Document Type : Original Article
Authors
1 Clinical Science Department, Faculty of Veterinary Medicine, Islamic Azad University, Urmia Branch, Urmia, Iran
2 Comparative Histology and Embryology, Faculty of Veterinary Medicine, Urmia University, Urmia, Iran
Abstract
Keywords
Chemotherapy and radiologic treatments are important,
effective methods for the treatment of
cancer. Meanwhile, oncology treatments are associated
with long-term infertility effects. Following
oncological treatment, remarkable toxicity occurs
in ovarian tissue which in turn leads to a severe
loss in the ovarian follicular bank, finally resulting
in fertility problems (
In order to protect the ovaries as well the ovarian
follicular banks from the side effects of the
above mentioned therapeutic methods, numerous
research has been conducted to remove ovarian
tissue, at least for a therapeutic period. The first
attempt for ovarian transplantation in animals and
humans dates from the 19th century. According
to an early report, following transplantation of a
mice ovary on the ovarian bursa several neonates
were born (
To avoid ischemic injury due to unstable blood
flow, attempts have recently been made to use
vascular anastomosis. The vascular anastomosis
of the ovary is remarkably smaller in comparison
to the other organs and transplantation with vascular
anastomosis requires high level techniques
known as super micro-surgery (
In this study all experiments which conducted on animals were in accordance with the guidance of Ethical Committee for research on laboratory Animals of Urmia University.
The present study used 20 mature female dogs obtained from West Azerbaijan, Urmia (Iran ecotypes). We divided the animals into the following groups. Test group 1 (T1=5) underwent ovarian tissue transplantation. Test group 2 (T2=5) underwent bilateral ovariectomies. In test group 3 (T3=5) unilateral ovariectomies were performed with transplants. The control-sham group (CS=5) did not have ovariectomies or transplants. In order to prevent oxidative stress, vitamin E (150 mg/ kg) was administrated directly to the serum during surgery and given intra muscularly, 2 days after surgery, every 24 hours for a period of 2 weeks.
Ovaries were divided in two equal pieces from the middle line of the ovarian tissue. Half of the ovaries were implanted in subserosa of the greater curvature area on the muscular layer of the stomach wall. The ovary was grafted intra-muscularly by inducing hemorrhage in the region and closing the stomach seromuscular flap with absorbable suture material in two layers in the form of a saclike cavity in order to directly expose the implanted ovaries to blood in the experimentally created blood sinus.
On day 60 following surgery, the ovaries were removed and fixed in formaldehyde acetic solution (IFAA, Germany) for 4 weeks. Ultimately, they were dissected free from per-ovarian tissues. Samples were processed through paraffin embedding and serially cut with a rotary microtome, and stained with the hematoxylin and eosin technique.
Follicular morphology was examined by microscope
(×400). All follicles in the test and controlsham
groups were counted and recorded depending
on their sizes. Follicles were classified as 100 and
101-200 μm (small or pre-antral follicles). Normal
follicles had a complete layer of flattened granulosa
cells, oocytes with cytoplasm, and a normal
nucleus. Abnormal follicles were classified as follows:
cytoplasmic damage, pyknotic nucleus, and
combination of damaged nucleus and cytoplasm.
Follicular number was estimated by counting follicles
in all slides (
Light microscopic investigation of the ovarian
medullar, cortical arteries and veins showed the
general histological structure of the tissue vessels.
The histological characteristics for normal and abnormal
arteries and veins were investigated (
Blood samples from corresponding animals were collected directly from the heart on days 10, 20, 30, 40, 50 and 60 after surgery, centrifuged (3000 rpm/5 minutes) and subjected to assays of serum progesterone and estradiol. Progesterone and estradiol were assessed by electrochemilunescence.
All results are presented as mean ± SD. Differences between quantitative histological and hematological data were analyzed with two-way ANOVA, followed by Bonferroni test, using Graph Pad Prism 4.00,. P<0.05 was considered significant. Correlation between total follicular number with survived follicles were analyzed on an Indigo-2 O2 work station (Silicon Graphics, Mountain View, CA) using Matlab (MathWorks, Inc., Natick, MA).
Histological observations demonstrated that in T1 animals total follicular numbers (per one ovary) decreased in comparison to T3 and controlsham animals. Analysis of correlation between total follicular numbers with follicular viability per one ovary showed that the number of total follicles that survived in T2 ovaries approximated T3 viable follicles. The data for follicular viability and number (per one ovary) are presented in figure 1.
Light microscopic investigations revealed that the
transplanted ovaries exhibited more damage in the
oocyte cytoplasm, nucleus and/or combination of
cytoplasm and nucleus (p≤0.05) in comparison
to the intact ovaries in the T3 and control-sham
groups (
Correlation between total follicular number (density) with percent of follicles that survived. Black spots are represent total follicular density. Smooth lines illustrate the percentage of follicles that survived. Total follicular number positively correlated with the percentage of 100 μm survived follicles, r2=0.068; p≤0.05 and 101-200 μm survived follicles r2= 0.79; p≤0.05.
Comparisons of <100 and 101-200 µm follicular oocyte damage between control-sham T1 and T3 groups
Groups | 100 µm intact follicles (%) | Cytoplasmdamage (%) | Nucleus damage (%) | Cytoplasm &nucleus damage (%) |
---|---|---|---|---|
98.09 ± 1.43 | 11.8 ± 1.30 | 1.95 ± 0.61 | 1.48 ± 0.39 | |
78.17 ± 1.32* | 14.6 ± 1.67* | 2.79 ± 0.14*a | 1.92 ± 0.16 | |
81.60 ± 2.07* | 14.4 ± 1.14* | 2.08 ± 0.05*a’ | 1.89± 0.23 | |
98.80 ± 1.64 | 7.00 ± 0.81 | 1.22 ± 0.43 | 0.77 ± 0.52 | |
79.75 ± 2.06*b | 11.25 ± 0.95*c | 2.55 ± 0.32*d | 1.67 ± 0.45*e | |
83.87 ± 0.95*b’ | 8.75 ± 0.50*c’ | 2.02 ± 0.05*d’ | 1.16 ± 0.21e’ | |
Stars indicate significant differences (p≤0.05) between T1 and T3 animals with control-sham in the same column.
Different letters and superscripts in the same column indicate significant differences (p≤0.05) between T1 and T3 animals. All data are presented as mean ± SD
In contrast to oocytes, analysis of the same damages
in granulosa cells showed no significant differences
(p≥0.05) between T1 and T3 groups (
Mean percentage of cytoplasmic damage (C.D), nuclear damage (N.D) and combined cytoplasmic+nuclear damage of granulosa cells. Stars indicate significant differences (p≤0.05) between T1 and T3 groups with the control-sham group. There are no statistically significant differences (p≥0.05) between T1 and T3 animals. All data are presented as mean ± SD.
Light microscopic analyses illustrated that
ovaries in the T1 group underwent light necrosis,
particularly in regions far from the blood
vessels. Necrotic cells were located in sub-capsular
regions and/or adjacent with the capsule.
The parenchyma close to new generated blood
vessels manifested with approximately normal
histological appearance. The intact remaining
ovaries in the T3 and control-sham groups had
no necrotic parenchyma. Blood vessels of the
T1 cases exhibited no fractures in any of the
animals but very low bloated vascular muscle
cells, endothelial cell damage and hypertrophy
as detected by light microscopic analyses
(
Reorganized endothelial cells were present in the
inner and outer medulla of the transplanted ovaries
(Figes
There were several histologically normal veins
and arterioles in the medulla and cortical regions
of the implanted ovaries. There were no statistically
significant differences (p≥0.05) between histologically
normal veins and arterioles of the T1
and T3 groups (
Blood serum analyses illustrated that the serum level of estradiol decreased in both T1 and T3 animals. Meanwhile, after day 40 there were no significant (p≥0.05) differences between T1 and T3 serum estradiol levels. T2 animals maintained constant levels of estradiol (20.78 ± 1.40) during 60 days.
Histological assessment of ovarian medulla and cortex arteries and veins endothelial bloating (EB), hypertrophy (EH) and degeneration (ED) in control-sham, T1 and T3 groups.
Ovaries | Medullar artery | Medullar vei | |||||
---|---|---|---|---|---|---|---|
BD | EH | BE | BD | EH | BE | ||
0 | 0 | 0 | 0 | 0 | 0 | ||
12.33 ± 1.03 | 10.50 ± 1.04* | 9.61 ± 0.49 | 8.75 ± 1.25 | 8.25 ± 0.95* | 8.00 ± 0.81 | ||
11.50 ± 1.37 | 9.11 ± 0.77* | 9.81 ± 1.00 | 8.10 ± 1.52 | 8.12 ± 0.77* | 7.63 ± 0.44 | ||
0 | 0 | 0 | 0 | 0 | 0 | ||
8.78 ± 0.42 | 6.39 ± 0.57 | 6.00 ± 0.63 | 5.37 ± 0.62 | 5.60 ± 0.61* | 4.21 ± 0.49 | ||
8.38±0.48 | 6.09±0.79 | 5.16±0.98 | 4.95±0.60 | 5.18±0.76 * | 4.26±0.60 | ||
Stars indicate significant differences (p≤0.05) between data in the same column. All data are presented as mean±SD.
Histological architecture from the transplanted ovary. A. Low magnification. Note the dark brown sites (arrow heads) close to the recovered blood vessels showing newly organized endothelial cells in order to generate new blood vessels. B. High magnification from outer medulla of the transplanted ovary. Note the dark brown stained endothelial cells aggregated abundantly close to light brown stained cells showing recovered endothelial cells (arrows). C. High magnification from inner medulla, dark brown stained endothelial cells (arrow heads) located adjacent to the endothelial cells with heterogeneous cytoplasm (arrows), endothelial cell staining (A, ×100; B and C, ×400).
Histological comparison of normal arterioles and veins in the medullar and cortical regions of the ovaries in T1 and T3 animals. Data are presented in percent (%). Note: N.M.A, normal medullar arteriole; N.C.A, normal cortical arteriole; N.M.V, normal medullar vein and N.C.V, normal cortical vein. There were no significant differences (p≥0.05) between data evaluated for T1 and T3. All values are presented as mean ± SD.
Although the serum level of progesterone was constant the first day after transplantation, a statistically significant (p≤0.05) decrease occurred until day 30 in both T1 and T3 groups. After day 30, the blood level of progesterone began to increase in both T1 and T3 groups, which was not statistically different (p≥0.05). Data for blood estradiol and progesterone levels are presented in Figures 5-A and 5-B. T2 group showed an approximately constant level of progesterone during 60 days (0.177 ± 0.008). A comparison of estradiol and progesterone blood levels in the T1 and T3 groups with control-sham animals showed lower serum levels of these hormones in the experimental groups compared with the controlsham group. Meanwhile, after days 30 and 40, serum levels of progesterone and estradiol began to stabilize.
Blood level of progesterone (A) and estradiol (B). Stars indicate significant differences (p≤0.05) for progesterone levels on days 1 to 30 and estradiols level on days 1 to 40 between T1 and T3 groups. There were no considerable differences (p≤0.05) between data for T1 and T3 progesterone and estradiol levels after days 30 and 40. There were remarkable differences (p≤0.05) between data for both T1 and T3 groups with control-sham animals during all days. All values are presented as mean ± SD.
In recent decades there have been striking advances
in the treatment of cancer by using chemotherapy
and/or radiotherapy methods. As survival
and cure rates rise, the focus is turning to
the late effects of treatments, of which the loss
of fertility and gonadal failure seem to be very
importance points. Nowadays various options
exist such as oocyte and sperm cryopreservation
in adults (
Although total follicular numbers decreased
in T1 animals, an evaluation of the percentage
of follicles that survived in correlation with
total follicular number showed that more than
half of the total follicular population survived
after transplantation in T1 animals. According
to animal and human preliminary studies, the
key factor responsible for follicular survival
seems to be post-graft ischemia. As the process
of revascularization can take more than a
day to complete, thus tissue ischemia can be
a problem for implants (
Comparing endothelial cell damage and bloating between T1 and T3 animals showed no considerable differences between these two groups. This situation suggests that the intact ovary in the T3 animals began to have compensatory revascularization following unilateral ovariectomy and therefore some alterations manifested in older endothelial cells. On the other hand, the transplanted ovaries of the T1 group showed the abovementioned alterations, the same as T3 animals, but these alterations were not statistically considerable in comparison to the T3 group. May be after implantation the ovaries started to reorganize the flow of blood by compensatory revascularization. Furthermore, the environmental condition (post implanting side effects) could be considered another reason for structural changes in T1 animal ovarian vessels.
It is well known that the ovary is responsible for
female hormonal (estrogen and progesterone)
secretion and fertility. Once ovarian function
disrupts, women and female animals experience
sexual difficulties and will probably have serious
problems in developing secondary sexual characteristics.
The ovarian transplantation method
used in the present study is expected to protect
endocrine function because of the preservation
of ovarian granulosa cells. Resumption of the
menstrual cycle after transplantation is regarded
as fertility in gynecology but this understanding
is not completely correct. In transplantation with
no vascular anastomosis, the menstrual cycle
may resume because some oocytes and granulosa
cells remain alive. However, the quality of
the follicles can severely spoil due to ischemic
injuries. Although the remaining poor quality
oocytes and granulosa cells maintain the sexual
cycle, fertility is expected to be low (
Here we report that survival of the ovaries after implantation depends on blood support and our study showed that direct adjustment of the blood during the first days after transplantation could help ovarian tissue survive from post implantation ischemia. Furthermore our findings showed that this method (experimentally inducing a blood sinus around the transplanted ovaries) is the type of reconstructive surgery for simultaneously conserving neovasculation by reorganization of blood vessels endothelial, muscular cells and fibrotic structure associated with protecting endocrine function of the grafted ovaries.