Hainan Reproduction vol 11 no 9 pp 2009-2013, 1996
Microvascular density in conditions of endometrial
atrophy
M.Hickey1'4, T.M.Lau2, P.Russell 3 ,1.S.Fraser 1 and
P.A.W.Rogers2
'Department of Obstetrics and Gynaecology, University of Sydney,
NSW 2001, ^ o n a s h University Department of Obstetrics and
Gynaecology, Monash Medical Centre, Clayton, Victoria 3168,
3
Department of Anatomical Pathology, Royal Pnnce Alfred
Hospital, Missenden Rd, Camperdown, NSW 2001, Australia
''To whom correspondence should be addressed
Irregular menstrual bleeding in users of hormonal contraception represents the single major reason for discontinuation of these contraceptive methods. The
mechanisms which underlie these bleeding disturbances
are poorly understood, but appear to be associated with
changes in the endometrial microvasculature following
abnormal patterns of sex steroid exposure. Endometrial
microvascular density is known to be increased in users
of the low-dose levonorgestrel contraceptive implant,
Norplant®. This study explores microvascular density in
other conditions of spontaneous (post-menopausal) and
induced (danazol and goserelin) endometrial atrophy.
Endometrial biopsies were fixed, paraffin-embedded and
sections were immunostained using anti-CD34 antibody to
identify vascular endothelial cells. The mean microvascular
density (± SEM) for control samples was 186 ± 8 vessels/
mm2. There were no statistically significant changes in
vascular density observed across the menstrual cycle. Mean
microvascular density in spontaneous and induced endometrial atrophy did not differ significantly from that
observed in the control population. The mean microvascular density was 230 ± 17 vessels/mm2 in 31 postmenopausal women, 269 ± 67 vessels/mm2 in 25 subjects
treated with danazol was and 191 ± 45 vessels/ mm2 in
nine subjects treated with goserelin. These findings suggest
that the mechanisms controlling microvascular density in
conditions of endometrial atrophy may vary according to
the nature of the atrophic stimulus.
Key words: atrophy/endometrium/vascular density
Introduction
The disruption of normal menstrual bleeding patterns by
long-acting progestogenic contraception presents a major
challenge to contraceptive providers. Non-hormonal contraceptive systems may also influence menstrual bleeding.
Copper-containing and inert lntrauterine devices (IUD) are
associated with an increased menstrual blood loss (Andrade
and Orchard, 1987).
© European Society for Human Reproduction and Embryology
Recent attention has focused on the role of endometrial
blood vessels in the initiation and control of menstrual blood
loss and breakthrough bleeding (BTB) (Fraser and Peek,
1992; Rogers et al., 1993). Histological and hysteroscopic
examination of the endometrium in women complaining of
BTB associated with steroid hormone contraceptives suggests
that the source of the bleeding may be capillaries and small
veins (Fraser and Peek, 1992; Hickey et al., 1996). Dilated
and disrupted small venules located close to the endometrial
surface following vaginal (Hourihan et al., 1986) and intrauterine (Sheppard et aL, 1987) progestogen exposure are a
potential site of irregular bleeding.
Angiogenesis (the formation of new blood vessels from
existing vessels) does not usually occur in the adult human
apart from during pathological processes such as retinopathy,
rheumatoid arthritis, tumour growth or wound repair. The
female reproductive tract is an exception, where there is
periodic growth and regression of blood vessels. The process
of angiogenesis and vascular regression in these tissues appears
to be hormonally regulated. However, the local mechanisms
that control these processes have yet to be determined.
Distinct periods of angiogenesis have been identified in
the endometnum during the menstrual cycle (Markee, 1940;
Ferenczy et al., 1979; Kaiserman-Abramof and Padykula,
1989). The mechanisms controlling vascular proliferation in
the endometrium are not fully understood and angiogenesis at
this site may occur by a different process from that in other
tissues (Goodger and Rogers, 1994).
Endometrial atrophy describes the loss of glandular and
stromal elements of the endometrium and may arise in response
to exogenous steroid hormones or following the withdrawal of
endogenous ovarian steroids. The response of the endometrial
vasculature to an atrophic stimulus may differ from that of the
glands and stroma (Hourihan et al., 1986; Rogers et al., 1993).
Two clinical treatments that may induce endometrial atrophy
are danazol and gonadotrophin-releasing hormone (GnRH)
agonists.
Danazol is an isoxazol derivative of 17oc-ethinyl testosterone.
It has numerous effects on the reproductive system; it alters
the pulsatile secretion of GnRH, inhibits the luteinizing
hormone (LH) and follicle stimulating hormone (FSH) surge,
causes abnormal follicular maturation and thus reduces
oestrogen production. Direct endometrial effects include die
suppression of oestrogen and progesterone receptors, rendering
the endometnum functionally hypo-oestrogenic and hypoprogestogenic (Jeppson et al., 1984). The endometrium
following prolonged danazol exposure is likely to be atrophic
(Greenblatt et al, 1971). Bleeding patterns following danazol
exposure are determined by treatment dose. At 800 mg/day
2009
M.Hickey et aL
the majority of patients became amenorrhoeic (Fraser, 1985),
whilst at lower doses (200 mg/day) they may continue to
menstruate regularly, but with a significant reduction in
menstrual blood loss (Chimbira et al, 1980). Between 200800 mg/day, the clinical response to danazol is varied and
bleeding patterns are generally irregular with scanty loss and
a dose-related increase in amenorrhoea (Fraser, 1985).
GnRH analogues have a higher binding affinity to GnRH
receptors than naturally-occurring GnRH, with a greatly
increased half-life. After an initial stimulatory effect,
prolonged use of these agonists down-regulates pituitary
gonadotrophic receptors, producing hypogonadotrophic hypogonadism (Meldrum et aL, 1982). The endometrium is generally thin and atrophic following prolonged GnRH agomst
exposure (Brooks et al., 1991). In normal ovulatory women
GnRH agonists will suppress ovulation and will cause amenorrhoea in up to 95% of women treated, depending on dose. The
rest will have bleeding that is lighter and less regular than
normal (Bergquist et ah, 1981).
This aim of this study was to quantify changes in microvascular density during conditions of endometrial atrophy due
to either danazol or GnRH agonist treatment, or following the
menopause.
Materials and methods
Subjects
Endometnal biopsies were obtained from 73 women exposed to
danazol or to goserelin, or from post-menopausal women not taking
hormone therapy. The latter group had no history of post-menopausal
bleeding and samples were taken from hysterectomy specimens
following vaginal hysterectomy for uterovaginal prolapse. Five specimens were excluded due to lack of endometnum for analysis, and
three due to inadequate patient details. This left 65 biopsies for
analysis: (i) 25 following exposure to 400-600 mg of danazol/day
for 6 weeks (400 mg given prior to endometrial ablation and 600 mg
in the treatment of endometriosis); (ii) nine following exposure to
goserelin for 12 weeks (prior to myoma resection); (iii) 31 postmenopausal women not exposed to hormone therapy.
Biopsies were obtained by outpatient curettage for 11/25 subjects
from the danazol group (Pipelle; Prodimed, Neully-en-Thielle,
France); by diathermy loop resection biopsy under general anaesthetic
for 14/25 danazol subjects and all goserelin subjects; or by hysterectomy specimens for uterovaginal prolapse (all post-menopausal
subjects). All subjects were recruited to the trial on the basis of fully
informed consent Tissue was collected in a number of research
centres m Australia, and local ethical approval for these studies was
obtained from ethics committees at Monash University and from
Family Planning, NSW, Australia.
Controls
Control data for this study were taken from a previously published
report where an identical methodology was used (Rogers et aL, 1993).
Normal menstrual cycle biopsies were collected as controls from 54
volunteers in Melbourne, Australia. This tissue was mainly taken
from women undergoing investigation for infertility, where the cause
of the infertility was shown clearly to be unrelated to uterine or
endocrine factors. Tissue was not collected from women who had
received exogenous sex steroid hormones or who had used an IUD
in the previous 3 months. Those with known utenne pathology were
2010
also excluded Endometnal biopsies were obtained by dilatation and
curcttage (D&C) or by Pipelle suction curette (Prodimed) from the
utenne fundus or corpus. The isthmus was always avoided. The date
of the last menstrual penod was known for all subjects.
Biopsy processing, histopathology and immunohistochemistry
All biopsies were immediately placed in 10% buffered formalin at
4°C for 4-6 h, and then nnsed and stored in phosphate-buffered saline
(PBS) at the same temperature. Endometrial tissue was processed by
routine paraffin embedding, and 5 um sections were cut for either
immunohistochemistry or haematoxylin and eosin (H&E) staining
for histopathological evaluation by an experienced gynaecological
pathologist (PR.). H&E stained sections of endometria were dated
according to the general classifications of Noyes et al. (1950).
The endometrial microvasculature was visualized using the mouse
monoclonal antibody against human CD34 antigen (Clone QBEND/
10; Serotec, Oxford, UK), which is expressed on the endothelial cell
membrane (Fina etal, 1990). Primary antibody binding was visualized
using a streptavidin-bioun horseradish peroxidase immunohistochemical staining kit (Zymed Laboratories, San Francisco, CA, USA).
Pnor to the primary antibody incubation, endogenous peroxidase
acuvity in the endometrial tissue was blocked by treatment with 3%
hydrogen peroxide (BDH Chemicals, Port Fairy, Victoria, Australia)
for 10 min at room temperature. Tissue sections were incubated with
the CD34 antibody for 60 min at 37°C at 1:25 dilution. Normal rabbit
serum (NRS 10%; Gibco, life Technologies Inc, Grand Island, NY,
USA) was added to the CD34 antibody to prevent non-specific binding.
Sections were mounted in Hydromount (National Diagnostics, New
Jersey, USA) without counterstaining to avoid interference during
image analysis. For negative controls, the primary antibody was
replaced by mouse immunoglobuhn (IgX^ (Silenus Laboratories,
Victoria, Australia) used at the same protein concentration, or the
diluent alone was used. In each staining run, a control section of
known immunohistochemical staining intensity was included as a
posiuve control and to ensure consistency between runs.
Microvascular profiles identified by CD34 staining were counted
using a Zeiss Axioskop microscope (Zeiss, GOttingen, Germany) with
a X10 magnification Zeiss Achroplan Lens, without a grid. A
Microcomputer Imaging Device (MCID); version 2, beta 2.4 (Imaging
Research Inc., Brock University, St Catharines, Ontario, Canada) was
used to accurately measure the field of view and count vessels stained
with anti-CD34. Black and white images were used to enhance
contrast between vessels and background staining. Image analysis
assessments of microvascular density were validated by regular
comparison with manual counts. Three fields of endometnal microvascular density (vessels/mm2) were calculated for each biopsy.
Statistical analysis
Statistical analyses were performed using the JMP program from
SAS on a Macintosh 6200/75 computer. Analysis of vanance was
used to assess whether there was a statistically significant difference
in means between two or more groups. When more than two groups
were included in the analysis of vanance, the Tukey-Kramer posthoc test was used to indicate which groups showed statistically
significant differences in means from other groups. Statistical significance was assumed when P <0.05. Values are means ± SEM unless
otherwise stated. The power of this study to detect significant
differences in sample size between the treatment groups was 52%.
Results
The mean age of the study population was 47 ± 9 years. Not
surprisingly, the mean age of subjects differed between the
Mlcrovascular density and endoraetrial atrophy
Table I. Histological classification of control cycle endometnal biopsies
Histological appearance
No of subjects
Menstrual
Early prohferanve
Early-mid prouferative
Mid prohferative
Mid-late proliferative
Late prohferanve
Early secretory
Mid secretory
Late secretory
Total biopsies
4
6
5
6
5
2
7
11
8
54
Table II. Endometnal histology in treatment group Figures in parentheses
give percentage of total
Histology
Post-menopause
Danazol
Goscrehn
Weakly prohferanve
Atrophic
Inactive
Total
6 (19)
15 (48)
10 (32)
31
1 (4)
13 (52)
11 (44)
25
0
6(66)
3(33)
9
Table HL Microvascular density m conditions of endometnal atrophy
comparative number of CD34 staining vessels
Treatment group
Microvascular density (CD34
lmmunostaining, mm2 ± SEM)
No of
subjects
Control (Rogers et aL, 1993)
Danazol
Post-menopause
Goserehn
186
268
230
191
54
25
31
9
±
±
±
±
8
64
17
45
groups. The average age of women in the post-menopausal
group was 62 ± 3 years. Average ages for the danazol and
goserelin groups were 39 ± 0.9 and 41 ± 0 6 years respectively.
The mean age of the control group was 34.8 ± 2 years.
Endometrial histology
Controls
Histopathological assessment of biopsies from 54 control
subjects (Noyes et al, 1950) are indicated in Table I.
Experimental group
Histological assessment was defined as follows: (i) atrophic,
with small sparse glands lined by inactive cuboidal epithelium
and densely hypercellular stroma; (ii) inactive, differing from
proliferative endometrium in the absence of mitoses in stroma]
and glandular cells; (iii) weakly proliferative with weak oestrogenic stimulation, with some irregularity of gland shape and
distribution but no evidence of hyperplasia. The distributions
of these different types are shown in Table II.
CD34 immunohistochemical staining results
CD34 antibody produced positive staining for the endothelial
lining of all identifiable blood vessels in the tissue samples.
The morphological appearance of the vessels did not differ
from the control population. Mean microvascular densities for
the different treatment groups are shown in Table HI.
Statistical analysis
Using analysis of variance, no statistically significant differences in vascular density were identified between the postmenopausal, danazol and goserelin subjects (F ratio = 0.47,
P = 0.62) or between these groups and the controls (F ratio =
1.74, P = 0.14).
No statistically significant difference in endometnal microvascular density was observed between those treated with
400 mg of danazol (mean density 310 ± 113 vessels/mm2)
and treatment with 600 mg danazol (mean density 215 ± 24
vessels/mm2; F ratio = 0.52, P = 0.47).
Table IV shows the endometrial vascular density of the
different treatment groups when further categorized into histological appearance.
No consistent association was observed between endometrial
histology and vascular density (F ratio = 0.30, P = 0.73).
Discussion
The results from this study suggest that the microvascular
density in these conditions of induced and spontaneous endometrial atrophy do not differ significantly from that of a control
population. The large SEM observed in the danazol and
goserelin treatment groups suggests that there is considerable
inter-individual variation in the response to these agents.
These findings differ from that observed in Norplant users
(Rogers et al., 1993) where a mean microvascular density of
294 ± 18 vessels/mm2 was seen, an increase of 158%
above the control population. This suggests that Norplant may
affect the control of endometrial vascular regression and/or
proliferation by mechanisms which differ from those of other
spontaneous and induced causes of endometrial atrophy. Shaw
et al. (1979, 1981) state that vascular density was increased
in IUD users, but it was not possible to compare our findings
with these publications since only relative changes in vascular
density between control and treatment groups are given. In
this study we are able to give precise values for vascular
density/unit area of endometrium. One of the advantages of
using an image analyser to measure vascular density is that it
allows an exact measurement of the tissue area observed.
In the normal menstrual cycle, mean microvascular density
remains static despite cyclical neovascularization and major
changes in growth, oedema and regression in glandular and
stroma] tissues (Shaw et al, 1979, 1981; Hourihan et al.,
1986; Rogers et al, 1993). This suggests that the mechanisms
controlling normal endometrial vascularization are tightly
regulated
The treatment group showing the highest mean vascular
density was that exposed to 400 mg danazol. This dose of
danazol is associated with incomplete suppression of ovarian
activity in many subjects (Fraser, 1985). Similarly, Norplant
is associated with a variable ovarian response, particularly
during the later months of use (Sivm, 1994). This suggests
that endogenous or exogenous sex steroid may act to 'uncouple'
the control of vascular and non-vascular elements in the
endometrium.
In users of high dose progestogens, Song et al. (1995)
observed a decrease in microvascular density, from 169 ± 9.3
2011
MHickey et aL
Table IV. Endometnal vascular density (mm 2 + SEM) and histological appearance
Histology
Post-menopause
Danazol
Goserekn
Mean
Weakly prohferauve
Atrophic
Inactive
Difference between groups groups (ANOVA)
266 + 32
217 + 27
224 ± 29
F ratio = 0 60
P = 0 55
31
92
325 ± 121
217 + 24
F ratio = 0 47
P = 0 62
25
—
210 + 45
153 ± 112
F ratio = 0 33
P •= 0.58
9
244 ± 35
257 + 48
211 + 20
Total
64
ANOVA = analysis of variance.
vessels/mm2 in the control population, to 103 ± 9.6 vessels/
mm2. High-dose progestogens are associated with marked
endometrial oedema, and this may account for the relative
reduction in microvascular density observed by these authors.
The results from this study should be interpreted with
caution, since other factors may have acted to influence
vascular density in these subjects. In the danazol group, 11
women were known to have endometriosis and were treated
with 600 mg of danazol for 4-6 weeks. The remainder (14)
had confirmed menorrhagia and were treated with 400 mg of
danazol for the same period. All those in the goserelin group
were known to have uterine myomata and were treated for 12
weeks. The post-menopausal group were between 2-20 years
since spontaneous or surgical menopause. Timing since
induction of endometrial atrophy may influence vascular
density. Longitudinal studies examining changes in microvascular density following the induction of endometrial atrophy
are in progress.
A wide range of histological appearances were observed in
all the groups studied, demonstrating individual endometrial
variation m response to similar 'atrophic' stimuli. It is possible
that these conditions may have acted independently to alter
endometrial microvascular density. Endometrial vascular
density did not differ significantly within histological groupings, although there was a tendency for atrophic endometrium
to be associated with a higher vascular density. The mean
vascular density for each histological group was similar to
that reported by Rogers et al. (1993) for Norplant users, and
these workers also failed to identify an association between
microvascular density and endometrial histology.
The clinical significance of these changes in vascular density
and morphology are unclear. There is no evidence at present
to suggest that changes in endometrial microvascular density
correlate directly with bleeding patterns (Rogers et al, 1993).
It is possible that new vessels or changes in the ratio of
endometrial vascular and non-vascular elements associated
with endometrial atrophy may result in small blood vessels
that are more 'fragile' than normal endometrial vessels and
that these may be associated with BTB. This may explain the
characteristic pattern of light, prolonged and irregular bleeding
in this population. Endometria exposed to Norplant, danazol
and goserelin have shown the highest vascular density values,
and these sex steroid hormones are more frequently associated
with irregular bleeding than the post-menopausal and highdose progestogen groups.
All biopsies in this study were obtained by blind techniques
from danazol and goserelin-exposed endometrium. Normal
2012
endometria] development (Dallenbach-Hellweg, 1987) and
vascular density (Shaw et al, 1979) are thought to be uniform
throughout the cavity. There is hysteroscopic evidence that
in women exposed to exogenous sex steroid hormones the
endometria] vascular distribution is irregular and patchy
(Brooks etal.,1991; Hickey et al., 1996). Directed endometrial
biopsies should help to clarify the relationship between density
and bleeding. Unfortunately, hysteroscopic equipment is not
available to provide directed biopsy specimens of adequate
size under out-patient conditions.
When endometrium cannot be obtained at endometnal
biopsy, there is evidence that these subjects may differ in
terms of ovarian steroids and bleeding patterns (Hadisaputra,
1996). This may undermine the value of studies where conclusions are drawn from a selected group of subjects with
sufficient tissue at biopsy.
The local mechanisms controlling these changes in endometrial vascular density are unclear, but may differ between
induced and spontaneous atrophy. Receptors for GnRH have
been identified in the endometrium (Bourgain et al, 1994),
and danazol is known to exert direct endometrial effects via
androgenic and progestogenic receptors (Barbieri, 1991). The
effects of sex steroid hormones on the endometrial vasculature
may depend upon the dose, duration and type of hormone,
and may also show individual variation.
The role of oestrogens in the regulation of microvascular
density is unclear, but Rogers et al. (1993) observed that
Norplant users with higher endogenous oestradiol concentrations had a tendency towards lower microvascular densities.
Oestrogen has been used to treat prolonged bleeding episodes
in Norplant (Diaz et al., 1990) and depot medroxyprogesterone
acetate (DMPA) (Fraser, 1983) users. Longitudinal studies
of changes in microvascular density following successful
management of bleeding problems with sex steroid hormones
are required. If oestrogen appears to reduce bleeding by
'normalizing' microvascular density in the endometrium, there
may be a role for other substances which oppose vascular
proliferation or promote vascular regression in the control of
BTB. Serum oestradiol values in this study might have provided
further useful information.
In conclusion, this study suggests that endometrial microvascular density following danazol and goserelin treatment and
in the post-menopause, does not differ significantly from that
of normal endometrium. Variations in endometrial histological
appearance were observed following exposure to danazol,
goserelin and in the post-menopause. Endometrial vascular
density did not differ significantly between histological groups.
Microvascular density and endometrial atrophy
These observations differ from those seen following Norplant
insertion, suggesting that the mechanisms of endometrial
vascular control may differ m response to this low dose
levonorgesterel implant.
Acknowledgements
Dr K.A.McCallum, and Western Diagnostics Pathology, Kalgoorhe,
WA, Australia and Dr C Laverty, Lavcrty's Pathology, Sydney,
Australia, for the donation of endometrial tissue for analysis
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Received on March 29, 1996, accepted on July 17, 1996
2013