Document Type : Original Article
The endometrium is an important part of the female
reproductive tract and plays a pivotal role in
uterine pathophysiology. Human endometrium is a
unique and dynamic tissue which has an intensive
period of proliferation, growth, angiogenesis, remodeling,
and destruction (
Sildenafil is a member of the 5-phosphodisterase
(5PDE) inhibitor, which hydrolyzes destructive
enzymes of cyclic guanosine monophosphate
(cGMP) and increases the intracellular level of
both cGMP and nitric oxide (NO) (
NO is a small, multi-faced molecule with a regulatory
role in many areas of biology. It diffuses the
cell membrane freely and controls the physiologic
and pathologic function of the cardiovascular, immune,
and nervous systems (
The biological role of NO was first detected
in the macrophages and neutrophils of rodents(
The role of NO in uterine biology and pathophysiology
is defined by the regulation and spontaneous
contraction of the myometrium during pregnancy.
Uterine circulation-induced NO synthetase enzyme
(NOS) is found in vessel walls, neurons, glandular
epithelium, endometrial stromal cells, myometrial
stromal cells, and mast cells (
Studies show that vaginal sildenafil improves
sexual response and endometrial receptivity, and it
can cure the sexual function of menopause women
After looking at other literature, there are no reports
on the effect of sildenafil on EEC. The aim of
this study, therefore, is to investigate the effect of
sildenafil on the numbers and morphology of EEC
and their NO secretion
In this experimental
Endometrial biopsies were washed in PBS that
contained a 2% antibiotic - antimycotic solution
(Sigma, Germany). The biopsies were chopped in a
2 mg/ml collagenase I solution (Sigma, Germany)
in DMEM/F12 media (Gibco, Denmark) and incubated
at 37°C for 90 minutes. Cell suspensions
were passed through 70 and 40 μm filter mesh
(cell strainer; Becton Dickenson Company, USA).
The 40 μm filter mesh was washed back to collect
endometrial glands (
The cells were divided into four groups. The
control group received DMEM/F12 media that
contained a 1% antibiotic–antimycotic solution
supplemented by 5% fetal bovine serum and 2
ìM L-glutamine. Experimental groups received
the same media and either 1, 10 or 20 ìM sildenafil
doses. These doses were selected based on
pervious work (
With a 6-10 second half-life, NO is very unstable
and rapidly converts to nitrite in media that contains
oxygen. NO concentration in the supernatant was
determined with the Greiss method (
Epithelial cell supernatants were collected each
time the media was changed and kept at -20° C.
The protein and phenol red of the supernatant were
deleted using Zinc sulfate (6 mg/400 μliter) (
Sodium nitrite (0.1 M) was used for the standard
curve, and increasing concentrations of sodium nitrite
(5, 10, 25, 50, 75, and 100 μM) were prepared.
The Greiss solution was added to all microplates
containing sodium nitrite and supernatant and was
read by an ELISA reader (stat fax100. USA) in
540 nm and 630 nm filters (
Data were analyzed by one way analysis variance and post hoc Tukey test. P<0.05 was considered significant.
Cell confluency was almost the same between the
control and case groups, with no significant difference
in final cell numbers (p=0.526). The 10 μM
dose showed the highest cell numbers (
EEC means number in control and experimental groups.
The cell viability assay with trypan blue staining
showed that the cells were alive at the end of the
study and sildenafil did not have a toxic effect on
them. The EECs were spheroid after collagenase
digestion of the endometrial tissue (
At the end of the first week, the EEC had a polygonal
to spindle shape, and during the second
week, they became long spindle shaped (
The means of NO were 70.17 in the control
group, 69.55 in the 1 ìM, 66.53 for 10 ìM, and
68.52 for 20 ìM doses of sildenafil. There was no
significant difference in NO secretion between the
control and case groups (p=0.761,
EEC (A) ×200 and epithelial glands (B) ×400 at beginning of the culture. Epithelial cell island (C) at the end of first week. Spindle EEC (D) during second week ×400.
EEC at the end of the study. Control group: (A) ×200. (B) ×400. 1 μM group: (C) × 200, (D) ×400, 10 μM, (E) ×200, (F) ×400.
NO concentration (μM) mean in control and experimental groups.
To our knowledge, the present study is the first
report on the effect of sildenafil on human EEC in
Some reports introduce sildenafil for the improvement
of endometrial thickness and receptivity (
One of the aims of the present work was to measure NO secretion by EEC using Greiss methods.
In the present work, sildenafil did not change NO
secretion. NO is an important regulator of the biology
and physiology of the reproductive system.
The complexity of its biological effects is a consequence
of its numerous potential interactions with
other molecules such as reactive oxygen species
(ROS), metal ions, and proteins (
The effects of NO are modulated by both direct
and indirect interactions that can be dose-dependant
and cell-type specific. NO can induce apoptosis
in some cell types and inhibit apoptosis in others.
Low NO concentration can inhibit apoptosis,
but a higher concentration of NO may be toxic and
can induce cell death, if not by apoptosis then by
More studies have to be performed to determine the exact mechanisms of sildenafil on EEC.
Sildenafil did not show inhibitory or excitatory effects on NO secretion by EEC and in lower doses, it exerted a proliferative effect.