1. Endometrial biopsies were collected from women undergoing gynaecological
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
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.
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
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
4. After incubation, primary cell medium was added and tissue was broken up using
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
8. Fresh medium was added and the density sedimentation was repeated. The
remaining 2 ml of medium contained glandular fragments that were centrifuged as
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
11. Medium was removed and the epithelial cell pellet was resuspended in 50%
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.
1. Noyes, R.W., Hertig, A.T. and Rock, J. (1950) Dating the endometrial biopsy.
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–
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.
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.
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.
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Universidade Federal do Ceará Pró-Reitoria de Pesquisa e Pós-Graduação PIBIC 2012/2013 - Edital 01/12 Desenvolvimento e validação de métodos analíticos e estudo de estabilidade para avaliação de fosfomicina em matéria-prima e granulado A fosfomicina trometamol possui um amplo espectro antimicrobiano contra bactérias gram-positivase gram-negativas comumente associadas