1 Departments of Obstetrics and Gynaecology and 2 Pathology, Klinikum Darmstadt Academic Teaching Hospital to the University of Frankfurt, 64283 Darmstadt, Germany
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Abstract |
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Key words: endometrialsubendometrial unit/immunocytochemistry/oestradiol and progesterone receptors/sperm transport/uterine peristalsis
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Introduction |
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The uterus is usually considered to be specialized, first, for the reception of the blastocyst by the endometrium and the continuous nourishment of the developing fetus and, second, for the eventual expulsion of the fetus. Furthermore, the uterine muscle is regarded to be normally functional for only a brief period following a lengthy gestation, unlike other smooth muscle organs (Garfield and Yallampalli, 1994; Romanini, 1994
). Recently, it became evident, however, that the uterus, especially the non-pregnant one, is not a quiescent organ but is rather actively involved in the very early processes of reproduction, in addition to providing the site of implantation. It could be demonstrated that the non-pregnant uterus acts as a peristaltic pump during the menstrual cycle with directed sperm transport as one of the main functions (Kunz et al., 1996
; Leyendecker et al., 1996
). Vaginal sonography of uterine peristaltic activity (Birnholz, 1984
; De Vries et al., 1990
; Lyons et al., 1991
; Kunz et al., 1996
; Leyendecker et al., 1996
) has shown that the uterine peristaltic waves, under physiological conditions, only involve the stratum subvasculare of the myometrium, thus attributing a special and separate function to this inner layer of the myometrium. Furthermore, there is indirect (Lyons et al., 1991
; Kunz et al., 1996
; Leyendecker et al., 1996
) and direct evidence (Kunz et al., 1998a
) that the uterine peristaltic activity is under the control of the ovarian dominant structure.
In view of these data it appeared to be necessary to re-examine the expression of the oestradiol and progesterone receptors in the different endometrial and myometrial layers during the menstrual cycle and in post-menopausal women. The data obtained were related to morphological, ontogenetic as well as phylogenetic data from the literature.
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Materials and methods |
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After resection, hysterectomy specimens were fixed with 4% buffered (phosphate-buffered saline, pH7.2; Merck, Darmstadt, Germany) formaldehyde for 24 h. At least two (left and right side) transmural uterine samples were taken from the mid-region of the uterine corpus. In some cases additional samples of the lower parts of the corpus were taken. From each of these samples 420 frontal sections were obtained that contained the whole uterine wall from the endometrium to the serosa or from the endocervix to the parametrium.
Immunostaining
Oestrogen and progesterone receptor immunocytochemistry was performed on paraffin-embedded sections (thickness 3 µm). After mounting, deparaffination and rehydration, unmasking of antigen was carried out using high-temperature techniques (Shi et al., 1991). The sections were then incubated with blocking serum (Vectastain Elite ABC kit, Vector Laboratories, Burlingame, CA, USA) for 30 min at 37°C. This was followed by overnight incubation at room temperature with primary anti-oestrogen-receptor mouse monoclonal antibody (Novocastra Laboratories, Newcastle upon Tyne, UK), developed against prokaryotic recombinant protein corresponding to the full-length oestrogen receptor molecule, at a dilution of 1:40 in Tris buffer (Siquia-Aldrich Chemicals, Deisenhofen, Germany) and primary anti-progesterone-receptor mouse monoclonal antibody (Novocastra Laboratories), developed against synthetic peptide corresponding to a site of predicted high antigenicity on the human progesterone receptor and binding to both known types of nuclear progesterone receptors (PR-A and PR-B; Viville et al., 1997
), dilution 1:40 in Tris buffer respectively. The sections were then incubated at 37°C with biotinylated secondary anti-mouse antibody followed by avidin biotin peroxidase complex (Vectastain Elite ABC, Vector Laboratories), each for 30 min in the first and 15 min in the second run and were washed with buffer after each step. The slides were flooded with freshly prepared diaminobenzidineimidazolehydrogen peroxide solution (Merck) for 45 min in the dark. After washing with water the sections were counterstained with haematoxylin, washed with water again, dehydrated (with ethylene alcohol and xylene; Merck) and mounted. Evaluation of this method was carried out by comparison of a series of 40 slides from the same specimen, which were treated under different unmasking conditions. Microwave heating at 600 W for 7 min was used, which gave the best morphological and staining results with the least cellular destruction. In order to obtain optimal staining differentiation, various concentrations of the primary antibodies were tested, with a dilution of 1:40 giving the best results.
Sections of receptor-positive mammary carcinomas served as positive controls; negative controls were made using sections of receptor-positive mammary carcinomas without incubation with primary antibody and several oestrogen-receptor-negative tissues (e.g. from tonsils and non-gynaecological carcinomas), which were prepared according to the standard protocol of oestrogen and progesterone receptor immunohistochemistry.
Evaluation
Results of specific staining were evaluated using a semiquantitative method. The staining intensity was graded as 0 = no, 1 = weak, 2 = moderate and 3 = strong staining respectively. The number of cells/area and the degree of positivity respectively for the endometrial glandular epithelium and stroma, as well as three myometrial regions, were compared. The myometrium of the stratum supravasculare (subserosal myometrium), of the stratum vasculare and of the stratum subvasculare (subendometrial myometrium) were counted out separately with high-power fields. The subserosal and the subendometrial endometrium were counted out completely. In the broad stratum vasculare at least 2025 high-power fields, which were evenly distributed along the transmural axis between the two other layers, were selected and counted out. The tissue was examined by a single observer and confirmed by a second observer. The immunoreactive score (IRS) was calculated using the following equation:
IRS = SPi (i + 1), where i = 1, 2 or 3, and Pi is the percentage of stained cells for each intensity, according to the method described by Lessey et al. (1988).
The immunoreactive score of positively stained cells per uterine region was determined by taking the arithmetic mean of the values of all counted high-power fields. There was a high reproducibility of the method in that, in a test series, the inter-assay variation over the full range of IRS never exceeded 10%.
The data obtained from individual uterine specimens of the pre-menopausal women were grouped according to phase of the menstrual cycle, with days 16 representing the early proliferative (n = 5), days 79 representing mid-proliferative (n = 6), days 1015 representing the peri-ovulatory (n = 5), days 1621 representing the early to mid-secretory (n = 5) and with days 2228 representing the mid- to late secretory phase (n = 6) of the cycle respectively.
Statistical analysis
Statistical analysis was performed with Student's t-test. Significance was assumed when P < 0.05.
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Results |
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Two specimens of the lower uterine portion were studied during the early follicular phase and two during the late secretory phase respectively, in addition to those specimens of the mid-region of the same uteri. There was no difference in staining characteristics of the layers along the longitudinal axis of the uterus.
Post-menopausal patients
There was maximal immunocytochemical expression in all layers for oestradiol and progesterone receptors.
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Discussion |
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The novel finding of this study, however, is the demonstration that there is no consistent pattern of steroid-receptor staining across the myometrial wall. While the expression of steroid receptors of the subendometrial myometrium paralleled the cyclic pattern of the endometrial epithelium and stroma, the outer part of the myometrium consisting of the stratum supravasculare and most of the stratum vasculare showed no cyclic pattern at all but rather strong staining throughout the whole cycle.
The high expression of both ER and PR in the uterine layers of post-menopausal women suggests that the expression of ER and PR is constitutive for uterine tissue and that the cyclic change of the receptor expression is, in addition to up-regulation by oestradiol, primarily a matter of down-regulation by changing progesterone concentrations (Kraus and Katzenellenbogen, 1993; Iwai et al., 1995
; Graham and Clarke, 1997
). The functional significance of cell-specific differential receptor regulation may be viewed in a suppression of hormone action in the respective tissue in the presence of high circulating concentrations of this hormone required for continuing action in another tissue (King et al., 1980
; McCormack and Glasser, 1980
; Lessey et al., 1988
; Shiozawa et al., 1996
).
In addition to the observation that cyclically changing uterine peristaltic activity is confined to the subendometrial myometrium (Lyons et al., 1991; Kunz et al., 1996
), the cyclically changing ER and PR pattern suggests strongly that the subendometrial myometrium is functionally distinct from the rest of the myometrium and is rather part of a functional unit of which the other components are the endometrial epithelium and the endometrial stroma. This conjecture is supported by both data from comparative morphology and human embryology.
Phylogenetic data of the uterine muscular wall show that, in all vertebrates listed (Table I), the muscular wall of the uterus or of the uterine part of the oviduct respectively is composed of a stratum subvasculare with a circular arrangement of the muscular fibres. While in birds (Van Tienhoven, 1961
) and monotremata (Van den Broek, 1933
) the stratum vasculare constitutes the only muscular layer of the uterus/oviduct, in marsupials (Lierse, 1965
) and rodents (Garfield and Yallampalli, 1994
) the outer stratum supravasculare with predominantly longitudinal muscular fibres has been acquired additionally. In the human, the loose mesenchymal mesh of the stratum vasculare, as observed in the rodent, has developed into a inner third muscular layer consisting of a three-dimensional mesh of irregular short muscular bundles and constituting the bulk of the uterine muscular wall (Werth and Grusdew, 1898
; Wetzstein, 1965
).
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The acquisition of uterine muscular layers in addition to the circular stratum subvasculare late in phylogeny corresponds to the late development of these layers during human embryology (Table II). The stratum vasculare and supravasculare respectively only develop in the third trimester of gestation or even postnatally (Werth and Grusdew, 1898
), whereas the anlage of the stratum subvasculare is already apparent at the beginning of the second trimester with a circular arrangement of mesenchymal cells around the primordial uterus and tubes. The ontogenetically early formation of the inner circular layer is also documented by a kind of fundo-cornual raphe of the stratum subvasculare (Werth and Grusdew, 1898
) (Figure 5
) that results from the fusion of the two paramesonephric ducts and their mesenchymal elements to form the primordial uterus. The fundo-cornual raphe appears to be an important structural detail with respect to uterine physiology. The bipartition of the circular subendometrial myometrium in the upper part of the uterine corpus and its separate continuation through the cornua into the respective tubes is apparently the morphological basis for directed sperm transport into the tube ipsilateral to the dominant follicle (Kunz et al., 1996
, 1998b
). Furthermore, the fundo-cornual raphe of the subendometrial myometrium might be a site of constant microtrauma due to uterine peristalsis and hyperperistalsis, from where endometriosis and adenomyosis may arise (Leyendecker et al., 1996
, 1998
). The cervical part of the uterus is morphogenetically strongly influenced by vaginal elements in that the inner paramesonephric component, the glandular epithelium and the circular muscular layer, is sheathed and blended with vaginal musculature and connective tissue (Werth and Grusdew, 1898
) (Figure 5
).
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Thus, the human uterus is composed, ontogenetically and phylogenetically, of two separate organs, the paramesonephric endometrialsubendometrial unit and the outer non-paramesonephric myometrium (Figure 5). Both the stratum vasculare and supravasculare subserve parturition. This requires growth and myometrial quiescence in a lengthy gestation (Soloff, 1989
), the constant action of both oestradiol and progesterone and, hence, the constant expression of their respective receptors (Katzenellenbogen et al., 1979
; Graham and Clarke, 1997
).
The functions of the endometrialsubendometrial unit are more complex. They consist in the cyclic preparation of the endometrium for implantation and in the cyclically changing uterine peristalsis with the main function of uterine sperm transport (De Vries et al., 1990; Kunz et al., 1996
; Leyendecker et al., 1996
). Inflammatory defence may also be regarded as a specific and phylogenetically old function of the endometrialsubendometrial unit (Leiva et al., 1994
; Surveyor et al., 1995
; Xu et al., 1995
; Loke and King, 1996
; Gipson et al., 1997
) since, in lower animals, the Müllerian ducts end in a cloaca and, in the human, the cervical mucus does not act as a barrier for passive ascension of inert particles and sperm in neither phase of the cycle (unpublished) (Faundes et al., 1981
; Kunz et al., 1996
). The cyclic changes in ER and PR expression in the endometrialsubendometrial unit apparently meet the requirements of a cyclic ovarian control over these functions.
In conclusion, immunocytochemistry of the whole uterine muscular wall and the endometrium revealed that the subendometrial myometrium exhibits a cyclic pattern of ER and PR expression that parallels that of the endometrium, whereas the outer portion of the uterine wall does not exhibit a cyclic pattern of ER and PR expression. The data were correlated with morphological, embryological and phylogenetic data of the myometrial wall, and it became evident that both endometrium and subendometrial myometrium form a functional unit with various cyclic reproductive functions in addition to those during gestation and parturition. This endometrialsubendometrial functional unit is phylogenetically an ancient organ within the uterus and could be termed the `archimetra' with reference to Werth and Grusdew (1898), who used the term archimyometrium to describe the ontogenetically old character of the stratum subvasculare of the myometrium.
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Notes |
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References |
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Submitted on June 8, 1998; accepted on October 1, 1998.