Endothelin and neutral endopeptidase in the endometrium of women with menorrhagia

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1 Human Reproduction vol.12 no.9 pp , 1997 Endothelin and neutral endopeptidase in the endometrium of women with menorrhagia M.M.Marsh 1, N.Malakooti 1, N.H.Taylor 2, described (Rees et al., 1984b) and increased prostaglandin-e 2 J.K.Findlay 1 and L.A.Salamonsen 1,3 receptors in myometrium have been correlated with MBL 1 (Adelantado et al., 1988). Although prostaglandin synthase Prince Henry s Institute of Medical Research, PO Box 5152, Clayton 3168, 2 Monash University Department of Obstetrics and inhibitors are able to reduce MBL in menorrhagia (Anderson Gynaecology, Monash Medical Centre, 246 Clayton Road, Clayton, et al., 1976; Fraser et al., 1981a) supporting a role for Victoria, 3168, Australia prostaglandins, changes in prostaglandin production associated 3 To whom correspondence should be addressed with menorrhagia have been difficult to demonstrate (Rees et al., 1984c). The mechanisms underlying excessive menstrual bleeding In view of its potent vasoconstrictor (Yanagisawa et al., or menorrhagia are not understood. In view of its potent 1988) and growth factor (Battistini et al., 1993) properties, vasoconstrictor and growth factor properties, endothelin endothelin (ET) is a potential aetiological factor in menorrhahas been proposed to have a potential paracrine role in gia. ET has been demonstrated in normal human endometrium the regulation of uterine blood flow and therefore could (Cameron et al., 1992; Economos et al., 1992; Salamonsen be a factor in menorrhagia. We compared the cellular et al., 1992; Ohbuchi et al., 1995) and its cellular expression localization of endothelin and its metabolizing enzyme, shown to vary across the normal menstrual cycle, with maximal neutral endopeptidase, in endometrial biopsies from women expression in the perimenstrual period (Salamonsen et al., with documented menorrhagia and in those with a normal 1992; Ohbuchi et al., 1995). In the endometrium of women menstrual cycle. Menorrhagia was documented by meas- using the long-acting progestogen contraceptive agent urement of menstrual blood loss, ml (median Norplant, often associated with menstrual bleeding disturb- SD). Endothelin and neutral endopeptidase were local- ances (Odlind and Fraser, 1990), ET has been shown be ized by immunohistochemistry, and the staining intensity reduced in the glandular and luminal epithelium (Marsh et al., was graded. Their immunostaining patterns were found to 1995). Neutral endopeptidase (NEP), a membrane bound differ in menorrhagia compared to the normal menstrual ectoenzyme which inactivates ETs (Casey et al., 1991; Head cycle. Endothelin was reduced in glandular epithelium in et al., 1993; Casey and MacDonald, 1996) was increased in menorrhagia and did not vary cyclically, while neutral the epithelium in the same endometrial biopsies, providing a endopeptidase was increased in the glandular epithelium. potential explanation for the finding of reduced immunoreactiv- In menorrhagia, stromal endothelin immunoreactivity was ity for ET (Marsh et al., 1995). Therefore, it could be postulated not different from the normal cycle and although neutral that an alteration in ET concentrations due to changes in endopeptidase immunostaining in stroma was similar to synthesis, processing or metabolism may lead to bleeding the secretory phase of normal endometrium, cyclical vari- disturbances. Investigation of ET in the endometrium of women ation was absent. The potential for increased metabolism with proven menorrhagia may contribute to our understanding of endothelin could be an explanation for the decreased of the role of ET in menstrual bleeding. The aim of these endothelin immunostaining in the glandular epithelium. studies was to compare the cellular localization of ET and its Key words: endometrium/endothelin-1/intrauterine device/ metabolizing enzyme, NEP, in endometrial biopsies from menorrhagia/neutral endopeptidase women with documented menorrhagia, and from those with a normal menstrual cycle. Introduction Menorrhagia, defined as a monthly menstrual blood loss (MBL) of 80 ml (Hallberg et al., 1966), is one form of abnormal uterine bleeding for which no cause can be identified in up to 50% of cases (Rees, 1987). The mechanisms underlying the excessive menstrual bleeding are not understood. Studies of endometrial morphology in menorrhagia have shown no correlation between MBL and endometrial/myometrial arterial density or endometrial glandular density (Rees et al., 1984a). In contrast, increased concentrations of prostaglandins F 2α and E 2 in menstrual fluid of women with menorrhagia have been Materials and methods Subjects and endometrial tissue collection Premenopausal women with regular ovulatory menstrual cycles who presented complaining of menorrhagia were assessed by measurement of MBL and enrolled in a collaborative study with the Monash University Department of Obstetrics and Gynaecology, Melbourne, Australia. Subjects without intrauterine pathology whose MBL was 80 ml per month on two occasions were included in the study following informed consent. This cohort of women included those previously reported (Critchley et al., 1994). All pads and tampons were collected during two menstrual periods and the MBL measured 2036 European Society for Human Reproduction and Embryology

2 Endometrial endothelin and NEP in menorrhagia by the alkaline haematin method (Hallberg and Nilsson, 1964). The MBL of the menorrhagic subjects varied between 80 and 600 ml per month with a median ( SD) of 146 ( 141) ml. Endometrial tissue (n 17) was obtained by pipelle biopsy at hysteroscopy, fixed in formalin, processed to paraffin and characterized as previously described (Salamonsen et al., 1992; Critchley et al., 1994). Proliferative (n 6), early secretory (n 3), mid-secretory (n 4) and late secretory phase (n 4) samples were studied. Unfortunately, no menstrual samples were obtainable from women with menorrhagia. A limited number of endometrial biopsies (n 4) were also obtained from women with intrauterine contraceptive devices (IUD). These subjects were not using the levonorgestrel IUD and did not complain of excessive menstrual bleeding. Control endometrial tissue (n 63) was obtained at dilatation and curettage from women who had given informed consent. These women were either of proven fertility undergoing tubal ligation or the partners of men with male factor infertility undergoing assessment of tubal patency before assisted fertility procedures. Control subjects did not complain of excessive menstrual bleeding and no menstrual blood loss measurements were available for the normal control or IUD subjects. Endometrium was dated from the woman s menstrual history and confirmed histologically by an experienced gynaecological pathologist. Protocols were approved by the Human Research and Ethics Committee at Monash Medical Centre. Immunohistochemistry Immunolocalization of ET and NEP was performed as previously described and the findings compared to the results obtained in the immunohistochemical study of endometrial biopsies from women with normal menstrual cycles (Salamonsen et al., 1992; Marsh et al., 1995). A small number of samples across the normal menstrual cycle (n 7) were examined for NEP to confirm the previously published data (Head et al., 1993). Sections (6 µm) were deparaffinized and rehydrated before immunolocalization was performed. ET was identified using polyclonal rabbit antiserum to human ET which crossreacted with ET-1, ET-2 and ET-3 (Cambridge Research Biochemicals, Northwich, UK) and streptavidin alkaline phosphatase (Salamonsen et al., 1992) and counterstained with Mayer s haematoxylin (Sigma Chemical Company, St Louis, MO, USA). NEP immunolocalization was performed using a mouse monoclonal primary antibody to human NEP [common acute lymphoblastic leukaemia antigen (CALLA; clone J5; Coulter Corporation, Hialeah, FL, USA)] and avidin biotin horseradish peroxidase (Marsh et al., 1995), and counterstained with Harris haematoxylin (1:10; Sigma). All sections were examined by light microscopy. Controls appropriate for each antibody were included in each series of sections examined, as well as a section from a single block of secretory endometrial tissue, enabling the comparison of the distribution and intensity of immunostaining in this section between each staining procedure. Negative controls for each section were included for ET and NEP; normal rabbit serum substituted in the same concentration as the primary antibody and mouse IgG substituted for the primary antibody respectively. Human placenta was used as a positive control. Immunostaining intensity was assessed by two independent obser- vers and scored semi-quantitatively from 0 (negative) to 4 (maximal staining intensity) in relation to the known positive and negative controls. For the analysis, these values were transformed into a percentage immunostaining intensity where negative 0 and maximal staining intensity 100%. Results were expressed as mean SEM. Haematoxylin and eosin stained sections were assessed and dated histologically by an experienced gynaecological histopathologist. Figure 1. Cellular localization of endothelin immunostaining intensity (% control; mean SEM) across the menstrual cycle in endometrial biopsies from subjects with (a) menorrhagia (histogram bar shading in the order: proliferative, early secretory, mid- secretory, and late secretory phases from left to right) and (b) normal menstrual cycles (histogram bar shading in the order: menstrual, proliferative, early secretory, mid-secretory, and late secretory phases from left to right). Numbers below each bar denote the number of samples examined in each cellular compartment. Results Endothelin The pattern of ET immunoreactivity in the endometrium of those women with documented menorrhagia (Figures 1a and 2c) was substantially different from that in endometrium from the same phase of the cycle in women with normal menstrual cycles (Figures 1b and 2a). In menorrhagia there was little variation in the immunostaining pattern for ET across the menstrual cycle. The intensity of staining in luminal and glandular epithelium in menorrhagia was less than at the same stage of the normal menstrual cycle, whereas stromal staining was similar (Figures 1 and 2). ET immunoreactivity was similar in predecidualized cells of the stroma in menorrhagic compared with the normal cycle subjects. In all the endometrial biopsies from subjects with IUD (n 4), ET immunoreactivity was reduced in the glandular epithelium ( ; mean SEM) to levels lower than at any time during the normal menstrual cycle, while stromal immunoreactivity ( ) was similar to the normal cycle (data not shown). The resulting staining pattern was similar to that observed in the menorrhagic subjects. Neutral endopeptidase The NEP immunostaining pattern in the endometrium in menorrhagia also differed from that of the normal menstrual 2037

3 M.M.Marsh Figure 2. Photomicrographs of human endometrium. Late secretory phase endometrium examined immunohistochemically for endothelin: (a) normal menstrual cycle, (c) menorrhagia, (e) negative control (positive staining pink/red and pale blue counterstain). Mid-secretory phase endometrium examined immunohistochemically for neutral endopeptidase (b) normal menstrual cycle, (d) menorrhagia and (f) negative control (positive staining brown and counterstain pale blue). Bar 50 µm. 2038

4 Endometrial endothelin and NEP in menorrhagia this method in the stroma does not play a key role in the menstrual bleeding differences observed. The more intense NEP immunostaining in glandular and luminal epithelium in menorrhagia could be an explanation for the reduced ET observed in this cellular compartment as a result of increased ability to metabolize ET and therefore reduced local ET concentration. In the limited number of endometrial biopsies examined from subjects with IUDs, the ET immunostaining pattern was similar to that observed in menorrhagia. Menstrual blood loss has been shown to be increased in users of IUDs (van Eijkeren et al., 1996) and the finding of reduced epithelial cell ET in this group provides further evidence that an alteration in epithelial cell ET is related to the disturbances in menstrual bleeding. In normal endometrium, NEP was expressed predominantly by stromal cells, with very little if any expressed by epithelial cells. Stromal immunoreactivity varied cyclically and peaked in the secretory phase, in agreement with the previously published data (Head et al., 1993). Previous studies have shown inactivation by NEP of a number of small peptides other than ET, including atrial natriuretic peptide, oxytocin, insulin-β, gastrin, cholecystokinin, interleukin-1α, opioids, substance P, neurotensin, neurokinins and enkephalins (Vijayaraghavan et al., 1990; Roques et al., 1993). NEP is widely distributed and expressed in other tissues including some epithelial cells (Roques et al., 1993). Although regulation of stromal expression of NEP by progestogens has been shown (Casey et al., 1991), factors responsible for epithelial cell expression are not known. The pattern of NEP immunostaining in menorrhagia was similar to that observed in the endometrium of Norplant users in whom synthetic progestogen concentra- tions are high. Therefore progestogens alone are unlikely to be responsible, as epithelial cell expression was not observed in the normal secretory phase when serum progesterone concen- trations are high (Marsh et al., 1995). No data were available on the endogenous progesterone concentrations in the control or menorrhagic subjects. Alterations in prostaglandin synthesis have been described and prostaglandin synthase inhibitors are capable of reducing MBL in menorrhagia (Anderson et al., 1976; Fraser et al., 1981a); however, no consistent changes in prostaglandin synthesis have been described. In short-term explant culture, ET-1 has been shown to activate phospholipase A 2 and phospholipase C in human proliferative endometrium, but not secretory endometrium (Ahmed et al., 1992), resulting in increased prostaglandin-f 2α release from normal human proliferative endometrium (Cameron et al., 1991). The finding of reduced ET would therefore potentially lead to a decrease in prostaglandin-f 2α production resulting in a net reduction in vasoconstrictor effect and unopposed action of the vasodilator prostaglandin-e 2 in the endometrium in menorrhagia. This sequence of events would be compatible with the results from previous studies of prostaglandins in the endometrium in menorrhagia, and represents a possible mechanism whereby ET could be involved in the control of menstrual bleeding via regulation of prostaglandin-f 2α -induced constriction of spiral arterioles. While these results would suggest a key role for epithelial 2039 Figure 3. Cellular localization of neutral endopeptidase immunostaining intensity (% control; mean SEM) across the menstrual cycle in endometrial biopsies from subjects with (a) menorrhagia (histogram bar shading in the order: proliferative, early secretory, mid-secretory, and late secretory phases from left to right) and (b) normal menstrual cycles (histogram bar shading in the order: proliferative, mid-secretory, and late secretory phases from left to right). Numbers below each bar denote the number of samples examined in each cellular compartment. cycle. The intensity of NEP was greater in epithelium of glandular and luminal origin in menorrhagic endometrium compared to the normal menstrual cycle, with the exception of the early secretory phase samples (Figures 2b, 2d and 3). Stromal immunostaining intensity remained high across the cycle in menorrhagia, and the staining was similar to that observed in the normal late secretory phase (Figure 3). Discussion This study has demonstrated clear differences in the immunostaining patterns of ET and NEP in the endometrium of women with menorrhagia compared to those with normal menstruation. Menorrhagia was documented by measurement of MBL, as a subjective history of heavy menstrual periods has been shown to be an unreliable indicator of actual MBL (Fraser et al., 1981b). In general, in menorrhagia ET immunostaining was decreased in the epithelium compared with normal, while its degradative enzyme, NEP, was elevated. Stromal ET immunostaining was similar in menorrhagia to the normal cycle and no changes were observed in the appearance of ET in the perivascular stromal cells previously described in the late secretory phase (Ohbuchi et al., 1995; Marsh et al., 1996) in the four late secretory menorrhagic samples. The lack of difference in stromal ET immunoreactivity between normal and menorrhagic endometrium suggested that ET detected by

5 M.M.Marsh cell ET in the control of menstrual bleeding, there was no Odlind, V. and Fraser, I.S. (1990) Contraception and menstrual bleeding disturbances. In d Arcangues, C., Fraser, I.S., Newton, J.R. and Odlind, V. correlation between the immunostaining pattern and number (eds), Contraception and Mechanisms of Endometrial Bleeding. Cambridge of bleeding days in the endometrial biopsies in users of University Press, Cambridge, UK, pp Norplant (Marsh et al., 1995). Together, these findings suggest Ohbuchi, H., Nagai, K., Yamaguchi, M. et al. (1995) Endothelin-1 and big endothelin-1 increase in human endometrium in menstruation. Am. J. Obstet. that although ET may play a role in the control of menstrual Gynecol., 173, bleeding, other factors are clearly involved in the processes Rees, M. (1987) Menorrhagia. Br. Med. J., 294, responsible for the initiation of bleeding. Rees, M.C.P., Dunnill, M.S., Anderson, A.B. et al. (1984a) Quantitative uterine histology during the menstrual cycle in relation to measured menstrual blood loss. Br. J. Obstet. Gynaecol., 91, Acknowledgements Rees, M.C.P., Anderson, A.B.M., Demers, L.M. et al. (1984b) Prostaglandins in menstrual fluid in menorrhagia and dysmenorrhoea. Br. J. Obstet. We thank Professor D.L.Healy of the Monash University Department Gynaecol., 91, of Obstetrics and Gynaecology and Drs H.O.D.Critchley and J.Kooy, Rees, M.C.P., Anderson, A.B.M., Demers, L.M. et al. (1984c) Endometrial formerly of the Monash University Department of Obstetrics and and myometrial prostaglandin release during the menstrual cycle in relation Gynaecology, for their collaboration in providing the menorrhagic to menstrual blood loss. J. Clin. Endocrinol. Metab., 58, endometrium and quantification of menstrual blood loss for these Roques, B.P., Noble, F., Daugé, V. et al. (1993) Neutral endopeptidase studies. 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