The Effect of Erythropoietin
in preventing
ischaemia-reperfusion injury
in ovarian tissue transplantation
Ali KOLUSARI1, Ayşe GÜLER OKYAY2, Evrim Arzu KOÇKAYA3
1 Yuzuncu Yil University, Faculty of Medicine, Department of Obstetrics and Gynecology, Van / Turkiye
2 Mustafa Kemal University, Tayfur Ata Sokmen Medical Faculty, Department of Obstetrics and Gynecology, Hatay/Turkiye
3 Gazi
University, The Higher Vocational Scool of Health Services, Ankara/ Turkiye
This study was conducted at Ankara University Animal Laboratory
after approval was taken from Ethics Committee of Yuzuncu Yil
University.
It is an experimental study.
Th study was supported financially by the Coordination of Scientific
Research Projects (SRP) of Yuzuncu Yil University
2
Objective
To evaluate the effects of recombinant human erythropoietin (EPO)
on tissue viability in autotransplanted rat ovaries.
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Erythropoietin (EPO)
•
•
•
•
A LMW glycoprotein hormone
Stimulates erythropoiesis
Synthesized in the fetal liver and in the adult kidney.
Ccytokine that exerts diverse biological effects such as
antioxidant
antiapoptotic
anti-inflammatory effects
• Extrahematopoietic functions
Angiogenesis
Antioxidative
Protective effects on the IR injury of ovary
4
Study Design-1
• 21 Wistar Albino type female rats at beginning
• 70 days-old and 160-180 gr
• 4 animals died, completed with 17 rats.
• 3 groups;
Sham control group (n=5)
EPO-treated group (n=6)
EPO-untreated group (n=6)
• Transplantation of ovaries in EPO-treated and untreated groups.
5
Transplanted ovaries in the pouch formed at anterior abdominal wall.
6
Study Design-2
• EPO-treated group………local 5000 U/kg EPO administration, 2 doses, 4 weeks interval
• EPO-untreated group………same amount of normal saline solution, locally to the
transplantation site
• 2 months after transplantation, ovaries were removed and blood samples were obtained.
• Tissues and blood samples were stored at -80˚C, gradual thawing on test day.
• Levels of VEGF, VEGF-C, LPO
activity of GPX, SOD and CAT
in blood and tissue samples
• Histopathological and morphometric analysis on tissue samples.
7
Study Design-3
Measurements made in rat sera:
After about 20 minutes rest, samples were centrifuged at 3500 XG for
15 minutes.
The serum was used to measure the level of
hemoglobin (Hb)
hematocrit (Htc)
VEGF, VEGF-C, LPO
the activities of GPX, SOD and CAT
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Study Design-4
• Biochemical tissue procedure:
The ovarian tissues (approximately 100 mg) were homogenized in
ice-cold Phosphate Buffered Saline (pH 7.4); 1:10 weight/volume
(w/v) with a homogenizer (IKA ultra turrax T 25 basic, IKA
Labotechnik, Staufen, Germany) at 16,000 rpm for 5 min. All
procedures were performed at 4˚C.
The homogenates were used to measure the level of VEGF, VEGF-C,
LPO, protein and the activity of GPX , SOD, and CAT.
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Study Design-5
• Histopathological Examinations
The ovarian tissues were fixed Bouin’s fluid and embedded in paraffin.
Sections (5 Micron) were stained with H&E, and then examined with
Olympus BX51 system light microscope for histopathological
evaluation.
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Study Design-6
• Morphometric Analysis
In morphometric analysis, ovarian follicle diameters (X axis=long axis, Y
axis=short axis) and heights of epithelium (in random 3 fields) of
Graafian follicle were measured at 200X magnification in ovary
sections.
All the measurements were made software Bab Bs200prop in Olympus
BX51 system light microscope.
In addition, the numbers of antral follicle were determined in the ovary
sections by light microscope.
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Study Design-7
• Statistical Analysis
SPSS (ver. 13) statistical program was used for all statistical
computations. Descriptive statistics for studied variables were
presented as median, mean, standard deviation, minimum and
maximum values. Kruskal-Wallis test was used to compare group
means for the studied variables. Dunn test was performed to
determine different group. Groups of data were compared with an
analysis of variance (ANOVA) followed Tukey’s multiple comparison
tests. Statistical significance level was assigned at the 0.05 level.
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Results-1
• Serum levels,
mean Catalase was significantly higher (p=0.003) and
mean SOD(p=0.033), LPO(p=0.050), VEGF(p=0.001), VEGF-C(p=0.024) were
significantly lower in EPO-treated group than in untreated group.
• Mean serum GPX levels were similar.
• Significantly higher levels of Estradiol were determined in EPO-treated
group than in untreated group.
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Groups
Sham Control
(n=5)
Mean±SD
Min-max
Treatment group
(n=6)
Mean±SD
Min-max
Untreated group
(n=6)
Mean±SD
Min-max
165,0-75,0
169,2±3,8
165,0±175,0
170,83
165,0±175,0
P
,652
Wbc (103/µL) 6,3±1,1 b
4,8-7,7
6,3±0,8 b
5,4±7,5
8,81 a
6,8±10,5
,010
Hb (g/dL)
Htc (%)
GPXser
(nmol/min/m
l)
CATser
(nmol/ml)
LPOser
(nmol/min/m
l)
13,1±0,2 b
39,3±0,8 b
12,8-13,4
38,3-40,4
15,0±0,6 a
44,9±0,7 a
14,2±15,8
43,8±45,8
12,74 b
38,17 b
11,9±13,5
37,3±39,2
,003
,001
0,4±0,1 a
0,3-0,5
0,29±0,1 b
0,2±0,4
,20 b
0,2±0,2
,002
64,4±17,2 a
49,0-90,0
44,2±12,0 b
32,0±60,0
27,33 c
18,0±32,0
,003
2,6±1,5 c
1,1-4,5
3,7±0,7 b
2,9±4,6
4,96 a
1,8±7,9
,050
11,0-16,0
19,0±5,3 b
11,0±25,0
26,17 a
13,0±36,0
,033
119,0±3,2 c
115,0-123,0
137,0±9,6 b
128,0±155,0
169,67 a
154,0±191,0
,001
28,4±4,8 b
21,0-34,0
30,8±12,7 b
14,0±46,0
46,83 a
35,0±62,0
,024
1,6±0,4 a
1,1-2,1,1
1,2±0,2 a
1,0±1,7
,75 b
0,6±1,0
,002
CATtis
67,4±5,7 a
(nmol/min/g)
62,0-74,0
53,2±7,3 b
41,0±62,0
30,67 c
22,0±38,0
,001
LPOtis
(nmol/min/m 1,9±0,7 c
l)
1,14-2,85
4,1±0,8 b
2,9±4,8
7,00 a
4,1±9,9
,002
SODtis (U/mg) 23,0±2,8 c
18,0-25,0
30,5±6,9 b
22,0±38,0
38,17 a
28,0±45,0
,014
114,0±3,4 c
110,0-119,0
143,7±10,8 b
131,0±161,0
165,33 a
154,0±179,0
,002
26,6±9,3 c
15,0-41,0
44,2±14,2 b
28,0±65,0
68,33 a
59,0±87,0
,003
Weight (gr)
169,0±4,2
SODser (U/ml) 13,6±2,1 c
VEGFser
(pg/ml)
VEGFCser
(pg/ml)
GPXtis
(nmol/mg)
VEGFtis
(pg/mg)
VEGFCtis
(pg/mg)
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Results-2
• Highest serum E2 levels were found in sham group (p=0.001).
• Tissue levels of GPX (1,23) and catalase (53,17) were significantly
higher in EPO group (p=0.002 and p=0.001, respectively).
• However, tissue levels of SOD and LPO, VEGF and VEGF-C levels were
significantly lower in EPO group than those in untreated group
(p=0.033, p=0.050, p=0.002 and p=0.003, respectively).
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Results-3
• In tissue examination, comparing X, Y axis and epithelial height, the
highest values were in sham group.
• Mean value of EPO group was found statistically significantly higher
than that of untreated group (p≤0.05).
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Results-4
Morphometric results of ovaries in all experimental groups.
Groups
Sham Control Group
(n=5)
Parameter
Treatment Group
(n=6)
Untreated Group
(n=6)
X Axis
583.96 ± 24.08
399.96 ± 47.89
335.11 ± 19.58 a,b
Y Axis
415.69 ± 16.50
313.16 ± 39.60
243.90 ± 13.46 a
62,49 ± 4,39
57,69 ± 3,84
34,62 ± 2,39 a,b
Epithelial height (micron)
Values are presented as mean ± SE (p≤0.05).
a Significantly different from Sham control group,
b Significantly different from treatment group.
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Results-6
• But, in untreated group, histopathological findings such as
hemorrhage, decreases in numbers of antral and Graffian follicle,
edema and atrophy in ovary were observed.
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Results-7
Slimming of follicular wall in ovary of untreated group, H&E, 200 X.
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Results-8
The number of antral follicle in ovary of rats in all experimental groups
Groups
Sham Control Treatment
Group (n=5) Group (n=6)
Number of antral
follicle
5.14 ±0.21
4.00 ±0.52
Untreated
Group
(n=6)
2.00 ± 030 a,b
Values are presented as mean ± SE (p≤0.05).
a Significantly different from Sham control group.
b Significantly different from treatment group.
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Discussion-1
• Since EPO has antioxidant, anti-apoptotic properties and contributes
angiogenesis, local EPO administration as 5000 U/kg was tried.
Antioxidant enzymes both in serum and ovarian tissue were
measured in order to understand antioxidant effects of EPO. VEGF
and VEGF-C were measured for angiogenetic effects of EPO. At the
same time ovarian tissue was histopathologically examined.
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Discussion-2
• Freezing may be accused of decreasing antral follicle count. However,
since primordial follicles have inactive metabolism, thawed ovarian
tissues are believed to be resistant to cryoinjury
• The main reason for the follicular loss after cryopreservation and
xenografting seems to be the ischemic effect which takes place after
transplantation rather than cryopreservation per se.
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• In this study we tried to demonstrate protective effects of EPO on
transplanted ovarian tissue. Because of antral follicle counts are a
good predictor of the number of mature follicles, we choosed this
parameter.
• In EPO-untreated group, autotransplantation of ovaries do decrease
the number of antral follicles and the height of the ovarian surface
epithelium. Elevated estrogen levels stimulate proliferation of ovarian
superficial epithelial cells by activating cellular estrogen receptors.
The histomorphologic and histomorphometric data in the EPOuntreated group seemed to support decreased E2 levels, number of
antral follicle and epithelial height of ovarium.
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• Various doses of EPO were used in studies. So, there isn’t any
consensus on the dose of EPO to be administered. Since we tried
5000 U/kg EPO dose previously in our experimental study on
ischemia-reperfusion of the ovary, in this study we again administer
the same dose locally two times with 4 weeks interval.
• Further studies with other doses of EPO are required to determine
the optimal dose and optimal way of administration.
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• VEGF-C is particularly unique among the VEGF family members. One
of the known functions of VEGF-C is lymphangiogenesis.
• According to the results of our study, ovarian tissue transplantation
caused increase in the serum and tissue levels of VEGF-C. Treatment
with EPO resulted a significant decrease in the levels of VEGF-C.
VEGF-C expression is induced by environmental stress such as
hyperthermia or oxidative stress. According to these data, VEGF-C
levels should demonstrate the level of environmental stress.
• In our study, VEGF-C levels both in serum and tissue were measured
and this study is the first one investigating VEGF-C in ovarian tissue
transplantation and EPO treatment.
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Conclusion
EPO improves the survival of follicles in ovarian grafts most likely
by reducing ischemic injury, by improving neoangiogenesis and
by its’ antioxidant effects.
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Thank you...
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