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Steroid hormone-dependent myometrial zonal differentiation in the non-pregnant human uterus
European Journal of Obstetrics & Gynecology and Reproductive Biology 81 (1998) 247–251
Steroid hormone-dependent myometrial zonal differentiation in the nonpregnant human uterus a, b c Jan J. Brosens *, Nandita M. de Souza , Fred G. Barker a b
Institute of Obstetrics and Gynaecology, Imperial College School of Medicine at Hammersmith Hospital, Du Cane Road, London W12 ONN, UK The Robert Steiner Magnetic Resonance Unit, Imperial College School of Medicine at Hammersmith Hospital, Du Cane Road, London W12 ONN, UK c Department of Pathology, Hillingdon Hospital, London, UK
Keywords: Myometrial zonal anatomy; MR imaging; Smooth muscle differentiation
1. Introduction It has long been recognized, but not always appreciated, that the myometrium is not an homogenous smooth muscle mass but a complex structure consisting of distinct zones. The zonal structure is recognized on magnetic resonance (MR) imaging where the disruption of the normal myometrial zonal anatomy is a feature of many uterine pathologies including endometrial cancer, fibroids, molar pregnancy and adenomyosis. This review examines the evidence for structural and functional differences between the inner and outer myometrial layers. The mechanisms that may govern myometrial zonal differentiation are also discussed.
2. Myometrial zonal differentiation
2.1. Macroscopy and microscopy of the non-gravid myometrium Classically, three distinct architectural layers are discerned in the adult human myometrium. There is a thin external layer that consists mainly of longitudinal fibers that pass over the fundus and converge at the cornua. The intermediate layer is thickest and consists of two groups of symmetrical smooth muscle, one arising from each cornu. The two systems interdigitate in the midsagittal plane of the uterus. Finally, there is an inner, circular layer of more transverse orientated smooth muscle fibers. A tripartite zonation of human uterine musculature can also be appreciated by light-microscopy: the subendometri*Corresponding author. Fax: 144 181 7437171.
al region or stratum submucosum is characterised by densely packed myocytes, in the middle layer or stratum vasculare the arcuate arteries branch through poorly orientated and loosely organised smooth muscle bundles and the thin outer layer or stratum subserosum consists of more dense and better orientated smooth muscle cells [1].
2.2. MR studies of the human myometrium The in vivo myometrial zonal anatomy was first described by Hricak et al. [2] in 1983 using MR imaging. In women of reproductive age, three layers can be distinguished in the myometrium on T2-weighted MR images: surrounding the high-signal intensity endometrial stripe there is a low-signal-intensity junctional zone followed by an outer intermediate-signal-intensity zone and a thin lowsignal-intensity subserosal zone (Fig. 1). MR imaging therefore not only confirms the concept of myometrium zonation as inferred from histological and anatomical observations, but also allows assessment of the changes in the myometrial zonal anatomy in response to physiological and pathological processes. The contrast in signal intensity between the outer myometrium (OM) and junctional zone (JZ) is a striking feature on T2-weighted scans and the reason for this difference in signal intensity has been the cause of considerable debate. Although morphometric studies have confirmed that the JZ represents the innermost layer of the myometrium the difference in signal intensities between the myometrial zones cannot be explained simply on the basis of myocyte density or orientation. Initially, it was postulated that the zonal polarity on MR images resulted from a higher vascular perfusion rate in the stratum
0301-2115 / 98 / $ – see front matter 1998 Elsevier Science Ireland Ltd. All rights reserved. PII: S0301-2115( 98 )00198-5
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J. J. Brosens et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 81 (1998) 247 – 251
Fig. 1. Sagital T2 weighted spin-echo (2500 / 80 ms [TR / TE]) image through the pelvis in a normal volunteer. The uterus is anteverted and the zonal anatomy can be distinguished. The endometrium is a high signal stripe (short arrow), surrounded by the low signal junctional zone (arrowhead). The outer myometrium is high signal (curved arrow) while the subserosal layer is low in signal (open arrow).
submucosum [3], but this was refuted by Scoutt et al. who demonstrated that the T1 and T2 values of unfixed myometrium were stable for at least 6 h after hysterectomy [4]. Using image analysis of histological sections the same group demonstrated that, in comparison with the outer myometrium, the JZ is characterised by a threefold increase in nuclear area and decreased extracellular matrix per unit volume. The increased nuclear area reflects both a higher smooth muscle density and an increased nucleocytoplasmatic ratio of the myocytes [4]. This latter observation is important as it demonstrates that there are not only architectural differences but also cellular differences between myocytes in the JZ and OM.
2.3. The myometrial zonal anatomy is sex steroid dependent Uterine zonal anatomy is dependent on gonadal hormones [5–7]. In premenarchal girls and postmenopausal women, the zonal anatomy is often indistinct with a comparatively low signal intensity from the myometrium. Ovarian suppression with gonadotrophin-releasing hormone analogues leads to an MR appearance of the uterus mimicking that of postmenopausal women; while hormone replacement therapy in postmenopausal women results in re-appearance of myometrial zonal anatomy [5]. Further evidence for hormonal responsiveness of the JZ is provided by the work of Wiczyk et al. [8] who performed MR scans on five volunteers with normal
ovulatory cycles on days 4, 8, 12, 16, 20 and 24 of the cycle. They demonstrated JZ thickness changes throughout the menstrual cycle in conjunction with endometrial thickness changes. The endometrium increased from 5.861.1 mm on day 4 to a mean peak of 10.361.7 mm on day 24 with the greatest growth occurring from day 8 to 16. Although the JZ showed substantially less growth, the pattern of growth was similar. Maximal increase of JZ thickness was observed between day 8 and 16 (5.160.7 mm to 6.760.7 mm) of the cycle which correlated with high circulating oestradiol levels. With the onset of the menstrual phase the JZ thickness decreases. These discreet cyclic variations in JZ thickness may be related to vascular changes in the inner myometrium or the presence of myofibroblasts at the endometrio–myometrial interface. Fuji et al. [9] reported that these immature myofibroblasts have more characteristics of smooth muscle cells in the luteal phase than in the follicular phase of the cycle, indicating that active metaplasia between the stromal cell and inner myocyte compartments may occur at the endometrio–myometrial junction throughout the menstrual cycle. The myometrial zonal anatomy changes profoundly during pregnancy. Focal disruption of the JZ has been observed as early as seven days after ovulation in a patient on whom serial MR scans were performed fortuitously during a conception cycle [10]. Interestingly, focal disruption of the JZ does not occur with ectopic pregnancies, suggesting that local factors released at the implantation site mediate this effect [11]. During pregnancy, the junctional zone increases in signal intensity and the zonal differences becomes indistinct. The normal zonal anatomy gradually reappears within six months of delivery [12].
3. Functional polarity of the human non-gravid myometrium Since the first studies on non-pregnant uterine contractility were carried out in 1889 by Heinricius, it has been recognised that the uterus contracts spontaneously throughout the menstrual cycle [13]. With the introduction of endovaginal ultrasonography, almost a century later, it has been observed that these contractions emanate only from the JZ and that their orientation, amplitude and frequency correlate with the phase of the menstrual cycle. In the follicular and periovulatory phases cervico-fundal subendometrial contractions can be seen, the amplitude and frequency of which increase notably towards ovulation. Short, asymmetrical myometrial waves are present during the luteal phase, but during menstruation propagated fundo-cervical subendometrial contractions waves are noted [14–16]. Kunz et al. used hysteroscintigraphy to demonstrate that rapid sperm transport through the female genital tract in the pre-ovulatory phase is provided by these subendometri-
J. J. Brosens et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 81 (1998) 247 – 251
al cervico-fundal contraction waves [16]. Others have postulated that the asymmetrical myometrial peristalsis during the luteal phase serves to maintain the developing blastocyst within the uterine fundus. Fundo-cervical contractions during menstruation are likely to limit retrograde menstruation, controlling menstrual flow and enhancing venous retrurn. Inner myometrial hyperperistalsis has been demonstrated in women suffering from excessive menstrual loss [17], and JZ dysfunction has also been implicated in the pathogenesis of pelvic endometriosis and subfertility [18–20]. A recent study found marked hyperperistalsis during the early and mid-follicular phase and dysperistalsis during the late follicular phase in women with endometriosis. Hysterosalpingoscintigraphy studies in women with endometriosis has shown that the hyperperistalsis is associated with a marked increase in the transport of inert particles from the vaginal depot to the peritoneal cavity while the peri-ovulatory dysperistalsis resulted in a significant decrease of uterine transport capacity in comparison with healthy controls [19]. In vitro studies also have confirmed the functional polarity of the myometrium. In non-pregnant uteri, sections taken from the OM show spontaneous strong regular contractions which can be amplified by adrenaline or, to a lesser extent by oxytocin. However, in contrast to the in vivo observations, muscle strips from the JZ display very few spontaneous contractions and are largely unresponsive to adrenaline and oxytocin stimulation [21].
4. Mechanism(s) controlling myometrial zonal differentiation
4.1. Structural differentiation of the JZ and OM The mechanisms that govern myometrial zonal differentiation are unknown. Ontogeneic studies of human myometrium suggest that cells from the inner mesechymal layer around the fused paramesonephric ducts may be the progenitors of both endometrial stromal cells and inner myometrial smooth muscle cells, while cells of the outer mesechymal layer are the precursors of the OM [21]. The independent cytodifferentiation of the myometrial zones in the foetus opens the possibility that smooth muscle proliferation and differentiation in both zones remain differentially regulated in the adult uterus. Alternatively, it is possible that factors released by the endometrium have a direct juxtacrine effect on the function and structure of the underlying myometrium. For instance the subendometrial myometrium expresses significantly higher levels of oestrogen receptor (OR), as measured by immunoassay and by immunocytochemistry, than the OM [22]. Interferon-gamma (IFN-g) has been shown to increase OR expression in ZR75-1 cells, an oestrogen responsive breast cancer cell line [23]. In the uterus, IFN-g is predominantly produced by activated CD3-positive T
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cells which are found in characteristic lymphoid aggregates in the endometrio–myometrial junction [24,25]. IFN-g also attenuates the action of certain cytokines, such as TGF-b, which have been implicated in myocyte proliferation [26] and endometrial stromal cell function [27]. Although the role of IFN-g and other cytokines released by the basal endometrium in the induction of a specific inner myometrial micro-environment remains speculative, the thickness of the JZ and its gradual blending with the OM is in keeping with an effect of locally produced factor(s) which diminishes with increasing distance from the site of production.
4.2. Inner myometrial peristalsis The mechanisms that govern the cycle-dependent contractions of the JZ in the non-gravid uterus are also not understood. However, it appears likely that the symmetrical, high-amplitude propagated contraction waves in the late follicular phase require electrical and mechanical coupling of myocytes in the JZ. Gap junctions are intercellular communication channels and their expression is required for coordinated synchronous myometrial contractions. In the myometrium, the major gap junction protein is connexin-43 (Cx-43) and its expression is thought to be under oestrogenic control [28,29]. The higher level of OR expression in the JZ in the proliferative phase may thus represent a preferential target for oestrogen regulation of Cx-43. In contrast, progesterone [30], human chorionic gonadotropin [31] and activators of the protein kinase A pathway [32] are thought to inhibit Cx-43 expression in myometrium. Analysis of the 59-flanking promoter region of the Cx-43 gene, however, showed no consensus binding sites for OR or progesterone receptor (PR) indicating that the effects of steroid receptor action may be mediated through interaction with other signalling pathways [33]. Transient transfection studies in myocytes using 2.8 kb of the Cx-43 promoter region linked to a reporter gene have demonstrated that transcriptional activation is dependent on binding of the activation protein-1 (AP-1), a composite transcription factor consisting of c-Jun and c-Fos, to AP-1 DNA binding motif in the proximal promoter region [33]. In human myocytes, cytokines and factors that activate the protein kinase C (PKC) pathway result in increased c-Fos and c-Jun levels and subsequent enhanced Cx-43 protein levels. Although OR activation can results in elevated c-Fos levels and in stabilisation of the AP-1 complex these effects appear cell specific [34]. For instance, Douall-Bell et al. [35] have demonstrated that in the bovine uterus Cx-43 expression in the inner but not outer myocytes can be blocked by pure anti-oestrogen. Although differential zonal regulation of Cx-43 has been demonstrated in the bovine myometrium [35], to the best of our knowledge, no studies have as yet addressed the regulation of the connexin proteins in the inner and outer human non-gravid myometrium.
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Although gap junctions are necessary, they alone are not sufficient to trigger coordinated contractions. Several cytokines have been identified which might modulate the myometrial peristalsis. For instance, epidermal growth factor is not only a potent mitogen for myometrial smooth muscle cells but it can also induce uterine contractions in vitro, both in intact tissue and isolated myometrial cells [36]. Specific binding sites for endothelins have also been found in the myometrium, and in tissue bath experiments, endothelin-1 markedly increased contractility of myometrial strips, an effect mediated through the endothelin-A receptor [37]. Interestingly, the greatest density of endothelin binding sites is found on glandular epithelium in the endometrio–myometrial junction and recent evidence suggests that endothelins in the endometrium can induce prostaglandin F2a and further endothelins release, in a paracrine and autocrine fashion [37]. These factors are potent uterotonins in non-gravid uteri and may therefore mediate the contractions of the underlying myometrium in a juxtacrine fashion.
[5] [6]
[7]
[8]
[9]
[10]
[11]
[12]
5. Conclusions [13]
A well-developed understanding of structure is often a prerequisite to the understanding of function. We have summarised the evidence for structural zonal differentiation of the human adult myometrium and emphasised the functional role of the inner myometrial contrractility in the non-gravid uterus. Although it is possible that myometrial zonal differentiation is the result of intrinsic cellular differences between the inner and outer myocytes, growing evidence indicate that a specific subendometrial microenvironment may be effected through complex interactions between ovarian sex steroid hormones and cytokines and uterotonins produced locally at the endometrio–myometrial interface. Clinically, disruption of the normal myometrial zonal differentiation process and function has been associated with a growing number of diseases such as dysfunctional uterine bleeding, adenomyosis, endometriosis and subfertility, emphasising the need to determine the exact regulatory pathways underpinning steroid hormone-dependent myometrial zonal differentiation.
References
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21] [22]
[1] Schwalm H, Dubrauszky V. The structure of the musculature of the human uterus: muscles and connective tissue. Am J Obstet Gynecol 1966;94:391–404. [2] Hricak H, Alpers C, Crooks LE, Sheldon PE. Magnetic resonance imaging of the female pelvis: initial experience. Am J Radiol 1983;141:1119–28. [3] Lee JKT, Gersell D, Balfe DM, Worthington JL, Picus D, Gapp G. The uterus: in vitro MR-anatomic correlation of normal and abnormal specimens. Radiology 1985;157:175–9. [4] Scoutt LM, Flynn SD, Luthringer DJ, McCauley TR, McCarthy SM.
[23]
[24]
Junctional zone of the uterus: correlation of MRI and histologic examination of hysterectomy specimen. Radiology 1991;179:403–7. Demas BE, Hricak H, Jaffe RB. Uterine MR imaging: effects of hormonal stimulation. Radiology 1985;159:123–6. McCarthy S, Tauber C, Gore J. Female pelvic anatomy: MR assessment of variations during the menstrual cycle and with use of oral contraceptives. Radiology 1986;160:119–23. Andreyko JL, Blumenfeld Z, Marshall LA, et al. Use of an agonistic analog of GnRH (NAFARELIN) to treat leiomyomata: Assessment by magnetic resonance imaging. Am J Obstet Gynecol 1988;158:903–10. Wiczyk HP, Janus CL, Richards CJ, Graf MJ, Gendal ES, Rabinwitz JG, Laufer N. Comparison of magnetic resonance imaging and ultrasound in evaluating follicular and endometrial development throughout the normal cycle. Fertil Steril 1988;49:969–72. Fuji S, Konishi I, Mori T. Smooth muscle differentiation at endometrio–myometrial junction: an ultrasound study. Virchows Archiv A Pathol Anat 1989;414:105–12. Turnbull LW, Manton DJ, Horsman A, Killick SR, Magnetic resonance imaging changes in uterine zonal anatomy during a conception cycle. Br J Obstet Gynaecol 1995;102:330–33. Barton JW, McCarthy SM, Kohorn EI, Scoutt LM, Lange RC. Pelvic MR imaging findings in gestational trophoblastic disease, incomplete abortion and ectopic pregnancy: are the specific?. Radiology 1993;186:163–8. Willms AB, Brown ED, Kettritz UI, Kuller JA, Semelka RC. Anatomic changes in the pelvis after uncomplicated vaginal delivery: evaluation with serial MR imaging. Radiology 1995;195:91–4. Heinricius G. En metod att grafiskt atergiva Kontraktioner hos en icke gravid livmoder. Finsk Lakaresallsk Handl 1889;31:349–53. de Vries K, Lyons EA, Ballard G, Levi CS, Lindsay DJ. Contractions of the inner third of the myometrium. Am J Obstet Gynecol 1990;162:679–82. Lyons EA, Taylor PJ, Zheng XH, Ballard G, Levi CS, Kredenster JV. Characterization of subendometrial myometrial contractions throughout the menstrual cycle in normal fertile women. Fertil Steril 1991;55:771–4. Kunz G, Beil D, Deininger H, Wildt L, Leyendecker G. The dynamics of rapid sperm transport through the femal genital tract: evidence from vaginal sonogrophy of uterine peristalsis and hysterosalpingoscintigraphy. Hum Reprod 1996;11:627–32. Chalubinski K, Deutinger J, Bernaschek G. Vaginosonography for recording of cycle-related myometrial contractions. Fertil Steril 1993;59:225–8. ´ E. Subendometrial contractility in menstrual Salamanca A, Beltran phase visualized by transvaginal sonography in patients with endometriosis. Fertil Steril 1995;64:193–5. Leyendecker G, Kunz G, Wildt L, Beil D, Deininger H. Uterine hyperperistalsis and dysperistalsis as dysfunctions of the mechanism of rapid sperm transport in patient with endometriosis and infertility. Hum Reprod 1996;11:1542–51. Ijland MM, Evers JLH, Dunselman GAJ, Volovics L, Hoogland HJ. Relation between endometrial wavelike activity and fecundability in spontaneous cycles. Fertil Steril 1997;67:492–6. Daels J. Uterine contractility patterns of the outer and inner zones of the myometrium. J Obstet Gynecol 1974;44:315–26. Richards PA, Tiltman AJ. Anatomical variation of the estrogenreceptor in normal myometrium. Virchows Arch A Pathol Anat 1995;427:303–7. Solary E, Prud’Homme JF, Maggelenat H, Calvo F. Modulation of proliferation, estrodial receptors and estrogen regulated protein PS2 / BCEI in human breast cancer cell lines by gamma interferon. J Br Regul Homeost Agents 1991;5:98–106. Stewart CJR, Farquharson MA, Foulis AK. The distribution and possible function of gamma interferon-immunoreactive cells in normal endometrium and myometrium. Virchows Arch A Pathol Anat 1992;420:419–24.
J. J. Brosens et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 81 (1998) 247 – 251 [25] Tabibzadeh S, Sun XZ, Kong QF, Kasnic G, Miller J, Satyaswaroop PG. Induction of a polarized micro-environment by human T cells and interferon-gamma in three-dimensional spheroid cultures of human endometrial epithelial cells. Human Reprod 1993;8:182–93. [26] Ishikawa O, LeRoy EC, Trojanowska M. Mitogenic effect of transforming growth factor b1 on human fibroblasts involves the induction of platelet-derived growth factor a receptors. J Cell Physiol 1990;145:181–6. [27] Arici A, MacDonald PC, Casey ML. Modulation of the levels of interleukin-8 messenger ribonucleic acid and interleukin-8 protein synthesis in human endometrial stromal cells by transforming growth factor-b1. J Clin Endocrinol Metab 1996;81:3004–9. [28] Garfield RE, Kannan MS, Daniel EE. Gap junction formation in myometrium: control by estrogen, progesterone and prostaglandins. Am J Physiol 1980;238:C81–9. [29] Petrocelli T, Lye SJ. Regulation of transcripts encoding the myometrial gap junction protein, connexin-43, by estrogen and progesterone. Endocrinology 1993;133:284–90. [30] Zhao K, Kuperman L, Geimonen E, Andersen J. Progestin represses human connexin-43 gene expression similarely in primary cultures of myometrial and uterine leiomyoma cells. Biol Reprod 1996;54:607–15. [31] Ambrus G, Rhao ChV. Novel regulation of pregnant human myometrial smooth muscle cells gap junctions by human chorionic gonadotropin. Endocrinology 1994;135:2772–9.
251
[32] Sakai N, Tabb T, Garfield RE. Studies of connexin 43 and cell-tocell coupling in cultured human uterine smooth muscle. Am J Obstet Gynecol 1992;167:1267–77. [33] Geimonen E, Jiang W, Ali M, Fisherman GI, Garfield RE, Andersen J. Activation of protein-kinase-C in human uterine smooth muscle induces connexin-43 gene-transcription through an AP-1 site in the promoter sequence. J Biol Chem 1996;271:23667–74. [34] Webb P, Lopez GN, Uht RM, Kusher PJ. Tamoxifen activation of the estrogen receptor /AP-1 pathway: potential origin for the cellspecific estrogen-like effects of antiestrogens. Mol Endocrinol 1995;9:443–56. [35] Doualla-Bell F, Lye SJ, labrie F, Fortier MA. Differential expression and regulation of connexin-43 and cell-cell coupling in myocytes from circular and longitudinal layers of the bovine myometrium. Endocrinology 1995;136:5322–8. [36] Gardner RM, Stancel GM. Blockade of epidermal growth factorinduced uterine contractions by indomethacin and nordihydroguaritic acid. J Pharmacol Exp Ther 1989;258:882–6. [37] Bacon CR, Morrison JJ, O’Reilly G, Cameron IT, Davenport AP. ETA and ETB endothelin receptors in human myometrium characterized by the subtype selective ligands BQ123, BQ3020, FR139317 and PD151242. J Endocrinol 1995;144:127–34.
Steroid hormone-dependent myometrial zonal differentiation in the nonpregnant human uterus a, b c Jan J. Brosens *, Nandita M. de Souza , Fred G. Barker a b
Institute of Obstetrics and Gynaecology, Imperial College School of Medicine at Hammersmith Hospital, Du Cane Road, London W12 ONN, UK The Robert Steiner Magnetic Resonance Unit, Imperial College School of Medicine at Hammersmith Hospital, Du Cane Road, London W12 ONN, UK c Department of Pathology, Hillingdon Hospital, London, UK
Keywords: Myometrial zonal anatomy; MR imaging; Smooth muscle differentiation
1. Introduction It has long been recognized, but not always appreciated, that the myometrium is not an homogenous smooth muscle mass but a complex structure consisting of distinct zones. The zonal structure is recognized on magnetic resonance (MR) imaging where the disruption of the normal myometrial zonal anatomy is a feature of many uterine pathologies including endometrial cancer, fibroids, molar pregnancy and adenomyosis. This review examines the evidence for structural and functional differences between the inner and outer myometrial layers. The mechanisms that may govern myometrial zonal differentiation are also discussed.
2. Myometrial zonal differentiation
2.1. Macroscopy and microscopy of the non-gravid myometrium Classically, three distinct architectural layers are discerned in the adult human myometrium. There is a thin external layer that consists mainly of longitudinal fibers that pass over the fundus and converge at the cornua. The intermediate layer is thickest and consists of two groups of symmetrical smooth muscle, one arising from each cornu. The two systems interdigitate in the midsagittal plane of the uterus. Finally, there is an inner, circular layer of more transverse orientated smooth muscle fibers. A tripartite zonation of human uterine musculature can also be appreciated by light-microscopy: the subendometri*Corresponding author. Fax: 144 181 7437171.
al region or stratum submucosum is characterised by densely packed myocytes, in the middle layer or stratum vasculare the arcuate arteries branch through poorly orientated and loosely organised smooth muscle bundles and the thin outer layer or stratum subserosum consists of more dense and better orientated smooth muscle cells [1].
2.2. MR studies of the human myometrium The in vivo myometrial zonal anatomy was first described by Hricak et al. [2] in 1983 using MR imaging. In women of reproductive age, three layers can be distinguished in the myometrium on T2-weighted MR images: surrounding the high-signal intensity endometrial stripe there is a low-signal-intensity junctional zone followed by an outer intermediate-signal-intensity zone and a thin lowsignal-intensity subserosal zone (Fig. 1). MR imaging therefore not only confirms the concept of myometrium zonation as inferred from histological and anatomical observations, but also allows assessment of the changes in the myometrial zonal anatomy in response to physiological and pathological processes. The contrast in signal intensity between the outer myometrium (OM) and junctional zone (JZ) is a striking feature on T2-weighted scans and the reason for this difference in signal intensity has been the cause of considerable debate. Although morphometric studies have confirmed that the JZ represents the innermost layer of the myometrium the difference in signal intensities between the myometrial zones cannot be explained simply on the basis of myocyte density or orientation. Initially, it was postulated that the zonal polarity on MR images resulted from a higher vascular perfusion rate in the stratum
0301-2115 / 98 / $ – see front matter 1998 Elsevier Science Ireland Ltd. All rights reserved. PII: S0301-2115( 98 )00198-5
248
J. J. Brosens et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 81 (1998) 247 – 251
Fig. 1. Sagital T2 weighted spin-echo (2500 / 80 ms [TR / TE]) image through the pelvis in a normal volunteer. The uterus is anteverted and the zonal anatomy can be distinguished. The endometrium is a high signal stripe (short arrow), surrounded by the low signal junctional zone (arrowhead). The outer myometrium is high signal (curved arrow) while the subserosal layer is low in signal (open arrow).
submucosum [3], but this was refuted by Scoutt et al. who demonstrated that the T1 and T2 values of unfixed myometrium were stable for at least 6 h after hysterectomy [4]. Using image analysis of histological sections the same group demonstrated that, in comparison with the outer myometrium, the JZ is characterised by a threefold increase in nuclear area and decreased extracellular matrix per unit volume. The increased nuclear area reflects both a higher smooth muscle density and an increased nucleocytoplasmatic ratio of the myocytes [4]. This latter observation is important as it demonstrates that there are not only architectural differences but also cellular differences between myocytes in the JZ and OM.
2.3. The myometrial zonal anatomy is sex steroid dependent Uterine zonal anatomy is dependent on gonadal hormones [5–7]. In premenarchal girls and postmenopausal women, the zonal anatomy is often indistinct with a comparatively low signal intensity from the myometrium. Ovarian suppression with gonadotrophin-releasing hormone analogues leads to an MR appearance of the uterus mimicking that of postmenopausal women; while hormone replacement therapy in postmenopausal women results in re-appearance of myometrial zonal anatomy [5]. Further evidence for hormonal responsiveness of the JZ is provided by the work of Wiczyk et al. [8] who performed MR scans on five volunteers with normal
ovulatory cycles on days 4, 8, 12, 16, 20 and 24 of the cycle. They demonstrated JZ thickness changes throughout the menstrual cycle in conjunction with endometrial thickness changes. The endometrium increased from 5.861.1 mm on day 4 to a mean peak of 10.361.7 mm on day 24 with the greatest growth occurring from day 8 to 16. Although the JZ showed substantially less growth, the pattern of growth was similar. Maximal increase of JZ thickness was observed between day 8 and 16 (5.160.7 mm to 6.760.7 mm) of the cycle which correlated with high circulating oestradiol levels. With the onset of the menstrual phase the JZ thickness decreases. These discreet cyclic variations in JZ thickness may be related to vascular changes in the inner myometrium or the presence of myofibroblasts at the endometrio–myometrial interface. Fuji et al. [9] reported that these immature myofibroblasts have more characteristics of smooth muscle cells in the luteal phase than in the follicular phase of the cycle, indicating that active metaplasia between the stromal cell and inner myocyte compartments may occur at the endometrio–myometrial junction throughout the menstrual cycle. The myometrial zonal anatomy changes profoundly during pregnancy. Focal disruption of the JZ has been observed as early as seven days after ovulation in a patient on whom serial MR scans were performed fortuitously during a conception cycle [10]. Interestingly, focal disruption of the JZ does not occur with ectopic pregnancies, suggesting that local factors released at the implantation site mediate this effect [11]. During pregnancy, the junctional zone increases in signal intensity and the zonal differences becomes indistinct. The normal zonal anatomy gradually reappears within six months of delivery [12].
3. Functional polarity of the human non-gravid myometrium Since the first studies on non-pregnant uterine contractility were carried out in 1889 by Heinricius, it has been recognised that the uterus contracts spontaneously throughout the menstrual cycle [13]. With the introduction of endovaginal ultrasonography, almost a century later, it has been observed that these contractions emanate only from the JZ and that their orientation, amplitude and frequency correlate with the phase of the menstrual cycle. In the follicular and periovulatory phases cervico-fundal subendometrial contractions can be seen, the amplitude and frequency of which increase notably towards ovulation. Short, asymmetrical myometrial waves are present during the luteal phase, but during menstruation propagated fundo-cervical subendometrial contractions waves are noted [14–16]. Kunz et al. used hysteroscintigraphy to demonstrate that rapid sperm transport through the female genital tract in the pre-ovulatory phase is provided by these subendometri-
J. J. Brosens et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 81 (1998) 247 – 251
al cervico-fundal contraction waves [16]. Others have postulated that the asymmetrical myometrial peristalsis during the luteal phase serves to maintain the developing blastocyst within the uterine fundus. Fundo-cervical contractions during menstruation are likely to limit retrograde menstruation, controlling menstrual flow and enhancing venous retrurn. Inner myometrial hyperperistalsis has been demonstrated in women suffering from excessive menstrual loss [17], and JZ dysfunction has also been implicated in the pathogenesis of pelvic endometriosis and subfertility [18–20]. A recent study found marked hyperperistalsis during the early and mid-follicular phase and dysperistalsis during the late follicular phase in women with endometriosis. Hysterosalpingoscintigraphy studies in women with endometriosis has shown that the hyperperistalsis is associated with a marked increase in the transport of inert particles from the vaginal depot to the peritoneal cavity while the peri-ovulatory dysperistalsis resulted in a significant decrease of uterine transport capacity in comparison with healthy controls [19]. In vitro studies also have confirmed the functional polarity of the myometrium. In non-pregnant uteri, sections taken from the OM show spontaneous strong regular contractions which can be amplified by adrenaline or, to a lesser extent by oxytocin. However, in contrast to the in vivo observations, muscle strips from the JZ display very few spontaneous contractions and are largely unresponsive to adrenaline and oxytocin stimulation [21].
4. Mechanism(s) controlling myometrial zonal differentiation
4.1. Structural differentiation of the JZ and OM The mechanisms that govern myometrial zonal differentiation are unknown. Ontogeneic studies of human myometrium suggest that cells from the inner mesechymal layer around the fused paramesonephric ducts may be the progenitors of both endometrial stromal cells and inner myometrial smooth muscle cells, while cells of the outer mesechymal layer are the precursors of the OM [21]. The independent cytodifferentiation of the myometrial zones in the foetus opens the possibility that smooth muscle proliferation and differentiation in both zones remain differentially regulated in the adult uterus. Alternatively, it is possible that factors released by the endometrium have a direct juxtacrine effect on the function and structure of the underlying myometrium. For instance the subendometrial myometrium expresses significantly higher levels of oestrogen receptor (OR), as measured by immunoassay and by immunocytochemistry, than the OM [22]. Interferon-gamma (IFN-g) has been shown to increase OR expression in ZR75-1 cells, an oestrogen responsive breast cancer cell line [23]. In the uterus, IFN-g is predominantly produced by activated CD3-positive T
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cells which are found in characteristic lymphoid aggregates in the endometrio–myometrial junction [24,25]. IFN-g also attenuates the action of certain cytokines, such as TGF-b, which have been implicated in myocyte proliferation [26] and endometrial stromal cell function [27]. Although the role of IFN-g and other cytokines released by the basal endometrium in the induction of a specific inner myometrial micro-environment remains speculative, the thickness of the JZ and its gradual blending with the OM is in keeping with an effect of locally produced factor(s) which diminishes with increasing distance from the site of production.
4.2. Inner myometrial peristalsis The mechanisms that govern the cycle-dependent contractions of the JZ in the non-gravid uterus are also not understood. However, it appears likely that the symmetrical, high-amplitude propagated contraction waves in the late follicular phase require electrical and mechanical coupling of myocytes in the JZ. Gap junctions are intercellular communication channels and their expression is required for coordinated synchronous myometrial contractions. In the myometrium, the major gap junction protein is connexin-43 (Cx-43) and its expression is thought to be under oestrogenic control [28,29]. The higher level of OR expression in the JZ in the proliferative phase may thus represent a preferential target for oestrogen regulation of Cx-43. In contrast, progesterone [30], human chorionic gonadotropin [31] and activators of the protein kinase A pathway [32] are thought to inhibit Cx-43 expression in myometrium. Analysis of the 59-flanking promoter region of the Cx-43 gene, however, showed no consensus binding sites for OR or progesterone receptor (PR) indicating that the effects of steroid receptor action may be mediated through interaction with other signalling pathways [33]. Transient transfection studies in myocytes using 2.8 kb of the Cx-43 promoter region linked to a reporter gene have demonstrated that transcriptional activation is dependent on binding of the activation protein-1 (AP-1), a composite transcription factor consisting of c-Jun and c-Fos, to AP-1 DNA binding motif in the proximal promoter region [33]. In human myocytes, cytokines and factors that activate the protein kinase C (PKC) pathway result in increased c-Fos and c-Jun levels and subsequent enhanced Cx-43 protein levels. Although OR activation can results in elevated c-Fos levels and in stabilisation of the AP-1 complex these effects appear cell specific [34]. For instance, Douall-Bell et al. [35] have demonstrated that in the bovine uterus Cx-43 expression in the inner but not outer myocytes can be blocked by pure anti-oestrogen. Although differential zonal regulation of Cx-43 has been demonstrated in the bovine myometrium [35], to the best of our knowledge, no studies have as yet addressed the regulation of the connexin proteins in the inner and outer human non-gravid myometrium.
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Although gap junctions are necessary, they alone are not sufficient to trigger coordinated contractions. Several cytokines have been identified which might modulate the myometrial peristalsis. For instance, epidermal growth factor is not only a potent mitogen for myometrial smooth muscle cells but it can also induce uterine contractions in vitro, both in intact tissue and isolated myometrial cells [36]. Specific binding sites for endothelins have also been found in the myometrium, and in tissue bath experiments, endothelin-1 markedly increased contractility of myometrial strips, an effect mediated through the endothelin-A receptor [37]. Interestingly, the greatest density of endothelin binding sites is found on glandular epithelium in the endometrio–myometrial junction and recent evidence suggests that endothelins in the endometrium can induce prostaglandin F2a and further endothelins release, in a paracrine and autocrine fashion [37]. These factors are potent uterotonins in non-gravid uteri and may therefore mediate the contractions of the underlying myometrium in a juxtacrine fashion.
[5] [6]
[7]
[8]
[9]
[10]
[11]
[12]
5. Conclusions [13]
A well-developed understanding of structure is often a prerequisite to the understanding of function. We have summarised the evidence for structural zonal differentiation of the human adult myometrium and emphasised the functional role of the inner myometrial contrractility in the non-gravid uterus. Although it is possible that myometrial zonal differentiation is the result of intrinsic cellular differences between the inner and outer myocytes, growing evidence indicate that a specific subendometrial microenvironment may be effected through complex interactions between ovarian sex steroid hormones and cytokines and uterotonins produced locally at the endometrio–myometrial interface. Clinically, disruption of the normal myometrial zonal differentiation process and function has been associated with a growing number of diseases such as dysfunctional uterine bleeding, adenomyosis, endometriosis and subfertility, emphasising the need to determine the exact regulatory pathways underpinning steroid hormone-dependent myometrial zonal differentiation.
References
[14]
[15]
[16]
[17]
[18]
[19]
[20]
[21] [22]
[1] Schwalm H, Dubrauszky V. The structure of the musculature of the human uterus: muscles and connective tissue. Am J Obstet Gynecol 1966;94:391–404. [2] Hricak H, Alpers C, Crooks LE, Sheldon PE. Magnetic resonance imaging of the female pelvis: initial experience. Am J Radiol 1983;141:1119–28. [3] Lee JKT, Gersell D, Balfe DM, Worthington JL, Picus D, Gapp G. The uterus: in vitro MR-anatomic correlation of normal and abnormal specimens. Radiology 1985;157:175–9. [4] Scoutt LM, Flynn SD, Luthringer DJ, McCauley TR, McCarthy SM.
[23]
[24]
Junctional zone of the uterus: correlation of MRI and histologic examination of hysterectomy specimen. Radiology 1991;179:403–7. Demas BE, Hricak H, Jaffe RB. Uterine MR imaging: effects of hormonal stimulation. Radiology 1985;159:123–6. McCarthy S, Tauber C, Gore J. Female pelvic anatomy: MR assessment of variations during the menstrual cycle and with use of oral contraceptives. Radiology 1986;160:119–23. Andreyko JL, Blumenfeld Z, Marshall LA, et al. Use of an agonistic analog of GnRH (NAFARELIN) to treat leiomyomata: Assessment by magnetic resonance imaging. Am J Obstet Gynecol 1988;158:903–10. Wiczyk HP, Janus CL, Richards CJ, Graf MJ, Gendal ES, Rabinwitz JG, Laufer N. Comparison of magnetic resonance imaging and ultrasound in evaluating follicular and endometrial development throughout the normal cycle. Fertil Steril 1988;49:969–72. Fuji S, Konishi I, Mori T. Smooth muscle differentiation at endometrio–myometrial junction: an ultrasound study. Virchows Archiv A Pathol Anat 1989;414:105–12. Turnbull LW, Manton DJ, Horsman A, Killick SR, Magnetic resonance imaging changes in uterine zonal anatomy during a conception cycle. Br J Obstet Gynaecol 1995;102:330–33. Barton JW, McCarthy SM, Kohorn EI, Scoutt LM, Lange RC. Pelvic MR imaging findings in gestational trophoblastic disease, incomplete abortion and ectopic pregnancy: are the specific?. Radiology 1993;186:163–8. Willms AB, Brown ED, Kettritz UI, Kuller JA, Semelka RC. Anatomic changes in the pelvis after uncomplicated vaginal delivery: evaluation with serial MR imaging. Radiology 1995;195:91–4. Heinricius G. En metod att grafiskt atergiva Kontraktioner hos en icke gravid livmoder. Finsk Lakaresallsk Handl 1889;31:349–53. de Vries K, Lyons EA, Ballard G, Levi CS, Lindsay DJ. Contractions of the inner third of the myometrium. Am J Obstet Gynecol 1990;162:679–82. Lyons EA, Taylor PJ, Zheng XH, Ballard G, Levi CS, Kredenster JV. Characterization of subendometrial myometrial contractions throughout the menstrual cycle in normal fertile women. Fertil Steril 1991;55:771–4. Kunz G, Beil D, Deininger H, Wildt L, Leyendecker G. The dynamics of rapid sperm transport through the femal genital tract: evidence from vaginal sonogrophy of uterine peristalsis and hysterosalpingoscintigraphy. Hum Reprod 1996;11:627–32. Chalubinski K, Deutinger J, Bernaschek G. Vaginosonography for recording of cycle-related myometrial contractions. Fertil Steril 1993;59:225–8. ´ E. Subendometrial contractility in menstrual Salamanca A, Beltran phase visualized by transvaginal sonography in patients with endometriosis. Fertil Steril 1995;64:193–5. Leyendecker G, Kunz G, Wildt L, Beil D, Deininger H. Uterine hyperperistalsis and dysperistalsis as dysfunctions of the mechanism of rapid sperm transport in patient with endometriosis and infertility. Hum Reprod 1996;11:1542–51. Ijland MM, Evers JLH, Dunselman GAJ, Volovics L, Hoogland HJ. Relation between endometrial wavelike activity and fecundability in spontaneous cycles. Fertil Steril 1997;67:492–6. Daels J. Uterine contractility patterns of the outer and inner zones of the myometrium. J Obstet Gynecol 1974;44:315–26. Richards PA, Tiltman AJ. Anatomical variation of the estrogenreceptor in normal myometrium. Virchows Arch A Pathol Anat 1995;427:303–7. Solary E, Prud’Homme JF, Maggelenat H, Calvo F. Modulation of proliferation, estrodial receptors and estrogen regulated protein PS2 / BCEI in human breast cancer cell lines by gamma interferon. J Br Regul Homeost Agents 1991;5:98–106. Stewart CJR, Farquharson MA, Foulis AK. The distribution and possible function of gamma interferon-immunoreactive cells in normal endometrium and myometrium. Virchows Arch A Pathol Anat 1992;420:419–24.
J. J. Brosens et al. / European Journal of Obstetrics & Gynecology and Reproductive Biology 81 (1998) 247 – 251 [25] Tabibzadeh S, Sun XZ, Kong QF, Kasnic G, Miller J, Satyaswaroop PG. Induction of a polarized micro-environment by human T cells and interferon-gamma in three-dimensional spheroid cultures of human endometrial epithelial cells. Human Reprod 1993;8:182–93. [26] Ishikawa O, LeRoy EC, Trojanowska M. Mitogenic effect of transforming growth factor b1 on human fibroblasts involves the induction of platelet-derived growth factor a receptors. J Cell Physiol 1990;145:181–6. [27] Arici A, MacDonald PC, Casey ML. Modulation of the levels of interleukin-8 messenger ribonucleic acid and interleukin-8 protein synthesis in human endometrial stromal cells by transforming growth factor-b1. J Clin Endocrinol Metab 1996;81:3004–9. [28] Garfield RE, Kannan MS, Daniel EE. Gap junction formation in myometrium: control by estrogen, progesterone and prostaglandins. Am J Physiol 1980;238:C81–9. [29] Petrocelli T, Lye SJ. Regulation of transcripts encoding the myometrial gap junction protein, connexin-43, by estrogen and progesterone. Endocrinology 1993;133:284–90. [30] Zhao K, Kuperman L, Geimonen E, Andersen J. Progestin represses human connexin-43 gene expression similarely in primary cultures of myometrial and uterine leiomyoma cells. Biol Reprod 1996;54:607–15. [31] Ambrus G, Rhao ChV. Novel regulation of pregnant human myometrial smooth muscle cells gap junctions by human chorionic gonadotropin. Endocrinology 1994;135:2772–9.
251
[32] Sakai N, Tabb T, Garfield RE. Studies of connexin 43 and cell-tocell coupling in cultured human uterine smooth muscle. Am J Obstet Gynecol 1992;167:1267–77. [33] Geimonen E, Jiang W, Ali M, Fisherman GI, Garfield RE, Andersen J. Activation of protein-kinase-C in human uterine smooth muscle induces connexin-43 gene-transcription through an AP-1 site in the promoter sequence. J Biol Chem 1996;271:23667–74. [34] Webb P, Lopez GN, Uht RM, Kusher PJ. Tamoxifen activation of the estrogen receptor /AP-1 pathway: potential origin for the cellspecific estrogen-like effects of antiestrogens. Mol Endocrinol 1995;9:443–56. [35] Doualla-Bell F, Lye SJ, labrie F, Fortier MA. Differential expression and regulation of connexin-43 and cell-cell coupling in myocytes from circular and longitudinal layers of the bovine myometrium. Endocrinology 1995;136:5322–8. [36] Gardner RM, Stancel GM. Blockade of epidermal growth factorinduced uterine contractions by indomethacin and nordihydroguaritic acid. J Pharmacol Exp Ther 1989;258:882–6. [37] Bacon CR, Morrison JJ, O’Reilly G, Cameron IT, Davenport AP. ETA and ETB endothelin receptors in human myometrium characterized by the subtype selective ligands BQ123, BQ3020, FR139317 and PD151242. J Endocrinol 1995;144:127–34.