1 Department of Obstetrics and Gynaecology, University of Cambridge, The Rosie Hospital, Cambridge, CB2 2SW, UK, 2 Department of Obstetrics and Gynecology, The Jones Institute for Women's Health, Eastern Virginia Medical School, Norfolk, VA 235071627, USA, and 3 Department of Pharmacology, NV Organon, Oss, The Netherlands
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Abstract |
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Key words: angiogenesis/bleeding/contraceptive/endometrium/endothelium
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Introduction |
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Steroid hormones produced by the ovary regulate the menstrual cycle including the changes in the endometrial vasculature. Differing levels, types and distribution of their specific receptors, i.e. oestrogen receptors (ER) and ß, and progesterone receptors (PR) A and B, presumably mediate their actions together with locally produced polypeptide factors, e.g. epidermal growth factor (EGF; Gordon et al., 1995). Vascular endothelial growth factor (VEGF) is a potent angiogenic growth factor (Ferrara et al., 1992
). It is produced in both glands and stromal cells of the endometrium, as well as smooth muscle cells of the myometrium (Charnock-Jones et al., 1993
). VEGF mRNA is up-regulated in the immature rat uterus in response to oestradiol, oestriol and progesterone (Cullinan-Bove and Koos, 1993
; Hyder et al., 1996
). VEGF is, therefore, a possible candidate as a mediator of steroid hormone action on the endometrial blood vessels. Alterations in the normal distribution of VEGF due to the administration of synthetic steroids may be responsible for any resulting aberrant bleeding.
The aims of the present study were to compare parameters which may be important in endometrial bleeding: VEGF, oestrogen receptors (ER), and progesterone receptors (PR) as reflected by immunohistochemistry and endothelial cell number in the endometrium; (i) throughout the normal menstrual cycle; and (ii) after using two different contraceptives, Implanon and Mircette. The effects of these parameters on the observed bleeding patterns and histology were analysed.
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Materials and methods |
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Endometrial biopsies were taken on an out-patient basis by Pipelle before treatment and ~12 months after commencement. Specimens were formalin fixed and embedded in paraffin (5657°C melting point, Surgipath Medical Industries, Richmond, IL, USA). Sections (5 µm) were mounted on 2% aminopropyltriethoxysilane (Sigma, St Louis, MO, USA) coated slides. One section was stained with haematoxylin and eosin for classification by a gynaecological histopathologist. These categories were normal proliferative, early secretory (i.e. days dated 1519), mid-secretory (days 2022) and late secretory (days 2328; Hendrickson and Kempson, 1980), atrophic, or oral contraceptive effect (i.e. consistent with exogenous progestin administration). Additional sections were used for immunohistochemistry as described below.
Immunohistochemistry
Vascular endothelial growth factor
Sections were stained for VEGF with an antibody directed against amino acids 120 of mature human VEGF and which therefore recognizes the 121, 145, 165, 189 and 206 amino acid splice variants. Sections were dewaxed, rehydrated and washed in phosphate-buffered saline (PBS). Endogenous peroxidase was quenched with 3% hydrogen peroxide in PBS (10 min), before application of primary antibody (without prior antigen unmasking): rabbit anti-human VEGF (A-20; Santa Cruz Biotechnology, Santa Cruz, CA, USA), diluted to 0.5 µg/ml in 1% bovine serum albumin (BSA)PBS, incubated for 1 h at 37°C. This was followed by a biotinylated secondary antibody: goat anti-rabbit immunoglobulin G (IgG; Zymed Laboratories, San Francisco, CA, USA), 1/100 in PBS (10 min); then horseradish peroxidasestreptavidin conjugate (Zymed) 1/400 in PBS (10 min). Colour was developed with aminoethylcarbazole (AEC) substrate chromogen mix (Zymed) for 510 min. Sections were counterstained with diluted Mayer's haematoxylin (1/20) for 1 min, then mounted in aqueous mounting medium (Clearmount Mounting Solution; Zymed). For negative controls 0.5 µg/ml normal rabbit IgG was substituted for the primary antibody.
Oestrogen and progesterone receptors
Separate sections were stained for ER and PR using mouse monoclonal antibodies: ER 1D5 (Dako, Glostrup, Denmark) and PR-AT 4.14 (ABR; Golden, Colorado, USA). Antigen retrieval was carried out by microwaving in 0.01 mol/l sodium citrate buffer (pH 6.0) for 10 min at 500 W. Antibodies were diluted 1/50 in protein blocking solution (1.5% normal horse serum, 2% BSA in PBSTween) and incubated overnight at 4°C after a 30 min pre-block. The secondary antibody was biotinylated horse anti-mouse (Vector Laboratories, Burlingame, CA, USA), diluted 1/200 for 45 min. This was followed by the Vectastain Elite ABC complex (1/200, 30 min), then metal-enhanced diaminobenzidine (Pierce Europe BV, Oud Beijerland, The Netherlands) in peroxidase buffer, 10 min and haematoxylin for 30 s. Sections were dehydrated and mounted in DePX (Merck, Poole, UK).
The glandular epithelium and stromal cell compartments from each stained section were scored subjectively on a scale of 04 by two or three independent observers. For VEGF, the scores were based on the staining intensity of the majority of cells. For ER and PR however, modified H-scores were assigned (Ravn et al., 1993). First an estimate was made for the fraction (%) of stained cells in each compartment: 0 = 09%, 1 = 1039%, 2 = 4069%, 3 = 7089%, and 4 = 90100%. Second, the staining intensity (I) was scored: 0 = no staining, 1 = weak but definite staining, 2 = moderate staining, 3 = pronounced staining and 4 = intense staining. The H-score was then calculated by the formula (%xI) / 4. The averaged subjective scores were analysed by the non-parametric Wilcoxon test for paired samples (before- versus after-treatment).
Endothelial cell number
Separate sections were stained to determine the number of endothelial cell (EC) nuclei per mm2. The method used was essentially as described previously (Goodger Macpherson and Rogers, 1994) and used an antibody which stains all endometrial vessels. Sections were treated as for VEGF above, except the primary antibody was mouse anti-human CD34 (QBEND 10; Serotec, Oxford, UK) diluted 1/25 in 1% BSAPBS incubated for 45 min at 37°C, and the secondary antibody was biotinylated rabbit anti mouse IgG (Zymed). All cell nuclei were stained with undiluted haematoxylin. From these sections, an estimate of the endothelial cell number was obtained, i.e. the number of EC nuclei per mm2. Each section was viewed under a microscope at x400, connected via a video camera to a personal computer and sampled with software using a uniform systematic random sampling method with a meander algorithm after outlining the section (Grid stereological software; Interactivision, Silkeborg, Denmark). In this way, 50 or 100 fields of known area per section were sampled and EC nuclei counted by one observer.
Bleeding records
Daily records of vaginal bleeding were kept by each participant for at least 13 cycles, with the following entries: no bleeding or spotting; spotting, requiring no more than one sanitary napkin or tampon per day; or bleeding, requiring more than 1 sanitary napkin or tampon per day. Records were analysed using a 90 day reference period immediately prior to the day of the after-treatment biopsy (biopsy day = day 91). Definitions used were as follows (Rogers et al., 1993): (i) a bleeding/spotting episode 1 or more consecutive days during which blood loss (bleeding or spotting) was recorded, each episode being bounded by two or more bleeding/spotting free days; a single bleeding/spotting free day within a bleeding/spotting episode being counted as part of the episode surrounding it; and (ii) a bleeding/spotting free interval 2 or more consecutive days during which blood loss had not occurred; each interval being bounded by bleeding/spotting days.
Bleeding during this 90 day reference period was categorized as: (a) amenorrhoea no bleeding/spotting; (b) prolonged one or more bleeding/spotting episodes lasting 10 days or more; (c) frequent more than four bleeding/spotting episodes; (d) infrequent less than two bleeding/spotting episodes; (e) irregular range of length of bleeding/spotting free intervals (i.e. greatest interval minus smallest interval) >17 days; (f) regular two to four bleeding/spotting episodes, no bleeding/spotting episode lasting 10 days or more, with a range of length of bleeding/spotting free intervals of 17 days or less.
The bleeding patterns in the women receiving the oral contraceptive were described as withdrawal bleeding, absence of withdrawal, early withdrawal, breakthrough bleeding or continued withdrawal. Statistical analysis was performed using non-parametric tests to look for possible relationships between the VEGF, ER, and PR glandular and stromal staining and endothelial cell number, and their influence on bleeding category and histological classification of the after-treatment biopsy.
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Results |
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Endothelial cell number
There were no significant changes in endometrial endothelial cell number across the normal cycle (n = 28 overall; proliferative phase n = 8; early secretory n = 7; mid-secretory n = 4; late secretory n = 9). See Table I for median scores and ranges.
Correlations between immunohistochemical parameters
In order to look for possible relationships between the various immunohistochemical parameters measured, Spearman rank correlation tests were used. There were significant positive correlations between glandular and stromal VEGF staining (r = 0.65, P < 0.0001), glandular ER and glandular PR (r = 0.57, P = 0.003) and stromal VEGF and endothelial cell number (r = 0.38, P = 0.046); and a negative correlation between glandular VEGF and glandular PR staining (r =0.49, P = 0.01).
Implanon study
Patients (n = 14) participated, with a median age of 26.0 years, range 1840 years. Detailed results showing averaged subjective scores for VEGF, ER and PR staining in the glands and stroma, together with their endothelial cell numbers, histological classification and bleeding summaries are shown in Table I.
Routine histology
All before-treatment biopsies were taken during the secretory phase of the cycle as judged by routine histology. Use of Implanon altered the histological appearance of the endometrium, resulting in reduced endometrial volume. A total of 11 after-treatment biopsies were judged to be atrophic, one weakly proliferative, one proliferative and one menstrual.
Immunohistochemistry
Seven of the after-treatment biopsies were judged to be inadequate, so these patients were excluded from analysis where appropriate. No staining was seen in negative controls. VEGF staining was always cytoplasmic. See Table I for median scores and their ranges for the immunohistochemical parameters in after-Implanon treatment biopsies. There was a significant reduction in VEGF staining in the glands with treatment (n = 6 before- versus after-treatment biopsy pairs, P = 0.031). There was no change in stromal VEGF staining with treatment. See Figure 1AD
for photomicrographs of examples of VEGF staining of endometrium from the same patient before and after Implanon treatment. Specific ER and PR staining was always nuclear and an example of this is shown in Figure 2AD
. The glands showed significantly increased PR staining with treatment (n = 7 pairs, P = 0.016) and a trend (although not significant) towards increased glandular ER staining (n = 6 pairs, P = 0.063). There was no change in stromal ER and PR staining. Endothelial cell number did not change with treatment.
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Menstrual bleeding pattern versus immunohistochemical parameters
Implanon disrupted the normal menstrual bleeding pattern in all subjects and resulted in amenorrhoea in only four out of 14. KruskalWallis analysis of variance showed that there were no significant differences between the observed bleeding categories during the 90 day reference period and any of the immunohistochemical parameters measured in the after-Implanon treatment biopsies.
Mircette study
Twenty patients participated, with a median age of 29.5 years (range 2347). Detailed results showing averaged subjective scores for VEGF staining in the glands and stroma, together with their endothelial cell numbers, histological classification and bleeding summaries are shown in Table I.
Routine histology
Eight before-treatment biopsies were taken during the proliferative phase of the cycle, 11 during the secretory phase, and one was judged to be inactive. Like Implanon, Mircette use altered the histological appearance of the endometrium. Of the after-treatment biopsies, three were judged to be proliferative, two secretory, seven atrophic, seven to show an exogenous progestogen effect, and one an exogenous progestogen effect with shedding.
Immunohistochemistry
One of the after-treatment biopsies was judged to be inadequate for immunohistochemical staining due to the small volume of tissue, so this patient was excluded from analysis where appropriate. No staining was seen in negative controls. See Table I for median scores and their ranges for the immunohistochemical parameters in the after-Mircette treatment biopsies. Statistical analysis showed that overall there was a significant reduction in VEGF staining in the glands with treatment (n = 19 before- compared with after-treatment biopsy pairs, P = 0.007). However, this reduction with treatment is not apparent when comparing only paired proliferative before-treatment biopsies against after-treatment samples (n = 7, P = 0.813), but is quite marked when comparing only paired secretory before-treatment biopsies against after-treatment samples (n = 11, P = 0.001). There was no significant difference in VEGF staining when comparing proliferative before-treatment biopsies (n = 8) with secretory before-treatment biopsies (n = 11, P = 0.129). Nor was there any significant change in stromal VEGF staining with treatment. See Figure 1EH
for photomicrographs of VEGF staining of endometrium from the same patient before and after Mircette treatment.
Overall, there were no changes in ER or PR staining with treatment in either the glands or stroma. There were no significant changes in glandular ER or PR when comparing only paired proliferative before-treatment biopsies against after-treatment samples, or paired secretory before-treatment biopsies against after-treatment samples. There were also no significant changes in stromal ER or PR when comparing only paired proliferative before-treatment biopsies against after-treatment samples. However, when comparing only paired secretory before-treatment biopsies against after-treatment samples, there was significantly reduced stromal PR staining with treatment (P = 0.027) and a trend towards increased stromal ER staining with treatment (P = 0.065). Endothelial cell number did not change significantly with treatment.
Correlations between immunohistochemical parameters
There was a significant positive correlation (Spearman rank correlation) between glandular EGF and stromal VEGF (r = 0.61, P = 0.006) after treatment. There were no other significant correlations between the immunohistochemical parameters measured in the after-Mircette treatment biopsies.
Histological category versus immunohistochemical parameters
KruskalWallis tests showed that the histological category of the after-treatment biopsy did not influence any of the immunohistochemical parameters.
Menstrual bleeding pattern versus immunohistochemical parameters
Mircette resulted in a regular bleeding pattern in the majority of subjects (11/19, 58%). Of the remaining subjects, seven experienced bleeding in addition to withdrawal bleeding and one woman failed to have regular withdrawal bleeding. Bleeding pattern data were analysed against each of the immunohistochemical parameters by the unpaired non-parametric test (MannWhitney) using two groups only: regular bleeders versus the rest; due to the very low subject numbers in each of several different bleeding categories. Results showed that there were no significant changes to the immunohistochemical parameters with bleeding pattern, however there was a trend towards increased stromal ER staining with non-regular bleeding (P = 0.051).
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Discussion |
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The present study showed that glandular PR staining was significantly reduced during the mid- and late secretory phases of the cycle compared to the proliferative phase. These results agree with those of others (e.g. Snijders et al., 1992; Critchley et al., 1993) and is consistent with down-regulation by progesterone (Chauchereau et al., 1991; Snijders et al., 1992
). The continued presence of PR in the stroma through the cycle also agrees with previous work (Critchley et al., 1993
). The lack of statistically significantly reduced glandular or stromal ER staining as the cycle progressed in the current study is probably due to insufficient subject numbers. Trends towards reduced ER staining in the secretory phase were, however, apparent.
The correlation between VEGF immunoreactivity in the glands and stroma suggests a similarity in regulation, although the variation in the glands is more pronounced. The relationship between glandular ER and glandular PR is reflected by similar regulation of each receptor by oestrogen and progesterone, i.e. oestrogen alone up-regulates ER and PR, whereas progesterone down-regulates ER and PR (McDonnell et al., 1995). The negative correlation between glandular VEGF and glandular PR is of interest, since this is consistent with the hypothesis that endometrial glandular VEGF is regulated by progesterone in vivo in the normal human adult. The finding that progesterone increases expression of the most abundant forms of VEGF in the uterus of immature rats is also consistent with this hypothesis (Cullinan-Bove and Koos, 1993
). The antibody used in this study does not distinguish between the different spliced forms of VEGF and it is suggested that the smaller 121 form of VEGF is the main variant expressed by stromal cells (Huang et al., 1998
). However, 165 is the main variant of glandular expression and like the 121 variant is regulated by ovarian steroids.
The correlation between stromal VEGF staining and endothelial cell number has not been previously reported. More work, with larger subject numbers, is required to investigate this interesting relationship further. As an angiogenic factor, it seems probable that VEGF could influence endometrial vascular density. In tumours, microvessel density has been used as a measure of tumour angiogenesis, with increased intratumoral microvessel density correlating with increased metastasis and/or decreased patient survival (Vartanian and Weidner, 1994). However, in the present and previous studies, neither endometrial endothelial cell number nor microvascular density (Rogers et al., 1993
; Goodger Macpherson and Rogers, 1994
) changed throughout the normal cycle and indeed changes in endothelial cell number do not necessarily indicate changes in angiogenic activity (Goodger Macpherson and Rogers, 1995
). The lack of change in intensity of stromal VEGF staining and endothelial cell number during the cycle suggest that these parameters may be regulated by non-steroidal mechanisms. However, this is at odds with the suggestion that glandular and stromal VEGF are regulated by similar mechanisms and that glandular VEGF is regulated by progesterone.
The contraceptives Implanon and Mircette, which contain the pro-form of the same progestagen in combination with ethinyl oestradiol, both significantly reduced endometrial glandular immunoreactive VEGF. After 12 months of use there was no difference in endothelial cell number compared to before-treatment, and there was no correlation between glandular VEGF staining and endothelial cell number for the after-treatment biopsies from either the Implanon or Mircette studies. In the light of the observation that stromal VEGF correlates with endothelial cell number during the normal cycle, however, the reduction in glandular VEGF with contraceptive treatment may be biologically unimportant. Indeed there was a similar positive correlation between stromal VEGF and endothelial cell number with Implanon treatment, but not Mircette treatment. The significantly increased glandular PR staining with Implanon use shows that etonogestrel alone fails to down-regulate the progesterone receptor in the same way that progesterone does. It is likely that the balance of agonist/antagonist actions and the progesterone receptor type contributes to the end result of progestin action. On the other hand, Mircette resulted in significantly reduced stromal PR compared with normal secretory phase values in response to progesterone. For both contraceptives, however, neither bleeding patterns during the 90 day reference period just prior to the after-treatment biopsy, nor histological category of the after-treatment biopsy showed any significant differences with VEGF, ER or PR staining, or endothelial cell number.
The lack of change in endothelial cell number with the two contraceptives used in this study contrasts with the action of three other contraceptives, for example, Norplant (an implant which releases 80 µg/day levonorgestrel); high doses of norethisterone and medroxypogesterone acetate (MPA). Norplant results in significantly increased microvascular and endothelial cell number compared with that seen during the normal cycle (despite significantly reduced endothelial cell proliferation), probably due either to increased regression of tissue surrounding the endometrial vessels, or a reduced rate of endothelial cell death (Rogers et al., 1993; Goodger Macpherson et al., 1994
; Hickey et al., 1999
). The increased vascular density did not seem to be directly related to increased endometrial bleeding. On the other hand, treatment with higher doses of norethisterone or MPA resulted in a significantly decreased endometrial vascular density (Song et al., 1995
). None of these studies, however, investigated VEGF immunoreactivity. Another study investigating endometrial microvascular density in various conditions of atrophy found no differences compared with the endometrium removed from normally cycling women (Hickey et al., 1996
). The present study confirms the apparent dissociation between endometrial endothelial cell number and endometrial bleeding. It has also been proposed that there may be considerable endometrial microvascular heterogeneity, particularly in response to Norplant treatment resulting in focal bleeding sites (Rogers, 1996
). The same may be true with other steroid treatments.
In conclusion, the present study shows that immunoreactive VEGF increases significantly in the glands of the endometrium during the normal menstrual cycle, while immunoreactive PR decreases. This study also shows that Implanon and Mircette significantly reduce glandular VEGF staining. This is not associated with changes in endothelial cell number, bleeding pattern, or histological category of the endometrium. Stromal staining for VEGF correlated positively with endothelial cell number although neither parameter changed significantly throughout the menstrual cycle, or influenced the endometrial bleeding pattern.
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Acknowledgments |
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Notes |
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References |
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Chauchereau, A., Savouret, J.F., and Milgrom, E. (1991) Control of biosynthesis and post-transcriptional modification of the progesterone receptor. Biol. Reprod., 46, 174177.[Abstract]
Critchley, H.O.D., Bailey, D.A., Au, C.L. et al. (1993) Immunohistochemical sex steroid receptor distribution in endometrium from long-term subdermal levonorgestrel users and during the normal menstrual cycle. Hum. Reprod., 8, 16321639.[Abstract]
Cullinan-Bove, K., and Koos, R.D. (1993) Vascular endothelial growth factor/vascular permeability factor expression in the rat uterus: Rapid stimulation by estrogen correlates with estrogen-induced increases in uterine capillary permeability and growth. Endocrinology, 133, 829837.[Abstract]
Ferrara, N., Houck, K., Jakeman, L. et al. (1992) Molecular and biological properties of the vascular endothelial growth factor family of proteins. Endocr. Rev., 13, 1832.[ISI][Medline]
Goodger Macpherson, A.M. and Rogers, P.A.W. (1995) Blood vessel growth and endothelial cell density in rat endometrium. J. Reprod. Fertil., 105, 259261.[Abstract]
Goodger Macpherson, A.M., and Rogers, P.A.W. (1994) Endometrial endothelial cell proliferation during the menstrual cycle. Hum. Reprod., 9, 399405.[Abstract]
Goodger Macpherson, A.M., Rogers, P.A.W. and Affandi, B. (1994) Endometrial endothelial cell proliferation in long-term users of subdermal levonorgestrel. Hum. Reprod., 9, 16471651.[Abstract]
Gordon, J.D., Shifren, J.L., Foulk, R.A. et al. (1995) Angiogenesis in the human female reproductive tract. Obstet. Gynecol. Surv., 50, 688697.[Medline]
Greb, R.R., Bukowski, R., Hsiu, J.G. et al. (1995) Vascular endothelial growth factor (VEGF) in primate endometrium. Immunohistochemical patterns during the cycle and after chronic RU 486 treatment in cynomolgus monkeys. Ann. N.Y. Acad. Sci., 761.
Hendrickson, M.R. and Kempson, R.L. (1980) Surgical Pathology of the Uterine Corpus. W.B.Saunders, Philadelphia, USA.
Hickey, M., Lau, T.M., Russell, P. et al. (1996) Microvascular density in conditions of endometrial atrophy. Hum. Reprod., 11, 20092013.[Abstract]
Hickey, M., Simbar, M., Markham, R. et al. (1999) Changes in vascular basement membrane in the endometrium of Norplant users. Hum. Reprod., 14, 716721.
Huang, J.C., Liu, D.Y., Dawood, M.Y. (1998) The expression of vascular endothelial growth factor isoforms in cultured human endometrial stromal cells and its regulation by 17ß-oestradiol. Mol. Hum. Reprod., 4, 603607.[Abstract]
Hyder, S.M., Stancel, G.M., Chiappetta, C. et al. (1996) Uterine expression of vascular endothelial growth factor is increased by estradiol and tamoxifen. Cancer Res., 56, 39543960.[Abstract]
Kaunitz, A.M. (1993) Combined oral contraception with desogestrel/ethinyl estradiol: Tolerability profile. Am. J. Obstet. Gynecol., 168, 10281033.[ISI][Medline]
Kuhl, H. (1996) Comparative pharmacology of newer progestogens. Drugs, 51, 188215.[ISI][Medline]
Lau, T.M., Affandi, B. and Rogers, P.A.W. (1999) The effects of levonorgestrel implants on vascular endothelial growth factor expression in the endometrium. Mol. Hum. Reprod., 5, 5763.
Li, X.F., Gregory, J. and Ahmed, A. (1994) Immunolocalisation of vascular endothelial growth factor in human endometrium. Growth Factors, 11, 277282.[ISI][Medline]
McDonnell, D.P., Clemm, D.L., Hermann, T., et al. (1995) Analysis of estrogen receptor function in vitro reveals three distinct classes of antiestrogens. Mol. Endocrinol., 9, 659669.[Abstract]
Ravn, V., Bruun Rasmussen, B., Hojholt, L. et al. (1993) Reproducibility of subjective immunohistochemical estrogen- and progesterone receptor determination in human endometrium. Pathol. Res. Prac., 189, 10151022.[ISI]
Rogers, P.A.W. (1996) Endometrial vasculature in Norplant users. Hum. Reprod., 11 (Suppl. 2), 4550.[ISI][Medline]
Rogers, P.A.W., Au, S.L. and Affandi, B. (1993) Endometrial microvascular density during the normal menstrual cycle and following exposure to long-term levonorgestrel. Hum. Reprod., 8, 13961404.[Abstract]
Shifren, J.L., Tseng, J.F., Zaloudek, C.J. et al. (1996) Ovarian steroid regulation of vascular endothelial growth factor in the human endometrium: Implications for angiogenesis during the menstrual cycle and in the pathogenesis of endometriosis. J. Clin. Endocrinol. Metab., 81, 31123118.[Abstract]
Snijders, M., De Goeij, A., Debets-Te Baerts, M.J.C. et al. (1992) Immunocytochemical analysis of oestrogen receptors and progesterone receptors in the human uterus throughout the menstrual cycle and after the menopause. J. Reprod. Fertil., 94, 363371.[Abstract]
Song, J.Y., Markham, R., Russell, P. et al. (1995) The effect of high-dose medium- and long- term progestogen exposure on endometrial vessels. Hum. Reprod., 10, 797800.[Abstract]
Torry, D.S., Holt, V.J., Keenan, J.A. et al. (1996) Vascular endothelial growth factor expression in cycling human endometrium. Fertil. Steril., 66, 7280.[ISI][Medline]
Vartanian, R.K. and Weidner, N. (1994) Correlation of intratumoral endothelial cell proliferation with microvessel density (tumor angiogenesis) and tumor cell proliferation in breast carcinoma. Am. J. Pathol., 144, 11881194.[Abstract]
Wilde, M.I. and Balfour, J.A. (1995) Gestodene A review of its pharmacology, efficacy and tolerability in combined contraceptive preparations. Drugs, 50, 364395.[ISI][Medline]
Submitted on April 1, 1999; accepted on September 2, 1999.