- Email: [email protected]
Uterine adenomyosis is associated with ultrastructural features of altered contractility in the inner myometrium
Uterine adenomyosis is associated with ultrastructural features of altered contractility in the inner myometrium Mohamed Khairy Mehasseb, M.D., Stephen Charles Bell, Ph.D., James Howard Pringle, Ph.D., and Marwan A. Habiba, Ph.D. Reproductive Sciences Section, Department of Cancer Studies and Molecular Medicine, Robert Kilpatrick Clinical Sciences Building, University of Leicester, Leicester, United Kingdom
Objective: To study the ultrastructure of the inner and outer myometrium, in the presence and absence of uterine adenomyosis. Design: Case control blinded comparison. Setting: University departments. Patient(s): Four premenopausal women with and six without uterine adenomyosis as the sole pathology. Intervention(s): Multiple samples were studied using transmission electron microscopy. Main Outcome Measure(s): Ultrastructure feature of the myometrium. Result(s): In uteri with adenomyosis, the myocytes exhibited cellular hypertrophy. The cytoplasmic myofilaments were less abundant. Abundant intermediate filaments formed cytoplasmic aggregates. The nuclei had a smooth outline with a clear ground substance, prominent nucleoli and peripherally arranged nuclear chromatin. There was occasional infolding of the nuclear envelope with entrapment of cytoplasmic organelles. The sarcolemmal bands were significantly longer and there were fewer caveolae. The perinuclear cell organelles were more distinct. The rough endoplasmic reticulum and Golgi apparatus were more prominent, denoting active protein synthesis, consistent with the observed cellular hypertrophy. All features were more prominent at the junctional zone. Conclusion(s): Smooth muscle cells from uteri with adenomyosis are ultrastructurally different from smooth muscle cells of normal uteri. These distinct features suggest a possible effect on myometrial contractility, together with hypertrophy. (Fertil Steril 2010;93:2130–6. 2010 by American Society for Reproductive Medicine.) Key Words: Electron microscopy, ultrastructure, uterus, adenomyosis, junctional zone, myometrium
Uterine adenomyosis is defined by the presence of endometrium within the myometrium. Microscopically, ectopic, nonneoplastic, endometrial glands and stroma are surrounded by hypertrophic and hyperplastic myometrium. Early description of the condition was made by Rokitansky in 1860 and by Von Recklinghausen in 1896 (1). Despite lack of agreement on the histological criteria, adenomyosis is frequently reported in hysterectomy specimens. Because the incidence of the disease in the general population is unclear, and because of its common association with other pathologies such as fibroids, the clinical significance of adenomyosis remains uncertain. Approximately 35% of women with adenomyosis are asymptomatic. Symptomatic women mostly exhibit menorrhagia (40%–50%), dysmenorrhoea (10%–30%), and metrorrhagia (10%–12%), and occasionally dyspareunia or dyschezia (2, 3). The extent and spread of adReceived September 9, 2008; revised January 17, 2009; accepted January 19, 2009; published online March 6, 2009. M.K.M. has nothing to disclose. S.C.B. has nothing to disclose. H.P. has nothing to disclose. M.A.H. has nothing to disclose. Reprint requests: Mohamed Khairy Mehasseb, M.D., Reproductive Sciences Section, Department of Cancer Studies and Molecular Medicine, Robert Kilpatrick Clinical Sciences Building, University of Leicester, Leicester, LE2 7LX, United Kingdom (TEL: þ44(0)116-2523149; FAX: þ44(0)116-2525846; E-mail: [email protected]).
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enomyosis may correlate with pelvic pain and dysmenorrhea and to a lesser degree, with menorrhagia and dyspareunia (4). Pelvic endometriosis coexists with adenomyosis in 2%–24% of cases, suggesting that the two conditions may be linked. In a cohort of infertile women, 126 of 160 (79%) with and 19 of 67 (28%) without endometriosis had adenomyosis (5). Endometriosis and adenomyosis may be variants of the same disease, involving dislocation of the basal endometrium (6). One theory is that adenomyosis results from the abnormal ingrowth and invagination of the basal endometrium into the subendometrial myometrium (junctional zone [JZ]) at the endometrial-myometrial interface (EMI) (7). During periods of regeneration, healing, and reepithelialization, the endometrium may invade a predisposed myometrium or a traumatized EMI. Hormonal, genetic, immunologic, and growth factors may play a role in this sequence of events. Mechanical damage and/or physical disruption of the EMI by dysfunctional uterine hyperperistalsis and/or dysfunctional contractility of the subendometrial myometrium (6, 7) or by sharp curettage during pregnancy (8) may allow for the dislocation of basal endometrium into the myometrium and the development of adenomyosis. The role of curettage during pregnancy is supported by the association of adenomyosis with a history of intrauterine procedures such as pregnancy termination (9), as well as by animal experiments in which unilateral
Fertility and Sterility Vol. 93, No. 7, May 1, 2010 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/10/$36.00 doi:10.1016/j.fertnstert.2009.01.097
TABLE 1 Clinical characteristics of participants. No.
Group
Age (y)
Parity
Indication for hysterectomy
Phase of cycle
Route of hysterectomy
1 2 3 4 5 6 7 8 9 10
Adenomyosis Adenomyosis Normal Normal Normal Normal Normal Normal Adenomyosis Adenomyosis
39 49 34 54 43 36 47 35 39 45
0 4, NVD 3, NVD 3, NVD 2, NVD 2, NVD 4, NVD 2, NVD 3, NVD 6, NVD
Menorrhagia, dysmenorrhea Menorrhagia, dysmenorrhea Menorrhagia, dysmenorrhea Menorrhagia Menorrhagia Menorrhagia Menorrhagia Menorrhagia Menorrhagia Menorrhagia, dysmenorrhea
Secretory Proliferative Proliferative Proliferative Secretory Secretory Secretory Proliferative Proliferative Proliferative
TAH TAH LAVH TAH LAVH TAH TAH TAH SH TAH
Note: TAH ¼ total abdominal hysterectomy; NVD ¼ normal vaginal delivery; LAVH ¼ laparoscopic assisted vaginal hysterectomy; SH ¼ subtotal hysterectomy. Mehasseb. Adenomyosis and myometrium contractility. Fertil Steril 2010.
adenomyosis is produced in pregnant rabbits by curetting one horn and tube while retaining a contralateral pregnancy (10). The EMI is also disturbed by the intramyometrial penetration of trophoblast during early pregnancy and this may underlie the higher incidence in parous women. It is possible that an underlying myometrial defect is responsible for, but could also be the effect of, both the invasion of the overlying endometrial stroma and glands and for the subsequent clinical manifestations. Although the gross and ultrastructure of the myometrium have been previously described in normal (11, 12) and leiomyomatous uteri (13, 14), there is no literature describing the myometrial structure in uterine adenomyosis. In this study, we compare the ultrastructure of the junctional zone and outer myometrium in the presence and absence of adenomyosis. MATERIALS AND METHODS The study was approved by the local research and ethics committee, and all participants provided written consent. Hysterectomy specimens from premenopausal women not using exogenous hormones were selected for this study. The uteri were removed by abdominal or laparoscopic assisted vaginal hysterectomy for menorrhagia with or without dysmenorrhea (Table 1). None of the participants had fibroids or endometrial abnormalities on preoperative ultrasound or hysteroscopy. Histopathologic examination proved a normal endometrium and absence of fibroids and confirmed adenomyosis (glands >2.5mm below EMI). None of the participants had endometriosis. All participants were parous except one woman in the adenomyosis group. None had a history of curettage, caesarean section or uterine surgery. There was no significant difference in mean age between the adenomyosis group (43 years) and the control group (40.4 years). Fertility and Sterility
FIGURE 1 Illustration of the way the anterior uterine wall was incised in the midline and toward the corneal ends. The area sampled was at the intersection of the three lines (i.e., the anterior wall of the uterus near the fundus).
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FIGURE 2 Ultrastructure of the myocytes at the JZ in normal and adenomyotic uteri. (A) Normal architecture of the JZ and the close proximity of the myocytes with no distinct bundle formation. The collagen fibers are prominent in the extracellular matrix. (B) The myocytes in adenomyotic uteri are more separated with less dense collagen. Nuclei have a smooth outline with a clear nuclear ground and peripherally arranged chromatin. (C and D) Attachment plaques (sarcolemmal bands; white arrows) and caveolae (black arrows) on the sarcolemmal membrane of normal myocytes. (E and F) Long sarcolemmal bands and sparse caveolae in adenomyotic myocytes (scale bars are shown on individual images).
Mehasseb. Adenomyosis and myometrium contractility. Fertil Steril 2010.
Uteri were obtained within 10 minutes of surgical removal and opened in the saggital plane. Multiple 3-mm3 samples were immediately obtained from the JZ (the first 5–8 mm underneath the endometrial/myometrial junction) and outer myometrium (the outer third of the myometrium). All samples were obtained from the anterior wall of the uterus near the fundus (Figure 1). Samples were primarily fixed in 2% glutaraldehyde in 0.1 M S€ orensens phosphate buffer for 48 hours at 4 C. The samples were then washed, post-fixed in buffered 1% osmium tetroxide for 30 minutes, rewashed, dehydrated in ethanol series, and treated with propylene oxide. The sam2132
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ples were then infiltrated with propylene oxide/LR white resin before final embedding and polymerization in LR White resin. Ultrathin 80-nm sections were cut from each sample using a Reichert Ultracut S ultramicrotome (Reichert-Jung, Vienna, Austria), collected on copper mesh grids, counter stained with 2% uranyl acetate and Reynolds’ lead citrate and examined using a JEOL 1220 transmission electron microscope (JEOL, Hertfordshire, United Kingdom) using an accelerating voltage of 80 kV. Digital images were recorded using a SIS Megaview III digital camera (Soft Imaging Systems, Munich, Germany) with Analysis Software.
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TABLE 2 Myocytes attachment plaques length (mm, mean ± SEM) and nuclear size (mm2, mean ± SEM): meaurements were based on 10 measurements per patient. Sarcolemmal plaques length
Normal group (n ¼ 6) Adenomyosis group (n ¼ 4)
Nuclear size
JZ
OM
JZ
OM
0.81 0.1 1.33 0.14a
0.66 0.06 1.3 0.08b
24.75 0.41 26.34 0.24a
23.66 0.34 24.71 0.32b
Note: JZ ¼ Junctional zone, OM ¼ Outer myometrium. a Significantly greater than normal junctional zone (P < 0.01). b significantly greater than normal outer myometrium (P < 0.05). Mehasseb. Adenomyosis and myometrium contractility. Fertil Steril 2010.
Parallel full uterine thickness samples were prepared for light microscopy, stained with hematoxylin and eosin, and used to confirm the menstrual cycle phase (proliferative or secretory)and the presence or absence of adenomyosis as well as to ensure that the samples are obtained from representative areas. A total of 10 uteri were examined: 4 with and 6 without adenomyosis. Measurement of the sarcolemmal dense bands (attachment plaques) length and the average nuclear size was made in 10 random fields from each area. Comparisons of means were made using Student’s t test for nonpaired samples, and differences were considered significant if P < 0.05.
RESULTS Myometrium Ultrastructure in the Absence of Adenomyosis The junctional zone was rich in myocytes closely interwoven in a dense connective tissue matrix with prominent collagen fibrils (Fig. 2A). The connective tissue-to-myocytes ratio was approximately 40:60. The cytoplasm was sparse and the typical trilaminar sarcolemmal (cell surface) membrane showed an even distribution of short dense plaques (sarcolemmal bands or attachment plaques), alternating with numerous prominent caveolae (Fig. 2C and D). We measured the length of the sarcolemmal bands in random fields from each specimen (Table 2), and the average was 0.81 0.1 mm (mean SEM). The cytoplasm contained an abundance of myofilaments, with their associated dense bodies, which were peripherally distributed in tight bundles, parallel to the sarcolemmal membrane. The nuclei were fusiform in shape with blunt ends, centrally placed in the myocyte, and with a crenated nuclear envelope (irregular outline) (Fig. 2A and C). The chromatin material was dense and finely dispersed in the nuclear ground substance. The average nuclear size was 24.75 0.41 mm2 (mean SEM; Table 2). The cells contained a normal prominent complement of perinuclear organelles (Fig. 3A), with no discernible structural abnormalities. In the normal outer myometrium, the myocytes were arranged in well-defined bundles, with narrow intercellular space, rich in collagen fibrils (Fig. 4). The individual bundles were widely spaced and separated by connective tissue, with a reversed connective tissue-to-myocytes ratio (60:40). Overall, the individual myocytes appeared similar to the JZ, with Fertility and Sterility
sparse cytoplasm rich in myofilaments lying parallel to the cell membrane. Numerous cytoplasmic aggregates of intermediate filaments and myelin bodies (lipolysosomes) were seen (Fig. 4B). The arrangement, frequency, and length of the attachment plaques (0.66 0.06 mm; mean SEM) and caveolae were comparable to the JZ. The nuclei maintained their elongated shape and size (23.66 0.43 mm2; mean SEM), with a crenated nuclear envelope, dense ground substance and finely dispersed chromatin material. We did not observe any differences between myometrial samples from different phases of the cycle (proliferative vs. secretory). Myometrial Ultrastructure in the Presence of Uterine Adenomyosis In the presence of uterine adenomyosis, the JZ myocytes were widely separated by a loose connective tissue matrix, with less prominent collagen fibrils. The cytoplasm was abundant, denoting cellular hypertrophy. The cytoplasmic myofilaments were less abundant, with less distinct bundling, but there was an abundance of intermediate filaments with a tendency to form cytoplasmic aggregates (Fig. 3E). Myelin bodies (lipolysosomes) were similarly more frequently observed (Fig. 3F). Compared to the normal JZ, the nuclei were more round and significantly enlarged (average nuclear size 26 0.24 mm2; P¼0.001; mean SEM), and the nuclear envelope lost its corrugated appearance exhibiting a smooth outline. The nuclei showed a clear ground substance with prominent nucleoli. The nuclear chromatin was peripherally arranged under the smooth nuclear envelope (Fig. 2B). Occasional infolding of the nuclear envelope with entrapment of cytoplasmic organelles was seen (Fig. 3C). The sarcolemmal bands were significantly longer (1.33 0.14 mm; P < 0.01; mean SEM; Table 2) with less prominent caveolae (Fig. 2E and F). With an abundant cytoplasm, the perinuclear cell organelles were more distinct. The mitochondria exhibited unfolding of the internal cristae (Fig. 3D). The rough endoplasmic reticulum and Golgi apparatus were more prominent, denoting active protein synthesis, which is consistent with the observed cellular hypertrophy (Fig. 3B). The outer myometrium showed features similar to the junctional zone. The bundle structure was preserved, but 2133
FIGURE 3 Myocyte abnormalities detected in the presence of adenomyosis. (A) Perinuclear organelles in normal myocytes compared to (B) Prominent cell organelles and endoplasmic reticulum in adenomyotic myocytes. (C) Abnormal invagination of the nuclear envelope entrapping organelles. (D) Abnormal mitochondria (M) with unfolded cristae. (E and F) Intermediate filaments aggregates (IF) and myelin bodies (MB) (scale bars shown on individual images).
Mehasseb. Adenomyosis and myometrium contractility. Fertil Steril 2010.
with increased intercellular space and less dense collagen fibrils. The nuclei were significantly larger than controls (24.71 0.32 mm2; P¼0.03; mean SEM), with abundant cytoplasm and the attachment plaques were significantly longer (1.3 0.08 mm; P < 0.01; mean SEM). DISCUSSION In this study, we described a range of ultrastructural abnormalities in the junctional zone and outer myometrial layers in women with uterine adenomyosis. As the lack of variation in ultrastructure of myocytes with the normal cycle has been described in the literature (14), we did not study each phase of the cycle separately. Enlargement of endoplasmic reticulum and an increase in free ribosomes have been described with 2134
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supraphysiologic doses of estrogen (15), and an increase in myofilament content and hypertrophy have been observed with synthetic progestogens (16). The coexistence of fibroids could have an effect on the host myometrium (14). To avoid confounding, we only studied uteri in which fibroids have been excluded by ultrasound, macroscopic and microscopic assessment. The inner and outer myometria are believed to be functionally and structurally distinct layers, based on magnetic resonance imaging (17), anatomic, and histologic studies (18, 19). We did not find significant ultrastructure differences between the inner and outer layer myocytes. However, the JZ had higher cellular density, with decreased extracellular space and no clear bundle arrangement. Overall, our description of the normal
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FIGURE 4 (A) Normal outer myometrium bundle arrangement and (B) high collagen-to-myocytes ratio. Note the intermediate filaments aggregates (IF) and the myelin bodies (MB) (scale bars shown on images).
Mehasseb. Adenomyosis and myometrium contractility. Fertil Steril 2010.
myometrium is in agreement with the previous studies examining the histology ultrastructure of the normal human myometrium (20–27). In the presence of adenomyosis, the observed loose connective tissue matrix with less prominent collagen fibrils may be explained by intercellular space expansion. Further research is needed to characterize the extracellular matrix components. The presence of myelin bodies (lipolysosomes) is suggested to be linked to cell injury, particularly ischemia (28). Nevertheless, the observation that outer myometrial cells from normal uteri contained an abundance of myelin bodies is unlikely to be explained by ischemia, because of the rich uterine blood supply. Thus, the cause of these myelin bodies in normal uteri remains speculative, but could possibly be related to postpartum involution. In line with the observations made by light microscopy (29, 30), our finding of enlarged nuclei and increased cytoplasm are consistent with cellular hypertrophy. In the presence of adenomyosis, the JZ showed cellular and nuclear hypertrophy, abnormal nuclear and mitochondrial shape, abundant myelin bodies and intermediate filaments aggregates, extensive endoplasmic reticulum, and lengthening of sarcolemmal plaques with reduced caveolae. The outer myometrium was similarly affected. The absence of localized changes indicates the presence of a uterus-wide pathology in both the JZ and outer myometrium. Whether these changes represent a primary myometrial defect or a phenomenon secondary to the presence of adenomyosis cannot be ascertained from our findings. The shape of the nucleus is affected by the contractile filaments of the cytoplasm. Contracted intestinal smooth muscle cells exhibited exaggerated nuclear envelope invaginations when examined using electron microscopy (31). Similarly, alterations of the intracellular ionic environment (especially bivalent cations; e.g., calcium) cause changes to the nuclear shape (32) and to the appearance of the chromatin Fertility and Sterility
material (33). Increased intracellular calcium is associated with nuclear envelope indentations and a condensed chromatin. Thus, the nuclear abnormalities in uteri with adenomyosis could potentially be explained by abnormal contractility. The increase in intracellular aggregates suggest an increase in intermediate filaments (14), as demonstrated by vimentin and desmin immunohistochemical staining (34). It is plausible that these filament aggregates could be related to an increased synthetic activity in the myocyte, as evidenced by the observed cellular hypertrophy, expanded cytoplasm, and by the increase in ribosomes and rough endoplasmic reticulum. The net effect can be an imbalance between the production and turnover of the cytoskeletal components. The sarcolemma of smooth muscle cells is divided into two structurally distinct regions: those bearing submembranous dense plaques and intervening zones that bear many vesicular invaginations or caveolae. The dense bands are junctions of the adherens type, serve as anchorage sites for actin cytoskeleton, and are typically marked by antibodies to vinculin (35). Caveolae have been implicated in a wide range of cellular functions. Caveolae contain a host of receptors, second messenger generators, G proteins, kinases, and ion channels in close proximity. Caveolae are often in close proximity to sarcoplasmic reticulum or mitochondria, and have been proposed to organize signaling molecules (36, 37). The increased length of the dense bands that anchor intracellular myofilaments could reflect an increase in cytoskeletal filaments (14). Abnormally shaped mitochondria with unfolded cristae suggest an abnormality in active cellular processes or the initiation of a degenerative process (14, 28). It is plausible that the main function of the JZ is concerned with preparation of the endometrium for implantation, sperm transport, and hemostasis during menstruation, with the outer myometrium being primary involved with parturition. It is also possible that the ultrastructural abnormalities observed with the sarcolemmal bands and caveolae may cause 2135
a disturbance in the normal calcium cycling in the affected myocytes, with a subsequent loss of normal rhythmic contractions. Chronic uterine dysfunctional peristalsis and hyperperistalsis have been proposed as causal factors for adenomyosis and endometriosis, because these result in dislocation of the basal endometrium in both the underlying myometrium and peritoneal cavity. Women with endometriosis displayed marked uterine hyperperistalsis that differs significantly from the peristalsis in unaffected women during the early, midfollicular, and midluteal phases (6). In conclusion, this study demonstrated that the myocytes of uteri harboring adenomyosis are ultrastructurally different from those of normal uteri. These ultrastructural changes suggest a possible defect in myometrial contractility. Dysfunctional contractility could be the result of the presence of adenomyosis or could contribute to its pathogenesis. Acknowledgements: We would like to thank Dr. Brian Eyden, (Consultant Histopathologist, Christie Hospital NHS Trust, Manchester, United Kingdom) for his helpful comments on the manuscript. We would also like to thank Natalie Allcock and Stefan Hyman (Electron Microscopy Services, University of Leicester, Leicester, United Kingdom) for technical assistance.
REFERENCES 1. von Recklinghausen F. Die adenomyomata und cystadenomata der uterus und tubenwandug: ihre abkunft von resten des Wolff’schen koerpers. August Hirschwald Verlag, Berlin, 1896. 2. Vavilis D, Agorastos T, Tzafetas J, Loufopoulos A, Vakiani M, Constantinidis T, et al. Adenomyosis at hysterectomy: prevalence and relationship to operative findings and reproductive and menstrual factors. Clin Exp Obstet Gynecol 1997;24:36–8. 3. Parazzini F, Vercellini P, Panazza S, Chatenoud L, Oldani S, Crosignani PG. Risk factors for adenomyosis. Hum Reprod 1997;12:1275–9. 4. Sammour A, Pirwany I, Usubutun A, Arseneau J, Tulandi T. Correlations between extent and spread of adenomyosis and clinical symptoms. Gynecol Obstet Invest 2002;54:213–6. 5. Kunz G, Beil D, Huppert P, Noe M, Kissler S, Leyendecker G. Adenomyosis in endometriosis–prevalence and impact on fertility. Evidence from magnetic resonance imaging. Hum Reprod 2005;20:2309–16. 6. Kunz G, Beil D, Huppert P, Leyendecker G. Structural abnormalities of the uterine wall in women with endometriosis and infertility visualized by vaginal sonography and magnetic resonance imaging. Hum Reprod 2000;15:76–82. 7. Leyendecker G, Herbertz M, Kunz G, Mall G. Endometriosis results from the dislocation of basal endometrium. Hum Reprod 2002;17:2725–36. 8. Curtis KM, Hillis SD, Marchbanks PA, Peterson HB. Disruption of the endometrial-myometrial border during pregnancy as a risk factor for adenomyosis. Am J Obstet Gynecol 2002;187:543–4. 9. Levgur M, Abadi MA, Tucker A. Adenomyosis: symptoms, histology, and pregnancy terminations. Obstet Gynecol 2000;95:688–91. 10. Lewinski H. Beitrag zur Frage der Adenomyosis. Zentralbl Gynakol 1931;55:2163. 11. Lowy J, Small JV. The organization of myosin and actin in vertebrate smooth muscle. Nature 1970;227:46–51. 12. Gompel C, Silverberg SG. Pathology in gynecology and obstetrics. Philadelphia: JB Lippencott Company, 1994:218–25. 13. Ferenczy A. Diagnostic electron microscopy in gynecologic pathology. Pathol Annu 1979;14:353–81.
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14. Richards PA, Richards PDG, Tiltman AJ. The ultrastructure of fibromyomatous myometrium and its relationship to infertility. Hum Reprod Update 1998;4:520–5. 15. Friederici HHR, DeCloux RJ. The early response of immature rat myometrium to estrogenic stimulation. J Ultrastruct Res 1968;22:402–12. 16. Dito WR, Batsakis JG. Norethynodrel-treated endometriosis: a morphological and histochemical study. Obstet Gynecol 1961;18:1–12. 17. Brosens JJ, Barker FG, de Souza NM. Myometrial zonal differentiation and uterine junctional zone hyperplasia in the non-pregnant uterus. Hum Reprod Update 1998;4:496–502. 18. Wetzstein R. Der Uterusmuskel: Morphologie. Arch Gynecol 1965;202: 1–13. 19. Noe N, Kunz G, Herbertz M, Mall G, Leyendecker G. The cyclic pattern of the immunocytochemical expression of oestrogen and progesterone receptors in human myometrial and endometrial layers: characterization of the endometrial-subendometrial unit. Hum Reprod 1999;14:190–7. 20. McCarthy S, Scott G, Majumdar S, Shapiro B, Thompson S, Lane R, et al. Uterine junctional zone: MR study of water content and relaxation properties. Radiology 1989;171:241–3. 21. Scoutt L, Flynn SD, Luthringer DJ, McCauley TR, McCarthy SM. Junctional zone of the uterus: correlation of MR imaging and histologic examination of hysterectomy specimens. Radiology 1991;179:403–7. 22. Brown HK, Stoll BS, Nicosia SV. Uterine junctional zone: correlation between histologic findings and MR imaging. Radiology 1991;179: 409–13. 23. Mark JST. An electron microscopy study of uterine smooth muscle. Anat Rec 1956;125:473–91. 24. Hashimoto M, Momori A, Kosaka M, Mori Y, Shimoyama T, Akashi K. Electron microscopic studies on the smooth muscle of the human uterus. J Jpn Obstet Gynecol Soc 1960;7:115–21. 25. Fujii S, Konishi I, Mori T. Smooth muscle differentiation at endometriomyometrial junction. An ultrastructural study. Virchows Arch A Pathol Anat Histol 1989;414:105–12. 26. Fujii S, Konishi I, Mori T. Ultrastructure of smooth muscle tissue in the female reproductive tract: uterus and oviduct. In: Motta PM, ed. Ultrastructure of smooth muscle. Boston: Kluwer, 1990:197–220. 27. Cole WC, Garfield RE. Ultrastructure of the myometrium. In: Wynn RM, Jollie WP, eds. Biology of the uterus. Vol. 455–504. New York: Plenum, 1989. 28. Cotran RS, Kumar V, Robbins SL. In: Robbins pathological basis of disease. Philadelphia: W.B. Saunders, 1989:2–3. 29. Loud AV, Olivetti G, Anversa P. Morphometric measurement of cellular hypertrophy. Lab Invest 1983;49:230–4. 30. Owens GK, Rabinovitch PS, Schwartz SM. Smooth muscle cell hypertrophy versus hyperplasia in hypertension. Proc Natl Acad Sci U S A 1981;78:7759–63. 31. Lane BP. Alterations in the cytologic detail of intestinal smooth muscle cells in various stages of contraction. J Cell Biol 1965;27:199–213. 32. Davies HG, Spencer M. The variation in the structure of erythrocyte nuclei with fixation. J Cell Biol 1962;14:445–58. 33. Barnicot NA. A study of newt mitotic chromosomes by negative staining. J Cell Biol 1967;32:585–603. 34. Eyden BP, Hale RJ, Richmond I, Buckley CH. Cytoskeletal filaments in the smooth muscle cells of uterine leiomyomata and myometrium: an ultrastructural and immunohistochemical analysis. Virchows Arch A Pathol Anat Histol 1992;420:51–8. 35. North AJ, Galazkiewicz B, Byers TJ, Glenney JRJ, Small JV. Complementary distributions of vinculin and dystrophin define two distinct sarcolemma domains in smooth muscle. J Cell Biol 1993;120:1159–67. 36. Fujimoto T. Calcium pump of the plasma membrane is localized in caveolae. J Cell Biol 1993;5:1147–57. 37. Goto Y, Yoshikane H, Honda M, Morioka S, Yamori Y, Moriyama K. Three-dimensional observation on sarcoplasmic reticulum and caveolae in myocardium of spontaneously hypertensive rats. J Submicrosc Cytol Pathol 1993;22:535–42.
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Objective: To study the ultrastructure of the inner and outer myometrium, in the presence and absence of uterine adenomyosis. Design: Case control blinded comparison. Setting: University departments. Patient(s): Four premenopausal women with and six without uterine adenomyosis as the sole pathology. Intervention(s): Multiple samples were studied using transmission electron microscopy. Main Outcome Measure(s): Ultrastructure feature of the myometrium. Result(s): In uteri with adenomyosis, the myocytes exhibited cellular hypertrophy. The cytoplasmic myofilaments were less abundant. Abundant intermediate filaments formed cytoplasmic aggregates. The nuclei had a smooth outline with a clear ground substance, prominent nucleoli and peripherally arranged nuclear chromatin. There was occasional infolding of the nuclear envelope with entrapment of cytoplasmic organelles. The sarcolemmal bands were significantly longer and there were fewer caveolae. The perinuclear cell organelles were more distinct. The rough endoplasmic reticulum and Golgi apparatus were more prominent, denoting active protein synthesis, consistent with the observed cellular hypertrophy. All features were more prominent at the junctional zone. Conclusion(s): Smooth muscle cells from uteri with adenomyosis are ultrastructurally different from smooth muscle cells of normal uteri. These distinct features suggest a possible effect on myometrial contractility, together with hypertrophy. (Fertil Steril 2010;93:2130–6. 2010 by American Society for Reproductive Medicine.) Key Words: Electron microscopy, ultrastructure, uterus, adenomyosis, junctional zone, myometrium
Uterine adenomyosis is defined by the presence of endometrium within the myometrium. Microscopically, ectopic, nonneoplastic, endometrial glands and stroma are surrounded by hypertrophic and hyperplastic myometrium. Early description of the condition was made by Rokitansky in 1860 and by Von Recklinghausen in 1896 (1). Despite lack of agreement on the histological criteria, adenomyosis is frequently reported in hysterectomy specimens. Because the incidence of the disease in the general population is unclear, and because of its common association with other pathologies such as fibroids, the clinical significance of adenomyosis remains uncertain. Approximately 35% of women with adenomyosis are asymptomatic. Symptomatic women mostly exhibit menorrhagia (40%–50%), dysmenorrhoea (10%–30%), and metrorrhagia (10%–12%), and occasionally dyspareunia or dyschezia (2, 3). The extent and spread of adReceived September 9, 2008; revised January 17, 2009; accepted January 19, 2009; published online March 6, 2009. M.K.M. has nothing to disclose. S.C.B. has nothing to disclose. H.P. has nothing to disclose. M.A.H. has nothing to disclose. Reprint requests: Mohamed Khairy Mehasseb, M.D., Reproductive Sciences Section, Department of Cancer Studies and Molecular Medicine, Robert Kilpatrick Clinical Sciences Building, University of Leicester, Leicester, LE2 7LX, United Kingdom (TEL: þ44(0)116-2523149; FAX: þ44(0)116-2525846; E-mail: [email protected]).
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enomyosis may correlate with pelvic pain and dysmenorrhea and to a lesser degree, with menorrhagia and dyspareunia (4). Pelvic endometriosis coexists with adenomyosis in 2%–24% of cases, suggesting that the two conditions may be linked. In a cohort of infertile women, 126 of 160 (79%) with and 19 of 67 (28%) without endometriosis had adenomyosis (5). Endometriosis and adenomyosis may be variants of the same disease, involving dislocation of the basal endometrium (6). One theory is that adenomyosis results from the abnormal ingrowth and invagination of the basal endometrium into the subendometrial myometrium (junctional zone [JZ]) at the endometrial-myometrial interface (EMI) (7). During periods of regeneration, healing, and reepithelialization, the endometrium may invade a predisposed myometrium or a traumatized EMI. Hormonal, genetic, immunologic, and growth factors may play a role in this sequence of events. Mechanical damage and/or physical disruption of the EMI by dysfunctional uterine hyperperistalsis and/or dysfunctional contractility of the subendometrial myometrium (6, 7) or by sharp curettage during pregnancy (8) may allow for the dislocation of basal endometrium into the myometrium and the development of adenomyosis. The role of curettage during pregnancy is supported by the association of adenomyosis with a history of intrauterine procedures such as pregnancy termination (9), as well as by animal experiments in which unilateral
Fertility and Sterility Vol. 93, No. 7, May 1, 2010 Copyright ª2010 American Society for Reproductive Medicine, Published by Elsevier Inc.
0015-0282/10/$36.00 doi:10.1016/j.fertnstert.2009.01.097
TABLE 1 Clinical characteristics of participants. No.
Group
Age (y)
Parity
Indication for hysterectomy
Phase of cycle
Route of hysterectomy
1 2 3 4 5 6 7 8 9 10
Adenomyosis Adenomyosis Normal Normal Normal Normal Normal Normal Adenomyosis Adenomyosis
39 49 34 54 43 36 47 35 39 45
0 4, NVD 3, NVD 3, NVD 2, NVD 2, NVD 4, NVD 2, NVD 3, NVD 6, NVD
Menorrhagia, dysmenorrhea Menorrhagia, dysmenorrhea Menorrhagia, dysmenorrhea Menorrhagia Menorrhagia Menorrhagia Menorrhagia Menorrhagia Menorrhagia Menorrhagia, dysmenorrhea
Secretory Proliferative Proliferative Proliferative Secretory Secretory Secretory Proliferative Proliferative Proliferative
TAH TAH LAVH TAH LAVH TAH TAH TAH SH TAH
Note: TAH ¼ total abdominal hysterectomy; NVD ¼ normal vaginal delivery; LAVH ¼ laparoscopic assisted vaginal hysterectomy; SH ¼ subtotal hysterectomy. Mehasseb. Adenomyosis and myometrium contractility. Fertil Steril 2010.
adenomyosis is produced in pregnant rabbits by curetting one horn and tube while retaining a contralateral pregnancy (10). The EMI is also disturbed by the intramyometrial penetration of trophoblast during early pregnancy and this may underlie the higher incidence in parous women. It is possible that an underlying myometrial defect is responsible for, but could also be the effect of, both the invasion of the overlying endometrial stroma and glands and for the subsequent clinical manifestations. Although the gross and ultrastructure of the myometrium have been previously described in normal (11, 12) and leiomyomatous uteri (13, 14), there is no literature describing the myometrial structure in uterine adenomyosis. In this study, we compare the ultrastructure of the junctional zone and outer myometrium in the presence and absence of adenomyosis. MATERIALS AND METHODS The study was approved by the local research and ethics committee, and all participants provided written consent. Hysterectomy specimens from premenopausal women not using exogenous hormones were selected for this study. The uteri were removed by abdominal or laparoscopic assisted vaginal hysterectomy for menorrhagia with or without dysmenorrhea (Table 1). None of the participants had fibroids or endometrial abnormalities on preoperative ultrasound or hysteroscopy. Histopathologic examination proved a normal endometrium and absence of fibroids and confirmed adenomyosis (glands >2.5mm below EMI). None of the participants had endometriosis. All participants were parous except one woman in the adenomyosis group. None had a history of curettage, caesarean section or uterine surgery. There was no significant difference in mean age between the adenomyosis group (43 years) and the control group (40.4 years). Fertility and Sterility
FIGURE 1 Illustration of the way the anterior uterine wall was incised in the midline and toward the corneal ends. The area sampled was at the intersection of the three lines (i.e., the anterior wall of the uterus near the fundus).
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FIGURE 2 Ultrastructure of the myocytes at the JZ in normal and adenomyotic uteri. (A) Normal architecture of the JZ and the close proximity of the myocytes with no distinct bundle formation. The collagen fibers are prominent in the extracellular matrix. (B) The myocytes in adenomyotic uteri are more separated with less dense collagen. Nuclei have a smooth outline with a clear nuclear ground and peripherally arranged chromatin. (C and D) Attachment plaques (sarcolemmal bands; white arrows) and caveolae (black arrows) on the sarcolemmal membrane of normal myocytes. (E and F) Long sarcolemmal bands and sparse caveolae in adenomyotic myocytes (scale bars are shown on individual images).
Mehasseb. Adenomyosis and myometrium contractility. Fertil Steril 2010.
Uteri were obtained within 10 minutes of surgical removal and opened in the saggital plane. Multiple 3-mm3 samples were immediately obtained from the JZ (the first 5–8 mm underneath the endometrial/myometrial junction) and outer myometrium (the outer third of the myometrium). All samples were obtained from the anterior wall of the uterus near the fundus (Figure 1). Samples were primarily fixed in 2% glutaraldehyde in 0.1 M S€ orensens phosphate buffer for 48 hours at 4 C. The samples were then washed, post-fixed in buffered 1% osmium tetroxide for 30 minutes, rewashed, dehydrated in ethanol series, and treated with propylene oxide. The sam2132
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ples were then infiltrated with propylene oxide/LR white resin before final embedding and polymerization in LR White resin. Ultrathin 80-nm sections were cut from each sample using a Reichert Ultracut S ultramicrotome (Reichert-Jung, Vienna, Austria), collected on copper mesh grids, counter stained with 2% uranyl acetate and Reynolds’ lead citrate and examined using a JEOL 1220 transmission electron microscope (JEOL, Hertfordshire, United Kingdom) using an accelerating voltage of 80 kV. Digital images were recorded using a SIS Megaview III digital camera (Soft Imaging Systems, Munich, Germany) with Analysis Software.
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TABLE 2 Myocytes attachment plaques length (mm, mean ± SEM) and nuclear size (mm2, mean ± SEM): meaurements were based on 10 measurements per patient. Sarcolemmal plaques length
Normal group (n ¼ 6) Adenomyosis group (n ¼ 4)
Nuclear size
JZ
OM
JZ
OM
0.81 0.1 1.33 0.14a
0.66 0.06 1.3 0.08b
24.75 0.41 26.34 0.24a
23.66 0.34 24.71 0.32b
Note: JZ ¼ Junctional zone, OM ¼ Outer myometrium. a Significantly greater than normal junctional zone (P < 0.01). b significantly greater than normal outer myometrium (P < 0.05). Mehasseb. Adenomyosis and myometrium contractility. Fertil Steril 2010.
Parallel full uterine thickness samples were prepared for light microscopy, stained with hematoxylin and eosin, and used to confirm the menstrual cycle phase (proliferative or secretory)and the presence or absence of adenomyosis as well as to ensure that the samples are obtained from representative areas. A total of 10 uteri were examined: 4 with and 6 without adenomyosis. Measurement of the sarcolemmal dense bands (attachment plaques) length and the average nuclear size was made in 10 random fields from each area. Comparisons of means were made using Student’s t test for nonpaired samples, and differences were considered significant if P < 0.05.
RESULTS Myometrium Ultrastructure in the Absence of Adenomyosis The junctional zone was rich in myocytes closely interwoven in a dense connective tissue matrix with prominent collagen fibrils (Fig. 2A). The connective tissue-to-myocytes ratio was approximately 40:60. The cytoplasm was sparse and the typical trilaminar sarcolemmal (cell surface) membrane showed an even distribution of short dense plaques (sarcolemmal bands or attachment plaques), alternating with numerous prominent caveolae (Fig. 2C and D). We measured the length of the sarcolemmal bands in random fields from each specimen (Table 2), and the average was 0.81 0.1 mm (mean SEM). The cytoplasm contained an abundance of myofilaments, with their associated dense bodies, which were peripherally distributed in tight bundles, parallel to the sarcolemmal membrane. The nuclei were fusiform in shape with blunt ends, centrally placed in the myocyte, and with a crenated nuclear envelope (irregular outline) (Fig. 2A and C). The chromatin material was dense and finely dispersed in the nuclear ground substance. The average nuclear size was 24.75 0.41 mm2 (mean SEM; Table 2). The cells contained a normal prominent complement of perinuclear organelles (Fig. 3A), with no discernible structural abnormalities. In the normal outer myometrium, the myocytes were arranged in well-defined bundles, with narrow intercellular space, rich in collagen fibrils (Fig. 4). The individual bundles were widely spaced and separated by connective tissue, with a reversed connective tissue-to-myocytes ratio (60:40). Overall, the individual myocytes appeared similar to the JZ, with Fertility and Sterility
sparse cytoplasm rich in myofilaments lying parallel to the cell membrane. Numerous cytoplasmic aggregates of intermediate filaments and myelin bodies (lipolysosomes) were seen (Fig. 4B). The arrangement, frequency, and length of the attachment plaques (0.66 0.06 mm; mean SEM) and caveolae were comparable to the JZ. The nuclei maintained their elongated shape and size (23.66 0.43 mm2; mean SEM), with a crenated nuclear envelope, dense ground substance and finely dispersed chromatin material. We did not observe any differences between myometrial samples from different phases of the cycle (proliferative vs. secretory). Myometrial Ultrastructure in the Presence of Uterine Adenomyosis In the presence of uterine adenomyosis, the JZ myocytes were widely separated by a loose connective tissue matrix, with less prominent collagen fibrils. The cytoplasm was abundant, denoting cellular hypertrophy. The cytoplasmic myofilaments were less abundant, with less distinct bundling, but there was an abundance of intermediate filaments with a tendency to form cytoplasmic aggregates (Fig. 3E). Myelin bodies (lipolysosomes) were similarly more frequently observed (Fig. 3F). Compared to the normal JZ, the nuclei were more round and significantly enlarged (average nuclear size 26 0.24 mm2; P¼0.001; mean SEM), and the nuclear envelope lost its corrugated appearance exhibiting a smooth outline. The nuclei showed a clear ground substance with prominent nucleoli. The nuclear chromatin was peripherally arranged under the smooth nuclear envelope (Fig. 2B). Occasional infolding of the nuclear envelope with entrapment of cytoplasmic organelles was seen (Fig. 3C). The sarcolemmal bands were significantly longer (1.33 0.14 mm; P < 0.01; mean SEM; Table 2) with less prominent caveolae (Fig. 2E and F). With an abundant cytoplasm, the perinuclear cell organelles were more distinct. The mitochondria exhibited unfolding of the internal cristae (Fig. 3D). The rough endoplasmic reticulum and Golgi apparatus were more prominent, denoting active protein synthesis, which is consistent with the observed cellular hypertrophy (Fig. 3B). The outer myometrium showed features similar to the junctional zone. The bundle structure was preserved, but 2133
FIGURE 3 Myocyte abnormalities detected in the presence of adenomyosis. (A) Perinuclear organelles in normal myocytes compared to (B) Prominent cell organelles and endoplasmic reticulum in adenomyotic myocytes. (C) Abnormal invagination of the nuclear envelope entrapping organelles. (D) Abnormal mitochondria (M) with unfolded cristae. (E and F) Intermediate filaments aggregates (IF) and myelin bodies (MB) (scale bars shown on individual images).
Mehasseb. Adenomyosis and myometrium contractility. Fertil Steril 2010.
with increased intercellular space and less dense collagen fibrils. The nuclei were significantly larger than controls (24.71 0.32 mm2; P¼0.03; mean SEM), with abundant cytoplasm and the attachment plaques were significantly longer (1.3 0.08 mm; P < 0.01; mean SEM). DISCUSSION In this study, we described a range of ultrastructural abnormalities in the junctional zone and outer myometrial layers in women with uterine adenomyosis. As the lack of variation in ultrastructure of myocytes with the normal cycle has been described in the literature (14), we did not study each phase of the cycle separately. Enlargement of endoplasmic reticulum and an increase in free ribosomes have been described with 2134
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supraphysiologic doses of estrogen (15), and an increase in myofilament content and hypertrophy have been observed with synthetic progestogens (16). The coexistence of fibroids could have an effect on the host myometrium (14). To avoid confounding, we only studied uteri in which fibroids have been excluded by ultrasound, macroscopic and microscopic assessment. The inner and outer myometria are believed to be functionally and structurally distinct layers, based on magnetic resonance imaging (17), anatomic, and histologic studies (18, 19). We did not find significant ultrastructure differences between the inner and outer layer myocytes. However, the JZ had higher cellular density, with decreased extracellular space and no clear bundle arrangement. Overall, our description of the normal
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FIGURE 4 (A) Normal outer myometrium bundle arrangement and (B) high collagen-to-myocytes ratio. Note the intermediate filaments aggregates (IF) and the myelin bodies (MB) (scale bars shown on images).
Mehasseb. Adenomyosis and myometrium contractility. Fertil Steril 2010.
myometrium is in agreement with the previous studies examining the histology ultrastructure of the normal human myometrium (20–27). In the presence of adenomyosis, the observed loose connective tissue matrix with less prominent collagen fibrils may be explained by intercellular space expansion. Further research is needed to characterize the extracellular matrix components. The presence of myelin bodies (lipolysosomes) is suggested to be linked to cell injury, particularly ischemia (28). Nevertheless, the observation that outer myometrial cells from normal uteri contained an abundance of myelin bodies is unlikely to be explained by ischemia, because of the rich uterine blood supply. Thus, the cause of these myelin bodies in normal uteri remains speculative, but could possibly be related to postpartum involution. In line with the observations made by light microscopy (29, 30), our finding of enlarged nuclei and increased cytoplasm are consistent with cellular hypertrophy. In the presence of adenomyosis, the JZ showed cellular and nuclear hypertrophy, abnormal nuclear and mitochondrial shape, abundant myelin bodies and intermediate filaments aggregates, extensive endoplasmic reticulum, and lengthening of sarcolemmal plaques with reduced caveolae. The outer myometrium was similarly affected. The absence of localized changes indicates the presence of a uterus-wide pathology in both the JZ and outer myometrium. Whether these changes represent a primary myometrial defect or a phenomenon secondary to the presence of adenomyosis cannot be ascertained from our findings. The shape of the nucleus is affected by the contractile filaments of the cytoplasm. Contracted intestinal smooth muscle cells exhibited exaggerated nuclear envelope invaginations when examined using electron microscopy (31). Similarly, alterations of the intracellular ionic environment (especially bivalent cations; e.g., calcium) cause changes to the nuclear shape (32) and to the appearance of the chromatin Fertility and Sterility
material (33). Increased intracellular calcium is associated with nuclear envelope indentations and a condensed chromatin. Thus, the nuclear abnormalities in uteri with adenomyosis could potentially be explained by abnormal contractility. The increase in intracellular aggregates suggest an increase in intermediate filaments (14), as demonstrated by vimentin and desmin immunohistochemical staining (34). It is plausible that these filament aggregates could be related to an increased synthetic activity in the myocyte, as evidenced by the observed cellular hypertrophy, expanded cytoplasm, and by the increase in ribosomes and rough endoplasmic reticulum. The net effect can be an imbalance between the production and turnover of the cytoskeletal components. The sarcolemma of smooth muscle cells is divided into two structurally distinct regions: those bearing submembranous dense plaques and intervening zones that bear many vesicular invaginations or caveolae. The dense bands are junctions of the adherens type, serve as anchorage sites for actin cytoskeleton, and are typically marked by antibodies to vinculin (35). Caveolae have been implicated in a wide range of cellular functions. Caveolae contain a host of receptors, second messenger generators, G proteins, kinases, and ion channels in close proximity. Caveolae are often in close proximity to sarcoplasmic reticulum or mitochondria, and have been proposed to organize signaling molecules (36, 37). The increased length of the dense bands that anchor intracellular myofilaments could reflect an increase in cytoskeletal filaments (14). Abnormally shaped mitochondria with unfolded cristae suggest an abnormality in active cellular processes or the initiation of a degenerative process (14, 28). It is plausible that the main function of the JZ is concerned with preparation of the endometrium for implantation, sperm transport, and hemostasis during menstruation, with the outer myometrium being primary involved with parturition. It is also possible that the ultrastructural abnormalities observed with the sarcolemmal bands and caveolae may cause 2135
a disturbance in the normal calcium cycling in the affected myocytes, with a subsequent loss of normal rhythmic contractions. Chronic uterine dysfunctional peristalsis and hyperperistalsis have been proposed as causal factors for adenomyosis and endometriosis, because these result in dislocation of the basal endometrium in both the underlying myometrium and peritoneal cavity. Women with endometriosis displayed marked uterine hyperperistalsis that differs significantly from the peristalsis in unaffected women during the early, midfollicular, and midluteal phases (6). In conclusion, this study demonstrated that the myocytes of uteri harboring adenomyosis are ultrastructurally different from those of normal uteri. These ultrastructural changes suggest a possible defect in myometrial contractility. Dysfunctional contractility could be the result of the presence of adenomyosis or could contribute to its pathogenesis. Acknowledgements: We would like to thank Dr. Brian Eyden, (Consultant Histopathologist, Christie Hospital NHS Trust, Manchester, United Kingdom) for his helpful comments on the manuscript. We would also like to thank Natalie Allcock and Stefan Hyman (Electron Microscopy Services, University of Leicester, Leicester, United Kingdom) for technical assistance.
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