a daily menstrualdiary for 90 days (the reference period) prior toendometrial biopsy.

The NET-EN subjects were recruited from RadenSaleh Clinic, Jakarta, Indonesia and had a mean± SEM age of 32.6 ± 1.2 years. They had allreceived two NET-EN injections (150 mg NET-EN) spaced 8 weeks apart, and were 6-8 weekspost the second injection. Endometrial biopsieswere taken using a Pipelle suction curette(Prodimed, 60530; Pipelle, Neuilly-en-Thele,France) or by microhysteroscopy.

Menstrual diary card analysis

Based on the daily menstrual diary, bleeding pat-terns were classified according to World HealthOrganization (Rogers et al., 1993) definitions asfollows: amenorrhoea (no bleeding/spotting daysduring the reference period); normal/regular bleed-ing (two to four bleeding/spotting episodes perreference period; no bleeding/spotting episodelasting 5s 10 days; range of lengths of bleeding/spotting-free intervals «£17 days); irregular bleed-ing (range of length of bleeding/spotting-free inter-vals >17 days over the reference period);infrequent bleeding (less than two bleeding/spot-ting episodes within the reference period); frequentbleeding (more than four bleeding/spotting epis-odes within the reference period); and prolongedbleeding (one or more bleeding/spotting episodeslasting 5*10 days) (Wonodirekso et al, 1993).

Tissue processing

Biopsies were fixed in phosphate-buffered formalin[phosphate buffer, pH 7.4, stock solution (Riedel-de Haen, Berlin, Germany) and 37% formalinstock solution (Riedel-de Haen)]. Biopsies werethen embedded in paraffin and 5 \im sections cut.Some of the sections were stained routinely usinghaematoxylin and eosin for histological evaluationby a pathologist. The dating was simply classifiedas early or late proliferative-like, early or latesecretory-like, premenstrual-like, atrophic or

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Table L Histopathology, cytokeratins 8, 18 and 19 immunohistochemical staining and bleeding patterns of subjects receivingnorethisterone enanthate contraception

No. Histological evaluation Staining intensity Staining distribution Bleeding pattern

1234567

Early secretory-likeEarly proliferative-likeLate proliferative-likeUnclassifiedEarly proliferative-likeUnclassifiedLate secretory-like

4344344

ApicalApicalBasal-apicalBasal-apicalBasal-apicalApicalApical

InfrequentFrequentProlongedFrequentProlongedInfrequentProlonged

'unclassified' (when the sample was inadequate).These classifications were based on the similaritiesbetween subject endometrium from our studyand the normal menstrual cycle. However, it isimportant to note that other histological findingswere also recorded, such as stromal haemorrhageor tissue breakdown in the endometrium with aproliferative-like appearance.

Immunohistochemical staining

The procedure followed for immunohistochemistrywas similar to that published previously(Wonodirekso et al., 1993). Sections were deparaf-finized in xylol (E.Merck, AG, Darmstadt,Germany), followed by rehydration using gradedalcohols through to deionized water. Sections werethen washed in 0.1 M phosphate-buffered saline(PBS; pH 7.6), and endogenous peroxidase activitywas quenched by treatment with 3% H2O2

(BioGenex, San Ramon, C.A., USA) for 10 minat room temperature. After washing with PBS,tissue antigen was unmasked with 1% trypsin for15 min at 37°C, followed by a PBS wash. Primaryantibody to cytokeratins 8, 18 and 19 (monoclonalantibody 5D3; BioGenex) was applied undiluted,direct from the kit, for 15 min at 37°C to bothNET-EN sections and positive controls. For nega-tive controls, serum was also applied direct fromthe kit for 5 min at 37°C. Antibody binding wasvisualized using chromogen (AEC; BioGenex),consisting of chromogen and substrate. After thor-ough rinsing with deionized water, mountingmedium (Entelan; Sigma Chemical Company,USA) was applied, without counterstaining, and acoverslip was applied to each section.

Evaluation of staining results

Results were evaluated semi-quantitatively basedon staining intensity as follows: 1 = weak staining,2 = moderate staining, 3 = strong staining, and4 = intense staining. Cytokeratin distribution wasalso observed and classified as: perinuclear cluster(cytokeratin packed around the nucleus), basal(cytokeratin distributed mainly in the basal regionof the epithelial cells), apical (cytokeratin distrib-uted mainly at the apical region of the epithelialcells) and basal-apical (cytokeratin distributedequally in both apical and basal regions). Resultswere compared with those from Norplant usersfrom an earlier study (Wonodirekso et al, 1993).

Results

Staining results are summarized in Table I. All ofthe proliferative and secretory endometria showedstromal haemorrhage, and one of the secretorybiopsies also had tissue damage. As shown inTable I, none of the subjects were amenorrhoeic,and there was no relationship between bleedingpatterns and histopathological findings. Regardlessof bleeding pattern or histopathological finding,epithelial tissues stained either strongly or intenselyfor cytokeratin, including isolated epithelial frag-ments from unclassified biopsies.

Neither surface or glandular epithelia showedany marked changes in cell shape. They consistedof a single layer of high columnar cells, with noobvious differences between proliferative-like andsecretory-like endometria (Figures 1 and 2). Incontrast, surface epithelial tissue from Norplantsubjects showed weaker immunostaining, and epi-

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Cytokeratin 8, 18 and 19 in endometrial epithelium

Figure 1. Immunostaining of early proliferative endometnaltissue from a norethisterone enanthate user. Note the intensestaining and basal-apical distribution of cytokeratin.Epithelial cells are columnar rather than cuboidal or rounded.

Figure 2. Immunostaining of late secretu:, .;irialtissue from a norethisterone enanthate user. Note the strong/intense staining anH distribution of cvtokeratin.

Figure 3. Immunostaining of Norplant tissue. This sectioncontains only surface epithelium; no glandular elements arepresent The surface epithelial cells are rounded and appearstratified. Cytokeratin is distributed mainly around the nuclei,arid overall staining is weaker than for norethisteroneenanthate subjects.

thelial cells were rounded and stratified (Figure 3).The two secretory-like NET-EN endometriashowed an apical cytokeratin distribution, two ofthe proliferative-like endometria showed a basal-apical distribution and one of the proliferative-likeendometria showed an apical distribution. The

other two samples which were unclassified showeda basal-apical and an apical distribution.

Discussion

Preliminary results from this study of cytokeratinexpression in endometrial biopsies from sevenwomen who had received NET-EN for between 3and 4 months prior to the study demonstrate thatthe expression of this intermediate filament isfar more typical of that seen during the normalmenstrual cycle, rather than that seen after 3-12months exposure to levonorgestrel (Wonodireksoetal, 1993). Despite the fact that the endometriumfrom the women receiving NET-EN was quitevaried in its histopathological appearance, the epi-thelial staining for cytokeratin was always strong.In most cases, the cellular localization of cytoker-atin was apical, although in some instances basalportions of the cell also stained strongly. There wasno relationship between either menstrual bleedingpattern or histology and cytokeratin staining intens-ity and distribution. Most epithelial cells werecolumnar in shape, and there was little evidencefor a regional variability within each endometrialbiopsy.

These preliminary results with NET-EN contrastsignificantly with our previous study of cytokeratinin the endometrium of women who had receivedNorplant for between 3 and 12 months. In thelatter group, cytokeratin expression was reducedsignificantly and the epithelial cell appearedcuboidal or even rounded in some instances. It ispossible that these dissimilarities arc caused by thedifferent lengths of exposure to progestin in thetwo groups. Clearly, endometrial biopsies fromwomen who have received NET-EN for >3—4months will be required to answer this question.

It has been hypothesized that a reduction inepithelial cell cytokeratin content could lead to aloss of mechanical integrity and epithelial celladhesive properties (Green and Jones, 1990). Suchalterations in epithelial integrity could causeincreased vulnerability of the endometrium to dam-age following physical trauma such as uterinecontractions, thus resulting in vascular damage andbleeding. However, to date, it has not been possibleto find any relationship between menstrual bleedingpatterns and cytokeratin staining in progestin-only

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contraceptive users (Wonodirekso et al., 1993).Despite this, the integrity of the surface epitheliumand glands in the endometrium could easily bean important factor in controlling the escape ofextravasated blood from the endometrial tissuesinto the uterine lumen, and subsequently outthrough the cervix. Thus, the light spotting andbleeding that is characteristic of progestin-onlycontraceptive breakthrough bleeding may well becontrolled if epithelial integrity is maintained.Minor vascular ruptures in the subepithelial regionwill more rapidly cease bleeding if the escapedblood is trapped within the endometrial tissues,rather that having an avenue of escape into theuterine lumen. The increased intratissue pressurecaused by the haematoma will help to stop bloodflow quite rapidly, as long as the epithelium is notbreached. Whether or not the integrity of theepidielium plays a role in stopping blood flowin this manner will require further investigation.However, it has been reported from hysteroscopicstudies that subepithelial haemorrhage is quitecommon in the endometrium of women on proges-tin-only contraception, regardless of whether ornot menstrual bleeding was reported just prior tohysteroscopy.

In interpreting the results of this study, it isimportant to note the small sample size and diefact diat die primary antibody against cytokeratins8, 18 and 19 used in this study (5D3; BioGenex)was different from that used in our earlierstudy (NCL-D53; Novocastra, Newcastle, UK)(Wonodirekso et al., 1993). Despite this, the obser-vations from NET-EN users reported here suggestthat after 2-3 months of exposure the epitheliumis not altered to the same degree as it is in Norplantusers. If diis observation is confirmed in longerterm users of NET-EN, it may be an importantdifference that could help to resolve the issue ofthe role of epithelial integrity in reducing theincidence of progestin-only endometrial break-through bleeding.

Future research

Further studies are planned to increased subjectnumbers and also to look at the longer termeffects of NET-EN on endometrial cytokeratin. Thepotential role of epithelial integrity in preventing

breakthrough bleeding may provide a new avenueof investigation into the treatment of diis problem.

Conclusions

The results from this study have shown that womenwho receive NET-EN for 3-4 months have anepithelial cytokeratin distribution more similar tothat seen during the normal menstrual cycle. Thisis in contrast to the endometrium from Norplantusers, in which cytokeratin expression is reducedand epithelial cells are more rounded. No relation-ship between cytokeratin expression and the break-through bleeding pattern was found. NET-EN andNorplant may act differently on endometrial epithe-lial cytokeratin.

AcknowledgementsThanks are due to Nurse Dra Rusminah for patientcoordination at Raden Saleh Clinic. This research wassupported by World Health Organization Research andTraining Grant no. 92154 to S.W.

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