Eutopic Endometrium From Women With Endometriosis Shows Altered Ultrastructure and Glycosylation Compared to That From Healthy Controls—A Pilot Observational Study

Endometriosis is defined as the extrauterine growth of endometrial glandular epithelial and stromal cells, generally found over pelvic peritoneal and visceral surfaces. It affects about 10% of women in the reproductive age group, 1 increasing to 30% in patients with infertility and up to 45% in patients with chronic pelvic pain, 2 and is one of the most common causes of infertility and chronic pelvic pain. Although existence of this disease has been known for over a century, our current knowledge of its pathogenesis, the pathophysiology of related infertility, and its spontaneous appearance is limited. Experimental models of endometriosis have been developed in rodents 3 , 4 and in the nonhuman primate 5 , 6 to overcome the ethical and practical issues limiting research in humans. Studies using a baboon model have elegantly demonstrated that intrapelvic injection of endometrial curetting caused pelvic endometriosis and decreased pregnancy rates. 7 , 8 The baboon model has also been used to investigate the aberrant expression of several genes in endometrial tissue. 9 – 11 More recently, we have used the primate model of induced endometriosis to examine the ultrastructure of eutopic and ectopic endometrium by light and electron microscopy as well as the expression of glycans by lectin histochemistry. The results indicated that endometriotic tissue is characterized by significant changes both in the gland architecture and in glycosylation compared to normal endometrium, 12 with the premature appearance of features normally associated with late stages of the cycle. Nevertheless, there remains a lack of knowledge of the ultrastructural and biochemical changes in endometrial tissues in humans. In the current pilot study, we extend our observations to biopsies obtained from a carefully selected group of women with visually and histologically proven endometriosis and controls without the disease, examining both the ultrastructure of the glandular tissue and the expression of glycans bound by Dolichos biflorus agglutinin 13 (DBA). In this way, we hope to assess whether accelerated maturation of gland architecture and biochemistry, which was described in the baboon model, is also a feature in the human. Indeed, the examination of this type of epithelial glycosylation offers a sensitive and unique assessment of normal tissue cycling and changes occasioned by pathology. It is hoped that inspection of tissue from this cohort of women may give some insight into the etiology of endometriosis by means of establishing whether there are any indications of the derivations of the various lesions.

The ultrastructure of normal glandular epithelium has been fully described in the literature 20 – 24 and so only a brief summary, restricted to main features, of the changes observed from day 9 onward through the menstrual cycle will be described here ( Figure 1 ) and in Table 1 . At 9 days, nuclei were basally situated and euchromatic with a smooth outline; several mitoses were visible (*, Figure 1A ). Small or elongated mitochondria and vertically orientated Golgi with narrow saccules and inconspicuous cisternae of endoplasmic reticulum were seen; lateral membranes were generally straight. Small aggregates of glycogen were beginning to form basally (G, Figure 1A ). Some secretory droplets and heterophagosomes 21 were present. By day 14, there was generally more subnuclear glycogen though mitoses were still evident in 1 specimen. Basal deposits of glycogen were seen in day 17 specimens (Gly, Figure 1B ), which had polarized cells, long parallel supranuclear cisternae of endoplasmic reticulum, and Golgi saccules. Some nuclei contained nucleolar channel systems ( Figure 1C ) typical of days 17 to 20, whereas giant mitochondria were sometimes present basally ( Figure 1D ). Desmosomes were well developed on lateral membranes, which were more tortuous. Large cytoplasmic projections (pinopods) generally devoid of organelles and characteristic of days 19 to 22 were seen ( Figure 1E ). By day 24, many of the nuclei were heterochromatic and glycogen was scattered throughout the cell ( Figure 1F ). Mitochondria were mainly basal and lateral membranes convoluted. Some Golgi saccules and secretory droplets but few endoplasmic reticulum cisternae were present. Later, cells became more cuboidal with basal nuclei and glycogen foci more scattered. Subnuclear fat droplets were present and there was increased lateral membrane interdigitations (arrow, Figure 1G ); by day 27, occasional large heteroly-sosomes were also present ( Figure 1H ). The proliferative phase eutopic endometrium from women with endometriosis (day 2 [stage II], day 5 [stage III], day 7 [stage I], and day 9 [stage II]) did not show any significant differences from that obtained from healthy controls. Mitotic figures were found (*, Figure 2A ) and some cells had small clumps of glycogen within. No secretions were observed. At day 9, there was some evidence of heterophagosome formation ( Figure 2B ) as in controls; by day 13, in a stage IV specimen (PL6), mitotic figures were still present but there was some heterogeneity in the nuclear morphology ( Table 2 ). The specimen from day 16 (PL8, stage I-II) was probably from the late proliferative phase ( Figure 2C ) with euchromatic, basal nuclei and mitotic figures. There were numerous apical secretory droplets, with scant glycogen production and no evidence of nucleolar channel formation. The 2 day 15 specimens were very different from each other, yet both showed later than expected phenotypes. One (PL1, stage I) had a very heterogeneous morphology, some glands exhibiting characteristics of a mid-secretory phenotype, with occasional pinopods normally seen on days 19 to 20 and glandular cells rich in dispersed glycogen ( Figure 2D ); other glands were more like controls with heavy basal deposits of glycogen. There were also dilated glands with cuboidal cells, suggestive of a later phenotype and heavy luminal secretions. Complex nucleoli showed some evidence of nucleolar channel systems. The other 15-day specimen (PL17, stage III), with only a few glands, had a much later phenotype with little intracellular glycogen, heterochromatic nuclei, and complex lateral membrane interdigitations ( Figure 2E ). The 3 day 17 specimens were variable in their morphology. Several mitotic figures were seen in 1 (PL21, Figure 2F ), despite the presence of masses of glycogen, suggesting asynchrony in differentiation though this specimen was only at stage I of the disease process. Some glands showed a more appropriate ultrastructure, with numerous Golgi stacks and cisternae of rough endoplasmic reticulum. PL5 (stage III) also had large aggregates of glycogen in some glands ( Figure 2G ), and both had some enlarged mitochondria though no fully formed nucleolar channel systems were seen. In PL5, there were some cuboidal epithelial cells in dilated glands with clusters of mitochondria, though nuclei were large and euchromatic and there was little evidence of protein biosynthesis ( Figure 2H ). The third day 17 case (PL27, stage III) also showed a mixture of features with narrow glands of columnar cells with oval, euchromatic nuclei, similar to those seen in the proliferative phase ( Figure 2I ). However, there were also late secretory features: dispersed glycogen, basal fat droplets, and lateral membrane interdigitations with intercellular spaces basally. Intraepithelial leucocytes were present with some heterophagosomes. The day 18 specimen showed occasional nucleolar channel systems and enlarged (though not “giant”) mitochondria. Even though this was at stage IV of the disease, mitotic figures were not seen and the appearance was consistent with that expected for the time in the menstrual cycle. A day 19 specimen (PL12, stage II) showed delayed maturation ( Figure 3A ) with glands of columnar cells with little glycogen and an absence of both nucleolar channel systems and giant mitochondria, while a day 20 specimen (PL23, stage III) contained both these features typical of days 17 to 20 ( Figure 3B ). Columnar cells had aggregates of glycogen basally and sometimes apically though often also finely dispersed. Of 3 day 21 specimens examined, 1 (PL28, stage II) showed features of a late secretory phenotype with dilated glands of cuboidal cells, though nuclei were round, euchromatic, and basally situated ( Figure 3C ), and there were only occasional aggregates of glycogen. Many supra-nuclear mitochondria and small apical vacuoles with amorphous contents were present with fat droplets basally. Lateral membranes were straight with few interdigitations, and there were some intercellular spaces. The other 2 specimens (PL36, stage I and PL31, stage III) were more typical for their dates, cells being columnar and with more heterochromatic nuclei, vertically orientated Golgi bodies, and occasional aggregates of glycogen (G, Figure 3D ). Cells containing large secondary lysosomes were seen in PL36 but no fat droplets were evident in either case. The day 22 (stage II) and one of the day 24 specimens (PL25, stage IV) showed features of a much earlier phenotype, with mitotic figures ( Figure 3E ) and abundant glycogen in subnuclear and supranuclear locations. Some nuclei were more irregular with condensed peripheral chromatin; in the day 22 biopsy, these were generally associated with dilated glands and cuboidal cells with small round mitochondria such as is found later in the cycle. Lateral membranes showed well-developed interdigitations basally. The cells of the second day 24 biopsy (PL26, stage III) were more typical of the mid-secretory phase, being columnar with heterochromatic nuclei and finely dispersed glycogen ( Figure 3F ). In dilated glands, the cells were more cuboidal, with irregular apical plasma membranes and scant microvilli. Mixed features were found in one of the day 25 specimens (PL30, stage III) with very regular, basal, euchromatic nuclei in columnar cells with dispersed aggregates of glycogen and clusters of fat droplets (F, Figure 3G ). The other (PL33, also stage III) contained more heterochromatic nuclei, though a minority were euchromatic and more regular. A nucleolar channel system was seen in 1 cell, while fat droplets typical of the late secretory phase were found in others ( Figure 3H ). Some glands contained scattered glycogen rosettes, while others had none. Intercellular spaces were often present in both cases with loose interdigitations between the cells ( Figure 3H ). The day 27 specimen (stage IV) was rather anomalous, with glands of rather low, cuboidal cells, euchromatic nuclei, and sparse organelles—mitochondria were not clustered basally as expected—nor were lateral membranes showing interdigitation as is usually found at that stage in the cycle ( Figure 4A ). The day 29 specimen (PL29, stage III) also showed mixed features with widely dilated glands of columnar rather than cuboidal cells and variable nuclear morphology. Glycogen was more aggregated than expected ( Figure 4B ). Apical vacuoles with diffuse contents, sometimes with a dense core, were present ( Figure 4C ), and large intracellular inclusions with heterogeneous contents were frequently seen ( Figure 4D ). Basal fat droplets and an irregular basal plasma membrane was present, though lateral membrane interdigitations were not prominent ( Figure 4B ). Binding of the lectin from Dolichos biflorus (DBA) to N -acetyl galactosamine-containing sequences, including GalNAcα1,3(LFucα1,2)Galβ1,3/4-GlcNAcβ1—is not generally found in the proliferative phase endometrial glands ( Figure 5A ) but occurs during the mid-secretory to late secretory phase of the menstrual cycle ( Figure 5B ). Occasional foci of stain can be seen on some cell apices as early as day 9, possibly because of adhesion of previously secreted products to cell surfaces. There was a general increase in binding during the course of the secretory phase, with stained glands often alternating with unstained (see Table 1 ). Specimens from women with stages I to III endometriosis showed extremely variable staining with DBA, with levels of staining generally associated with the late secretory phase sometimes appearing much earlier in the cycle (eg, day 17) and weak staining when more binding would normally be expected. Biopsies from women with grade IV endometriosis, however, completely failed to express any DBA-binding glycans and apart from 1 day 13 specimen (PL6), which was not expected to show staining, was invariably associated with a gland pattern suggestive of an earlier phase of the cycle, with narrow tubular glands composed of an epithelium with basally situated nuclei ( Figure 5C and E; see Table 2 ).

In this pilot study, we have demonstrated that eutopic endometrium from women with endometriosis may have significant biochemical and ultrastructural differences from normal control tissue and these changes appear to become marked particularly in the mid-secretory part of the cycle. One of the most striking and important abnormalities was the complete absence of DBA-binding glycans in endometrium from women with stage IV endometriosis, and in 2 of the 3 cases from the midsecretory to late secretory phase, this was in keeping with morphological evidence of a proliferative phase phenotype and delay in maturation. More specimens with stage IV endometriosis are, of course, required to confirm these initial findings. The specimen from day 24 (PL25) of the cycle had mitotic figures at the ultrastructural level; none were seen in the day 27 endometrium (PL22), but some glands contained nuclei that were round and euchromatic, suggestive of a newly divided state, and the ultrastructure of the cells was unusual for that stage in the cycle. Women with stages I to III generally showed DBA binding appropriate or even increased for the time in the cycle, apart from a case with stage II disease, which had negligible binding on day 22 of the cycle associated with the presence of mitotic figures. These morphological changes in women with endometriosis are in agreement with the alterations in gene expression patterns that have been recently reported. 25 These studies demonstrated that in women with moderate-to-severe disease, there is an abnormal transition from the proliferative to early secretory phase, which is associated with the inability of progesterone to downregulate DNA synthesis and cellular proliferation. Thus, our results provide a morphological correlation with the molecular evidence for the attenuation of the action of progesterone in the eutopic endometrium of women with endometriosis. It is thus plausible to speculate that such results at stage IV disease are indicative of progesterone resistance. This has been reported in endometriosis 26 together with deficiency in 17-β-hydroxysteroid dehydrogenase type 2 in glandular epithelial cells. 27 Aromatase activity, which catalyses the conversion of C 19 steroids to estrogens, has also been found to be elevated in eutopic endometrium from women with endometriosis 28 and has been thought to be a factor that sustains the endometriotic lesions, partially or totally mediated by estrogen receptor a-positive stromal cells 29 present in lesions. 30 Kao and colleagues 31 found dysregulation of glycodelin, which is under the regulation of progesterone, in women with endometriosis and suggested that this, too, is consistent with progesterone resistance and proposed that its underexpression may contribute to the reported low implantation rates and reduced fertility. It is interesting to observe, however, that despite abnormalities in the distribution of steroid receptors and concomitant changes in both structure and function of uterine glands, menstruation and cycle length appear to be unaffected. The absence of changes in cycle length in both baboons 32 and women with endometriosis may reflect the ability of progesterone to prevent the endometrium from regression because of the presence of a sufficient number of progesterone receptors for basal action but an insufficient responsiveness to progesterone to induce appropriate morphological transformation or gene expression during the secretory phase. 33 , 34 Previous studies on the endometrium in women with and without endometriosis have suffered from the lack of objectivity and well-fixed material, hence making the interpretation of data difficult and controversial. We were fortunate in obtaining well-preserved eutopic endometrial tissue that has enabled fine detail of the cells to be examined. The influence of the cycle length and the dysfunction of the hypothalamus-pituitary-ovary axis were not an issue as the main inclusion criteria for the study were regular cycles and no hormonal therapy for a period of at least 3 months before sampling. Although it was not possible to test the women for the LH (luteinizing hormone) surge, morphometry was used to confirm the dating of samples as well as ultrastructural markers such as the presence of the nucleolar channel system and giant mitochondria, which helped to offset the variation in gland ultrastructure seen in the secretory phase. Nuclear morphology was found to be very variable, and some control and endometriotic specimens showed very euchromatic nuclei in the mid to late secretory phase, which was a surprising finding as most reports of endometrial ultrastructure describe heterochromatic nuclei in the late secretory phase. 20 – 22 Eutopic endometrium from women with endometriosis did not show any specific ultrastructural abnormalities compared to that from normal women, and in the proliferative phase, there were no significant differences in the ultrastructural features of the 2 tissues. Later in the cycle, however, features normally confined to particular stages appeared over a much wider period of time. Hence, mitotic figures—not usually seen beyond days 14 to 16 of the cycle—were observed in 3 cases on days 17, 22, and 24, those cells in the process of cell division on days 17 and 24 being associated with intracellular glycogen, a feature never seen in control cases where cell division is normally segregated chronologically from glycogen biosynthesis. Likewise, giant mitochondria were observed later than in the controls. Euchromatic nuclei were a consistent finding throughout the secretory phase but, unlike the controls, were not always accompanied by areas of glands with heterochromatic nuclei later in the cycle, and glycogen deposits also tended to be more prominent later in the cycle compared to controls, which started to show diminished amounts after day 17. Other specimens had phenotypes more characteristic of midsecretory to late secretory phase. Sometimes nuclei were heterochromatic, as in a day 19 specimen but often they were euchromatic, a feature seen sometimes in controls but more generally associated with the proliferative phase. As can be seen from Table 2 , there was also considerable variation between specimens obtained on the same day of the cycle, for instance, on days 15, 17, and 24, with respect to the incidence of mitoses and deposition of glycogen. The presence of late mitoses is consistent with the recent findings of Burney and colleagues 25 who observed the maintenance of a proliferative molecular profile in the early secretory endometrium from women with endome-triosis, with an enrichment of genes involved in cell cycle regulation. They concluded that the pathways governing the transition from the proliferative to differentiated state were dysfunctional in the women with endometriosis. They suggested that nonsteroidal signaling pathways may also be involved. Dysregulation of cell cycle genes such as GOS2 and SALP (Sam68-like phosphotyrosine protein a) has also been described in endometrium from women with endometriosis 31 and this, too, may contribute toward alteration in mitotic activity. The presence of dividing cells well into the secretory phase is also consistent with findings of decreased apoptosis in eutopic endometrium from women with endometriosis compared to healthy controls, 35 indicating abnormal survival of eutopic endometrial cells throughout the entire menstrual cycle. Higher expression of Ki67 was also reported in endometriotic glands in the proliferative phase and early-and mid-secretory phases, 36 while transforming growth factor (TGF)-β1 remained unchanged in these endometria, unlike normal glands that showed an increase. In conclusion, our study has demonstrated significant differences in the biology of eutopic endometriotic and normal endometrium, both in the tissue architecture and the biochemistry. In advanced disease, the gland epithelium in the secretory part of the cycle showed a delay in maturation both morphologically and also in the expression of DBA-binding glycans. Several researchers have shown that pelvic endometriosis is heterogeneous in its clinical manifestations and response to treatment 37 – 39 ; however, these findings may explain some of the fertility problems associated with endometriosis, as receptivity is intimately bound up with the morphology and especially glycosylation of the uterine tissues. Previous studies have shown that treatment of women with RU484 is associated with a block in the normal expression of the glycans bound by DBA 40 and, in the cases examined here, defects in steroid receptor may be eliciting the same phenomena. Further studies are therefore required to examine the distribution of these receptors in suitably fixed material.

A total of 38 women of reproductive age with regular menstrual cycles (28–32 days) and without any history of autoimmune disease were recruited. Of these participants, we had 24 women (study group, mean age 34, range 22–45 years) with visually and biopsy-proven endo-metriosis who had undergone endometrial curettage and laparoscopic excision of endometriotic deposits and 14 fertile women (control group, mean age 34, range 25–44 years) without endometriosis who had undergone endometrial curettage only for the purpose of the study. The demographic details of the 2 groups were comparable (data not shown). All women with endometriosis complained of pelvic pain, deep dyspareunia, and/or sub-fertility. None of the participants had an intrauterine device in situ or had received hormonal therapy in the 3 months preceding laparoscopy and endometrial sampling. Endometriosis at the time of laparoscopy was staged according to the revised American Fertility Society (rAFS) scoring system 14 and confirmation of dating of control specimens was undertaken by one of us (I.M.I.) using stereological techniques on ultrathin sections (see below). As no significant ultrastructural abnormalities were apparent in 4 endometriotic biopsies taken between days 2 and 9, only those cases from day 13 onward will be described in detail. The study was approved by the Local Research Ethics Committee (Ref No 06/Q1407/173) as well as by the Universities of Manchester, United Kingdom, and Padua, Italy. All women gave written informed consent to participate in the study. Tissue was received from 2 sites: Department of Obstetrics and Gynaecology, University of Padua, Italy (from P. Litta, specimens coded PL), and Department of Reproductive Medicine, Central Manchester and Manchester Children’s University Hospitals NHS Trust, Manchester, United Kingdom (from L. G. Nardo, specimens coded NE). All chemicals and reagents were obtained from Sigma (UK or Italy) unless otherwise specified. Tissue from Padua was fixed in half-strength Karnovsky fixative (2% w/v parafor-maldehyde and 2.5% glutaraldehyde [v/v] in 0.1 M phosphate buffer pH 7.2) for 24 to 48 hours, then rinsed in buffer and transported to the United Kingdom. The material from Manchester was either fixed in half-strength Karnovsky fixative (as above) if available or in neutral buffered formalin and then refixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer pH 7.2 (Agar Scientific Ltd, Stansted, UK) for 2 to 3 hours when received in the laboratory. Both sets of tissue were diced into 1-mm thick slices before further processing and divided into 2 parts. One part was postfixed in 1% osmium tetroxide (Agar Scientific Ltd)/1.5% potassium ferrocyanide in 0.05 M sodium cacodylate buffer pH 7.2 for 1 hour in the dark, rinsed twice in buffer, dehydrated, and processed into Taab epoxy resin (TAAB Laboratories Equipment Ltd, Aldermaston, UK) for electron microscopy. The other part was not osmicated but otherwise processed in the same way for lectin histochemistry. Semithin 0.5-µm sections were cut on a Reichert Ultracut microtome using a diamond knife, stained with 1% toluidine blue in 1% borax on a hotplate, and suitable areas containing glands and/or surface epithelium selected for further examination. Ultrathin sections of the osmicated material, 70-nm thick, were mounted on 200 mesh copper grids and contrasted with uranyl acetate (Agar Scientific Ltd)/lead citrate and examined in a Philips CM10. Digital images of optimally orientated glandular epithelium were captured using a Deben camera and stored as 4-MB TIFF files. Digital TEM images acquired were loaded onto Histometrix , a stereology software (Histometrix MIL6, Kinetic Imaging Ltd, UK) installed on an IBM compatible personal computer. The software was used to estimate the parameters below. The volume density, V f , of nucleus in cytoplasm of surface epithelia was estimated by point counting. 15 , 16 Briefly, this was obtained by generating a grid with an array of random points on the image and counting the total number of points, ∑ P falling within the nucleus (∑ P nuc ) and dividing it by the number of points falling within the whole cell ( ∑ P cell ). The volume fraction of nucleus in the cell, V f(nuc) , is given by V f ( nuc ) = ∑ P nuc ∑ P cell . Surface epithelial cell nucleus volume was determined by point-sampled intercept method. 17 On 4 randomly selected images showing whole cells from each sample, an unbiased counting frame with superimposed random test points was applied. If a test point falls on a nucleus profile, a line, l 0 , in an isotropic uniform direction (IUR) was drawn through the point to the nuclear boundary. The volume of the nucleus, V̅ nuc , was then estimated from the following equation: V ¯ nuc = π 3 × ∑ l 0 3 . Estimates of surface epithelial volume, V cell , was obtained using the volume density of the nucleus and nuclear volume (see above): V cell = V ¯ nuc V f ( nuc ) . Sections 0.75-µm thick were cut and mounted on 3-aminopropyltriethoxysilane-coated slides, 18 dried in an oven at 50°C for 2 days, and then stained with biotinylated Dolichos biflorus lectin as previously described. 12 Epoxy resin was removed with saturated sodium ethoxide diluted 1:1 with absolute ethanol, followed by washes in ethanol and distilled water, then endogenous peroxidase was blocked with 10% (v/v) hydrogen peroxide (BDH, Poole, UK) before exposure to 0.03% trypsin (w/v; type II-S) in 0.05 M Tris-buffered saline (TBS) pH 7.6 for 4 minutes at 37° C. After washing, sections were incubated with 10 µg/mL biotinylated lectin from Dolichos biflorus in 0.05 MTBS containing 1-mmol/L calcium chloride for 1 hour at 37°C, washed in the same buffer, then treated with 5 µg/mL avidin peroxidase in 0.125 M TBS, pH 7.6, with 0.347 M sodium chloride for 1 hour at 37°C. 19 Sections were washed and sites of lectin binding revealed with 0.05% (w/v) diaminobenzi-dine tetrahydrochloride dihydrate in 0.05 M TBS, pH 7.6, and 0.015% (v/v) hydrogen peroxide (100 volumes) for 5 minutes at 18 ± 0.5°C. Sections were rinsed, airdried, and mounted in neutral synthetic mounting medium (BDH). Negative controls were carried out by substitution of 0.05 M TBS pH 7.6 with added calcium for the lectin to identify any nonspecific binding of avidin peroxidase to the tissue section or the presence of residual endogenous peroxidase activity. Sections were also incubated with the lectin in the presence of 0.2 M N -acetyl galactosamine to block binding of the lectin. Sections were assessed using a semiquantitative ranking system of analysis, where staining intensity was allocated a grade from – (negative) to ++++ (intense staining).