Human Reproduction vol.11 no.5 pp.1067-1072, 19%

Endothelial cell proliferation in the endometrium of women with menorrhagia and in women following endometrial ablation

J.Kooy, N.H.Taylor, D.L.Healy and P.A.W.Rogers1 Monash University Department of Obstetrics and Gynaecology, Monash Medical Centre, 246 Clayton Road, Clayton 3168, Victoria, Australia

Local endometrial aberrations are thought to be the major contributing factor to essential menorrhagia. Here we have examined the role of endometrial angiogenesis, the growth of new blood vessels, in essential menorrhagia. Our study tested two hypotheses: firstly that angiogenesis is disturbed in the endometrium of women with menorrhagia,* and secondly that when menstrual blood loss is decreased following endometrial ablation, an endometrial environment favouring normal angiogenesis has returned. Angiogenesis was measured by endothelial cell proliferation. Proliferating endothelial cells were identified by an immunohistochemical double staining technique. A total of 57 women participated in this study, of whom 19 were controls, 20 had menorrhagia and 18 were 3-6 months post-ablation. There was a significant increase in endothelial cell proliferation in the endometrium of patients with menorrhagia compared with the control endometrium. Conversely, post-ablation endometrium showed a nonsignificant decrease in endothelial cell proliferation. The increased endothelial cell proliferation in the endometrium of patients with menorrhagia was not the result of a general increase in endometrial cellular proliferation and did not result in a change in endothelial cell concentration compared with control endometrium. These results support the hypothesis that angiogenesis is disturbed in the endometrium of patients with menorrhagia and normalized in post-ablation endometrium. Key words: angiogenesis/endometrium/endothelial cells/ menorrhagi a/proliferation

Introduction Menorrhagia, i.e. excessive menstrual bleeding, is experienced by 9-14% of all women (van Eijkeren et al, 1992). In 50% of cases, the menorrhagia has no underlying pathology and is termed essential (Rees, 1987). The endometrial mechanisms contributing to essential menorrhagia are poorly understood. There are a number of medical treatments for essential menorrhagia which may result in a substantial reduction in menstrual blood loss (van Eijkeren et al., 1992). However, until recently, the only permanent cure for menorrhagia was hysterectomy. There are two new and effective treatments for menorrhagia, © European Society for Human Reproduction and Embryology

Menstruation involves both tissue breakdown, resulting in blood loss from damaged vessels, and subsequent tissue repair. Angiogenesis, the growth of new blood vessels, is a normal component of tissue growth and repair following menstruation. There are a number of steps in the process of angiogenesis, including the breakdown of existing vascular basement membranes, coordinated migration and proliferation of the endothelial cells, the formation of new capillaries and their subsequent functional maturation (Findlay, 1986). Peaks of angiogenic activity appear to occur in normal human endometrium during post-menstrual repair, in the mid- to late proliferative phase and possibly during the mid-secretory phase (Rogers et al., 1992). The process of angiogenesis is complex and is tightly regulated by cytokines (Klagsbrun and D'Amore, 1991; Giudice, 1994). We propose that, in endometrium from patients with menorrhagia, the process of angiogenesis is disturbed. Furthermore, we propose that when menstrual blood loss is decreased following endometrial ablation, an endometrial environment which favours normal angiogenesis has returned. In this study we have used endometrial endothelial cell proliferation as a measure of angiogenesis. To identify proliferating endothelial cells, a immunohistochemical double staining system was used [Goodger (Macpherson) and Rogers, 1994]. Sections were stained with monoclonal antibodies to CD34, an endothelial cell marker, and the proliferating cell nuclear antigen (PCNA), a proliferation marker, to visualize all prolifer1067

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'To whom correspondence should be addressed

the levonorgestrel-releasing intrauterine device (LNG-IUD) and endometrial ablation. The LNG-IUD has been reported to reduce menstrual blood loss by 86% after 3 months and 97% after 12 months of use (Andersson and Rybo, 1990); however, this product is not available in Australia. Endometrial ablation has become a popular surgical alternative to hysterectomy in Australia (Molloy and Taylor, 1994). Despite its widespread use for the treatment of menorrhagia, there are few data regarding endometrial function in women following ablation. The endometrium has a remarkable regenerative capacity and is therefore likely to regrow following endometrial ablation if the uterine surface is free of fibrosis and adhesions. Indeed, we have reported previously the presence of histologically normal endometrium post-ablation (Critchley et al, 1995). Many women still menstruate post-ablation but at a greatly reduced volume (McClure etal., 1992). Therefore post-ablation endometrium must be different to that present before endometrial ablation. A knowledge of the structural and functional differences before and after endometrial ablation will help elucidate the local endometrial mechanisms that contribute to menorrhagia. As these local endometrial mechanisms are manifested at menstruation, an understanding of the events surrounding this process is of vital importance.

J.Kooy et al.

Materials and methods Subjects Menorrhagia has been defined as an objective menstrua] blood loss of 5*80 ml per menstrual cycle (Hallberg et al., 1966). A total of 57 women (age range 30-51 years) of reproductive age participated in our study. The women were divided into three groups: those with a subjective normal blood loss (n = 19); those with objective menorrhagia (n = 20); and those who were 3-6 months post-ablation in = 18). This study was initially designed to be longitudinal, with the major group being women suffering from objective menorrhagia who had decided to undergo endometrial ablation on the advice of their gynaecologist According to the original study protocol, a menstrual blood loss measurement and an endometrial biopsy were to be obtained from these women both before, and 3 months after, endometrial ablation. However, for a number of clinical and practical reasons this study design proved to be difficult to follow in most cases. This protocol was followed for four subjects. An endometrial biopsy and an objective measure of menstrual blood loss were obtained for each subject in the menorrhagia and post-ablation study groups. In addition, for 15 of the 18 post-ablation subjects an objective measure of menstrual blood loss before ablation was also determined. As a comparison for the endothelial cell proliferation part of the study, endometrial biopsies from 19 control subjects collected as part of a number of other studies were also utilized. Control biopsies were collected from women undergoing tuba! ligation or treatment for infertility caused by a male factor. Objective menstrual blood loss measurements were not available for these subjects; however, none of these women complained of bleeding problems and all had normal cycle lengths. Before endometrial ablation, patients were prescribed either Danazol (200 mg twice daily, n = 12; Winthrop, Ennington, Australia), Goserelin (two 3.6 mg s.c. implants, 1 month apart, n = 5; ICL, Melbourne, Australia) or medroxyprogesterone acetate (200 mg/day, n = 1; Upjohn, Rydalmore, Australia) to reduce endometrial thickness. All 18 endometrial ablations were performed at the Monash Medical Centre (Clayton, Victoria, Australia) using electrocautery as described by McClure et aL (1992). The small and large rollerballs were operated at 50 and 80 W respectively, while the cutting loop was used at 100 W. The endometrial ablations were performed by one of three experienced surgeons. 1068

Tissue collection and processing Biopsies were taken at random throughout the menstrual cycle and it is not known whether the cycles were ovulatory. Endometrial biopsies were collected at outpatient hysteroscopy by Pipelle (Prodimed, Neuilly-en-Thelle, France). Biopsies were fixed in 10% buffered formalin for 4—6 h at 4°C, and then washed in phosphatebuffered saline (PBS) and processed through to wax. Sections (5 |im) were cut using a microtome and placed onto 3-aminopropyltriethoxysilane (Sigma, St Louis, MO, USA)-coated slides. The sections were then either used for immunohistochemistry or stained with haematoxylin and eosin. All biopsies were dated by an experienced histopathologist into one of nine stages of the menstrual cycle (Noyes et al, 1950; Rogers et al., 1992). Measurement of menstrual blood loss All patients with menorrhagia and the post-ablation subjects collected their menstrual blood loss during at least one menstrual period, which was measured using the alkaline haematin method (Hallberg and Nilsson, 1964). Where two or more menstrual blood loss measurements were taken per subject, the mean menstrual blood loss was determined. Immunohistochemistry Sections were double stained with monoclonal antibodies to CD34 (Serotec, Oxford, UK), an endothelial cell marker, and PCNA (Novocastra, Newcastle, UK), a proliferation marker, to visualize and quantify all proliferating endothelial cells. A serial section was stained for CD34 alone and counterstained with haematoxylin to visualize and quantify the total number of endothelial cells. The staining protocols have been described previously by Goodger (Macpherson) and Rogers (1994), and all reagents used in the staining protocol were obtained from Zymed, San Francisco, CA, USA, unless otherwise stated. Briefly, for the double staining, sections were dewaxed, hydrated and blocked for endogenous peroxidase with 3% H 2 O 2 (in methanol) for 10 min at 37°C. The sections were then incubated for 20 min with normal rabbit serum at room temperature, followed by an incubation with anti-PCNA monoclonal antibody for 60 min at 37°C. Sections were then incubated sequentially at room temperature with biotinylated rabbit anti-mouse immunoglobulin (Ig), horseradish peroxidase-streptavidin and aminoethyl carbazole chromogen, to give a red colour. Sections were treated with double staining enhancer for 30 min at room temperature, followed by a 20 min incubation with normal rabbit serum. The sections were then incubated with a monoclonal antibody to the endothelial cell surface marker CD34 for 60 min at 37°C. Sections were incubated sequentially at room temperature with biotinylated rabbit anti-mouse Ig, alkaline phosphatase-streptavidin and fast blue chromogen, to give a blue colour. In each staining run, a tissue of known staining intensity was included as a positive control and an internal standard. This same tissue was also used for a negative control, in which isotype-matched irrelevant monoclonal antibodies were substituted for the two primary monoclonal antibodies (mouse IgG2a for the anti-PCNA antibody and mouse IgGl for the antibody to CD34). Quantification of the number of proliferating endothelial cells in each section was performed using a microscope linked via a colour video camera (MW-F15E Panasonic; Matsushita Communication Industrial Co., Japan) to a personal computer (Commodore Amiga 2000; Lane Cove, NSW, Australia) with stereology software (Grid, Graffiti Data; Medico Soft Division, Silkeborg, Denmark). All counting was carried out using a X40 objective, and counts were taken from the computer screen. The numbers of endothelial cells in the section were determined by sampling ~10% of the section using a

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ating endothelial cells. CD34 is a glycosylated transmembrane protein of unknown function which is expressed by a limited number of cells. In non-lymphohaemopoietic tissues (such as the endometrium), this molecule is confined to endothelial cells (Silvestri et al, 1992). PCNA is an auxiliary protein to DNA polymerase-8 (Bravo et al, 1987) and is required for DNA synthesis (Tan et al, 1986; Prelich et al, 1987). PCNA is first expressed in proliferating cells during the late G] phase (Danova et al, 1990) and is also expressed in cells in which unscheduled DNA repair is taking place (Celis and Madsen, 1986; Toschi and Bravo, 1988). The validity of using PCNA as a proliferation marker in human endometrium has been established previously by our laboratory [Goodger (Macpherson) and Rogers, 1994]. The aim of this study was to test two hypotheses: firstly that angiogenesis is disturbed in the endometrium of patients with menorrhagia; and secondly that when menstrual blood loss is decreased following endometrial ablation, an endometrial environment favouring normal angiogenesis has returned.

Menorrhagia: endometrial angiogeneds

Table I. Objective menstrual blood loss (mean ± SE) in patients with menorrhagia and post-ablation subjects Study group

Sample size

Menstrual blood loss (ml)

Menorrhagia Post-ablation Histologically normal endometrium Necrotic tissue No tissue

20

123.8 ± 7.6

10

50.7 ± 15.3

5 3

4.0 ± 4.0 10.3 ± 5.2

Table II. Endometrial endothelial cell proliferation in controls, women with mennorhagia and post-ablation Study group

Sample size

Endothelial cell proliferation (%) (Mean ± SE)

Control Menorrhagia Post-ablation Histologically normal endometrium

19 20

7.0 ± 1.2 14.5 ± 2.1

10

8.6 ± 1.4

4

9.2 + 5.6

Necrouc tissue

Statistical analysis of the data All data were analysed statistically using the non-parametric MannWhitney test Results with a P value of =SO.O5 were considered significant Data were expressed as mean ± SE.

Results Histology of endometrial biopsies Biopsies obtained from control subjects and patients with menorrhagia contained histologically normal endometrium. The post-ablation endometrial biopsies were divided into three groups based on their histology. In all, 10 biopsies contained endometrium that was histologically normal. Five biopsies contained necrotic material; of these, four contained Intact tissue, while the fifth necrotic biopsy contained isolated cells rather than intact tissue. The three remaining biopsies contained blood, mucus or both blood and mucus but no endometrial tissue. Objective menstrual blood loss of patients with menorrhagia and post-ablation subjects The mean ± SE menstrual blood loss of the patients with menorrhagia was 123.8 ± 7.6 ml (Table I). In the postablation subjects with histologically normal endometrium, the mean ± SE menstrual blood loss was 50.7 ± 15.3 ml. Of the necrotic biopsies, the four containing intact tissue were obtained from women who were amenorrhoeic. The fifth necrotic biopsy was obtained from a woman with a menstrual blood loss of 20 ml. Therefore, the mean ± SE menstrual

blood loss of the women from whom necrotic biopsies were obtained was 4.0 ± 4.0 ml. The three subjects from whom biopsies with no tissue were obtained had a mean ± SE menstrual blood loss of 10.3 ± 5.2 ml. Of the 18 subjects, eight (44%) were amenorrhoeic at 3-6 months post-ablation. Menstrual blood loss in post-ablation subjects with histologically normal, necrotic and no endometrial tissue at biopsy were all significantly lower than that in the patients with menorrhagia (P = 0.001, 0.0007 and 0.007 respectively). In addition, the mean menstrual blood loss for the post-ablation study groups was in all cases 0.05). In addition, there was no significant difference between the proliferation indices of the two post-ablation study groups and that of the control endometrium (P > 0.05). A 1069

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motorized stage (MS 316; Lang GMBH & Co., Hflttenberg, Germany). An endothelial cell proliferation index for each section was then determined by dividing the number of proliferating endothelial cells by the total number of endothelial cells and then expressing the result as a percentage. The samples were analysed blind to their origin by one researcher. Cellular proliferation in endometrial stroma, glands and surface epithelium was scored semi-quantitatively. Staining in each compartment was scored according to two criteria: the degree of staining (0 = no staining, 1 = 1/250 cells stained, 2 = 1/50, 3 = 1/10 and 4 >l/10) and the intensity of staining (0 = no staining, 1 = mild, 2 = moderate and 3 = intense). These scores were then multiplied together to obtain an immunohistochemical staining score (Bergqvist et al, 1993). No score was given in the absence of a particular compartment If a particular compartment was present, but no staining was evident, a score of 0 was given and included in the calculation of the mean. The samples were analysed blind to their origin by one researcher.

J.Kooy et aL

regression analysis between the objective menstrual blood loss and the endometrial endothelial cell proliferation index for the 20 patients with menorrhagia showed that there was no significant correlation (r = -0.154). Endothelial cell proliferation indices for the control, menorrhagia and histologically normal post-ablation study groups were not significantly different when biopsies were divided into secretory or proliferative phases of the menstrual cycle. Therefore, the mean endometrial endothelial cell proliferation index for the whole menstrual cycle was calculated for each study group. Cellular proliferation in endometrial stroma, glands and surface epithelium To determine whether any changes in endothelial cell proliferation were a result of a general change in endometrial cellular proliferation or were confined only to the endothelial cells, cellular proliferation in other endometrial compartments was also assessed. The compartments examined were the stroma, glands and surface epithelium. The proliferation scores for each of these compartments are shown in Table HI. Endometrial stroma was present in all of the biopsies examined; however, endometrial glands and surface epithelium were not always present It was found that there was no significant change in cellular proliferation in endometrial stroma, glands or surface epithelium for the four study groups. Therefore it appeared that the increase in endothelial cell proliferation seen in the endometrium of patients with menorrhagia was the result of a specific increase in endothelial cell proliferation rather than a general increase in endometrial cellular proliferation. Endometrial endothelial cell concentration The increase in endothelial cell proliferation in the endometrium of patients with menorrhagia was not accompanied 1070

Table IIL Cellular proliferation in endometrial stroma, glands and surface epithelium (values are mean ± SE). Proliferation scored according to two criteria, the degree of staining* and the intensity of stainingb, which were multiplied together to give a semi-quantitative measure Study group

Stroma

Glands

Surface epithelium

Control Menorrhagia Post-ablation Histologically normal endometrium Necrotic tissue

6.5 ± 0.8 (19) 8.0 ± 0.4 (20)

7.4 ± 0.8 (19) 7.9 ± 0.8 (20)

3.5 ± 1.0 (12) 4.6 ± 1.3 (14)

6.6 ± 1.1 (10)

9.2 ± 1.0 (9)

7.0 ± 1.8 (8)

4.5 ± 2.6 (4)

NP

12(1)

Values in parentheses are numbers of samples. NP = not present. •0 = no staining, 1 = 1/250 cells stained, 2 = 1/50, 3 = 1/10 and 4 > 1/10. b 0 = no staining, 1 = mild, 2 •= moderate and 3 = intense.

Table rv. Endometrial endothelial cell concentration measured as mean ± SE endothelial cell number/mm2 Study group Control Menorrhagia Post-ablation Histologically normal endometrium Necrotic tissue

Sample size

Endothelial cell concentration

19

20

210.3 i 17.0* 203.2 ± 19.7"

10

317.7 ± 42.4 b

4

160.3 ± 19.4*

••''Values with different superscripts were significantly different (P < 0.05).

by an increase in endothelial cell concentration because there was no significant difference in the mean endothelial cell number/mm2 between control and menorrhagic endometrium (Table IV). There was also no significant difference in endothelial cell concentration between the necrotic post-ablation endometrium and either the normal or menorrhagic endo-

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Figure 1. Photomicrograph of post-ablation endometrial tissue stained for the endothelial cell marker CD34 (blue) and the proliferating cell nuclear antigen (red). Actively dividing cells are observed in a normal endometrial stroma. This biopsy was collected 3 months after ablation during the secretory phase of the menstrual cycle. Bar = 20 (lm.

Menorrhagia: endometrial angiogenesis

metrium. In contrast, endothelial cell density was significantly higher in the histologically normal post-ablation endometrium compared with control, menorrhagic and necrotic post-ablation endometrium (P = 0.01, 0.02 and 0.03 respectively). Discussion

It is clear from this and other studies (Reid et al., 1992; Critchley et al, 1995) that the endometrium can regenerate following endometrial ablation. This regrowth has been reported to occur as 'islands' (Perrella et al, 1992). Although for three of the 18 post-ablation subjects no endometrial tissue was obtained at biopsy, a mean menstrual blood loss of 10 ml was obtained. This suggests that 'islands' of endometrium were missed at biopsy. However, a recent report suggests that menstrual bleeding can occur even in the absence of endometrium (Ewen and Sutton, 1993). There are three major clinical implications associated with the potential regrowth of post-ablation endometrium. Firstly, women should be informed that their post-ablation menstrual blood loss is likely to be substantially less but that a total absence of menstrual bleeding cannot be guaranteed (Critchley et al, 1995). Indeed, only eight of the 18 subjects in this study were amenorrhoeic at 3-6 months post-ablation. Secondly, as blastocyst implantation in residual post-ablation endometrium may still occur, it is essential to advise women of reproductive age to take contraceptive measures. Indeed, a number of pregnancies have been reported in patients after endometrial ablation (Wood and Rogers, 1993; Goldberg, 1994). Thirdly, to reduce the risk of endometrial cancer, post-ablation women on hormone replacement therapy should be prescribed a combination of oestrogen and progestogen. It is unclear from this study why increased endothelial cell proliferation is associated with increased menstrual blood loss. However, any aberration in the process of angiogenesis, such as increased endothelial cell proliferation, could alter the normal development of endometrial blood vessels. Such alterations may result in weakened blood vessels from which excessive amounts of menstrual blood may be released. Alternatively, increased endothelial cell proliferation in such endometrium may occur to compensate for another mechanism that has structurally weakened endometrial blood vessels. Here we have shown that patients with menorrhagia have high endometrial endothelial cell proliferation indices. Conversely, post-ablation subjects with a normalized menstrual blood loss showed a trend towards lower endometrial endothelial cell proliferation indices. These results support our hypothesis that angiogenesis is disturbed in the endometrium of patients with menorrhagia and normalized in post-ablation endometrium. Disturbed angiogenesis may be a direct cause of excessive menstrual bleeding. 1071

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In this study we have shown that endothelial cell proliferation was enhanced significantly in the endometrium of patients with menorrhagia compared with that in control subjects. In contrast, necrotic and histologically normal post-ablation endometrium from patients with a normalized menstrual blood loss showed a trend towards lower endothelial cell proliferation. Although there was no significant correlation between objective menstrual blood loss and endometrial endothelial cell proliferation in patients with menorrhagia, these two factors may be related. Indeed, a previous study has shown that tissue from patients with menorrhagia may have elevated angiogenic potential (Subakir et al, 1992). Alternatively, increased endothelial cell proliferation could be a measure to compensate for other aberrant functioning that occurs in menorrhagic endometrium. The decrease in endothelial cell proliferation in post-ablation endometrium, although not statistically different to that of menorrhagic endometrium, may reflect a normalization of the angiogenic process. We found that there was no significant difference in endothelial cell proliferation when comparing the proliferative and secretory phase biopsies within each study group. This result was in agreement with the study of Goodger (Macpherson) and Rogers (1994), which showed that there were no significant peaks in endothelial cell proliferation across the menstrual cycle. These results do not necessarily mean that there are no differences in endothelial cell proliferation between proliferative and secretory phase biopsies, but rather they indicate that the variability between women is great There was no significant difference in cellular proliferation in the stroma, glands and surface epithelium of endometrium from controls, patients with menorrhagia and post-ablation subjects. This indicates that the increase in endothelial cell proliferation in endometrium from patients with menontiagia is specific and not the result of a general increase of cellular proliferation in the endometrium. Despite the increase of endothelial cell proliferation in endometrium from patients with menorrhagia, there was no change in endothelial cell concentration. It is important to realize that this study only looks at one step in the process of angiogenesis. It does not take into account other factors that may influence endothelial cell numbers. For example, this study does not measure the rate of endothelial cell turnover. The processes surrounding blood vessel growth are complex and involve the interplay of many factors. This is reflected in the apparent increase in endothelial cell number in histologically normal post-ablation endometrium despite the apparent trend towards lower endothelial cell proliferation. It is possible that in post-ablation endometrium, the turnover of endothelial cells is slower than in endometrium from patients with menorrhagia. A recent study by Wingfield et al (1995) revealed that endothelial cell proliferation was increased in the endometrium

of patients with endometriosis compared with controls. This increase was most marked during the proliferative phase of the menstrual cycle and was accompanied by an increase in the level of cellular proliferation in endometrial glands, stroma and surface epithelium (Wingfield et al, 1995). Therefore, it appears that in patients with endometriosis, the increase in endothelial cell proliferation is not specific, but rather occurs as a result of a general increase in endometrial cell proliferation during the proliferative phase. This increase in endothelial cell proliferation does not appear to be associated with increased menstrual blood loss because none of the patients complained of excessive menstrual bleeding (M.Wingfield, personal communication).

J.Kooy et al.

Acknowledgements We wish to acknowledge Mr Peter Paterson, Dr Maxwell Michael and Dr Mark Lawrence, Department of Obstetrics and Gynaecology, Monash Medical Centre, for providing the opportunity to collect endometrial biopsies from the patients in this study. We are grateful to Sister Pamela Mamers for her help with patient coordination. We thank Dr Beatrice Susil, Department of Anatomical Pathology, Monash Medical Centre, for dating the endometrial biopsies, and Dr Nigel Wreford, Institute for Reproduction and Development, Monash University, for providing the facility for use of his equipment for the quantification of endothelial cell proliferation. This study was supported by the Australian National Health and Medical Research Council. J.K. was the recipient of the RACOG Arthur Wilson Memorial Scholarship.

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Received on May 17, 1995; accepted on March 13, 1996

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