Tissue collection
1. Endometrial biopsies were collected from women undergoing gynaecological procedures for benign conditions.
2. All
women reported regular menstrual cycles (25–35 days) and had not
received any form of hormonal treatment in the 3 months preceding
biopsy.
3. Biopsies were dated from the patient's last menstrual period (LMP).
4. Histological
dating according to published criteria and circulating sex steroid
concentrations were consistent with the date of LMP.
5. Written
informed consent was obtained from all patients prior to biopsy
collection and ethical approval was received from Research Ethics
Committee.
6. Tissue samples were collected in primary cell medium and were subsequently divided into two portions.
7. Endometrium
was: (i) fixed in 10% neutral buffered formalin overnight at 4°C,
stored in 70% ethanol and then wax embedded; and (ii) separated into
glandular and stromal compartments for cell culture.
Separation of endometrial biopsies into glandular and stromal compartments
1. This method for separation of glandular and stromal compartments of endometrium was adapted from that of Reference.
2. Several modifications were made and the details of the method used in this study are described below.
3. Endometrial
biopsies were washed twice in phosphate-buffered saline, sliced into
small fragments, immersed in collagenase/DNAase (1 and 0.1 mg/ml) and
incubated for 80 min at 37°C.
4. After incubation, primary cell medium was added and tissue was broken up using a syringe.
5. This yielded single cells and larger, glandular fragments.
6. This
suspension was centrifuged (450 g, 3 min) and then cells/fragments were
resuspended in fresh medium and allowed to separate by density
sedimentation.
7. After 5 min the supernatant (stromal compartment) was removed leaving 2 ml of medium.
8. Fresh
medium was added and the density sedimentation was repeated. The
remaining 2 ml of medium contained glandular fragments that were
centrifuged as above.
9. Medium was discarded and the epithelial fragments were incubated with collagenase/DNAase for 2 h at 37°C.
10. After incubation, medium was added and the cell suspension was centrifuged (see above).
11. Medium was removed and the epithelial cell pellet was resuspended in 50% Matrigel.
Cell culture
1. Primary endometrial epithelial cells were grown in
Matrigel in primary cell medium supplemented with 10% fetal calf serum,
penicillin (50 μg/ml; Sigma), streptomycin (50 μg/ml; Sigma),
gentamycin (5 μg/ml; Sigma), epidermal growth factor (25 ng/ml),
vascular endothelial growth factor (1 ng/ml), basic fibroblast growth
factor (5 ng/ml;) and estradiol (10–7 mol/l).
2. These growth
factors were included in the primary culture medium as there is evidence
that endometrial epithelial cells express their receptors and hence
they are likely to be involved in modulation of cell growth.
References
1. Noyes, R.W., Hertig, A.T. and Rock, J. (1950) Dating the endometrial biopsy. Fertil. Steril., 1, 3–25.
2. Osteen,
K.G., Hill, G.A., Hargrove, J.T. and Gorstein, F. (1989) Development of
a method to isolate and culture highly purified populations of stromal
and epithelial cells from human endometrial biopsy specimens. Fertil.
Steril., 52, 965–972.
3. Li, X.F., Gregory, J. and Ahmed, A. (1994)
Immunolocalisation of vascular endothelial growth factor in human
endometrium. Growth Factors, 11, 277–282.
4. Zhang, L., Rees, M.C.
and Bicknell, R. (1995) The isolation and long-term culture of normal
human endometrial epithelium and stroma. Expression of mRNAs for
angiogenic polypeptides basally and on oestrogen and progesterone
challenges. J. Cell Sci., 108, 323–331.
5. angha, R.K., Li, X.F.,
Shams, M. and Ahmed, A. (1997) Fibroblast growth factor receptor-1 is a
critical component for endometrial remodeling: localization and
expression of basic fibroblast growth factor and FGF-R1 in human
endometrium during the menstrual cycle and decreased FGF-R1 expression
in menorrhagia. Lab. Invest., 77, 389–402.
6. Meduri, G., Bausero, P.
and Perrot-Applanat, M. (2000) Expression of vascular endothelial
growth factor receptors in the human endometrium: modulation during the
menstrual cycle. Biol. Reprod., 62, 439–447.