Several potential options are available to
preserve fertility in patients that face premature
ovarian failure, including immature and
mature oocyte, and embryo cryopreservation.
Each direction has its own benefits and limitations.
In cases where chemotherapy cannot be
postponed, cryopreservation of ovarian tissue
is an option (
Autotransplantation of cryopreserved ovarian
tissue offers the possibility of restoring ovarian
function in women and children after highly gonadotoxic
cancer treatment. Theoretically, orthotopic
autotransplantation can restore normal
reproductive function, which leads to natural
conception. A live birth in a primate following
a fresh ovarian tissue transplant and the first
live birth in a human after orthotopic transplantation
of cryopreserved ovarian tissue have
been reported in 2004 (
In this experimental study, all chemicals were purchased from Sigma company (Germany), except those mentioned below.
Animal experiments were performed according to the Declaration Helsinki and the Guiding Principles in the Care and Use of Animals (DHEW publication, NIH, 80-23).
For the experiments, we used female NMRI mice that were housed, bred and kept at a temperature of 20-25°C and 50% humidity under light-controlled conditions (12 hour light/12 hour dark) and provided with sterile food and water in the Central Animal House of Royan Institute according to national standards.
We euthanized two-week-old mice, after which their left ovaries were removed and immediately placed in α-MEM medium at room temperature. The fat tissue surrounded the ovaries were removed under a loop (5 minutes). Then, we immediately collected the ovaries to perform the cryopreservation procedure. After two weeks of cryopreservation, whole ovaries were thawed and autotransplanted to the same mouse from which the ovary had been removed. Three weeks after transplantation of thawed ovaries, the seven-week-old mice were killed and the grafted ovaries on the gluteus muscle tissue were collected. These ovaries were considered the experimental group (Vit-trans group). In addition to the collection of grafted ovaries, we also collected the right ovaries from the same mice, which were considered to be the opposite ovaries (Opp) group. For the control group, both ovaries of age matched (seven-week-old) female mice were collected (7 week-fresh group).
Following ovariectomy, the ovaries were immediately
vitrified. During vitrification, ovaries were
immersed in an equilibration solution composed
of 7.5% DMSO and 7.5% ethylene glycol (EG) in
HEPES-buffer TCM 199 (Gibco, USA; pH=7.4) supplemented with 20% human serum albumin
(HSA) (Octapharma, Switzerland) for 15 minutes
at room temperature. Ovaries were subsequently
transferred into the vitrification solution that consisted
of 15% EG, 15% DMSO, and 0.5 M sucrose
in HEPES-buffer TCM 199 (pH=7.4) supplemented
with 20% HSA for 30 minutes. Each whole ovary
was placed on the Cryotop (Kitazato Co., Ltd.,
Fujinomiya, Japan) polyester sheet with a minimum
volume of the vitrification solution (
For thawing, after pulling up the cryotops from liquid nitrogen, the sheets were placed directly in a thawing solution composed of 1 M sucrose in HEPES-buffer TCM199 (pH=7.4) supplemented with 20% HSA for 10 minutes at room temperature. The ovaries were detached from the sheet and transferred to α-MEM medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) and an antibiotic solution composed of penicillin G (100 IU) and streptomycin (100 IU) after which ovaries were incubated for 30 minutes.
Whole mouse ovary on the Cryotop polyester sheet.
Thawed ovaries were immediately transplanted back into the same female mouse which had her left ovary removed two weeks prior. At this time, we anesthetized four-week-old mice with intraperitoneal injections of 10% ketamine (100 mg/ kg; Alfasan, Woerden, Holland) and 2% xylazine (10 mg/kg; Alfasan, Woerden, Holland). The concentrations of 100 mg/ml Ketamine and 20 mg/ml Xylazine were diluted in 8.5 ml physiological serum; 0.1 g body weight was injected.
After a full incision through the dermal tissue and the placement of an incision along the gluteus superficialis muscle fibers, the ovary was inserted within the muscle incision. The muscle fiber was sutured with 5-0 non-absorbable vicryl surgical thread (Ethicon, Belgium) in order to allow detection of the transplantation site. The skin was sutured with 6-0 absorbable threads (prolene; Ethicon, Belgium). Following the grafting procedure and recovery, animals were kept in the animal room.
About 72 hours prior to ovary removal, we injected 7.5 IU/ml PMSG (Folligon, Intervet) in mice from both the experimental and control groups; 48 hours later, both groups received 7.5 IU/ml hCG (Pregnyl, Organon) and after 14 hours we collected the ovaries.
At three weeks following transplantation, mice were killed by cervical dislocation. Grafted and Opp were removed from the experimental groups and fresh ovaries were removed from intact mice for oocyte isolation.
Each ovary was mechanically dissected by a 26- gauge needle in α-MEM droplets supplemented with 10% v/v FBS and antibiotic solution (penestrep). Released oocytes and COCs from the ovary were selected for IVM or IVF by the following criteria. Those oocytes and COCs that lacked polar bodies were chosen and divided into two groups: germinal vesicle (GV) and germinal vesicle breakdown (GVBD). Both groups were transferred to IVM culture medium.
The oocytes and COCs that contained polar bodies were selected and transferred to IVF medium. For selection, the oocytes had to be visible and round in shape.
IVM medium was composed of α-MEM supplemented with 5% FBS, 100 mIU/ml rhFSH (GONAL-f, Serono) and 7.5 IU/ml hCG (Pregnyl, Organon). After a 16 hour incubation period, we observed oocytes and COCs (after pipetting to remove granolusa cells) to determine which had released first the polar bodies. These were assessed as metaphase II (MII) stage oocytes, which were subsequently transferred to IVF medium.
We used 7- to 14-week-old adult male mice to obtain sperm. The caudae epididymides were cut in several zones and placed in a 1 ml droplet of T6 medium supplemented with 15 mg/kg BSA. Droplets were covered with mineral oil and incubated for at least 30 minutes at 37°C in a humidified atmosphere with 5% CO2 for sperm capacitation. About 10 mature MII oocytes were added to the 100-150 µL droplet of sperm suspension at a concentration of 0.8 × 106 sperm per ml and then incubated for at least 4 hours. The oocytes were pipetted to remove the attached sperm and monitored under a microscope for the presence of a second polar body or two pronuclei (2PN) to confirm fertilization.
About 10 fertilized oocytes were cultured in a 20 µl droplet comprised of T6 medium supplemented with 4 mg/ml BSA. Droplets were covered with mineral oil. The developmental stages of the embryos were observed at 24, 48, 72 and 96 hours after fertilization.
All numbers are presented as percentages. Analyses and comparison of significances were performed by the Chi-square test. P<0.05 was considered to be statistically significant.
All Vit-trans mouse ovaries (n=15) were recovered
five weeks following vitrification and transplantation.
We collected the Opp ovaries (n=6) and
age-matched 7w-fresh mouse ovaries (n=10). There
were 66 oocytes, which included 16 COCs and 50
denuded oocytes collected from dissected Vit-trans
ovaries. There were 106 (47 COCs, 59 denuded
oocytes) oocytes collected from the Opp ovaries
and 83 (53 COCs, 30 denuded oocytes) oocytes collected
from 7w-fresh ovaries (
We recorded the IVM rates of oocytes recovered
from transplanted and OPP ovaries for each female
Oocyte development after
|Denuded oocytes||50||18.0||32.0b, c||38.0a||70.0a||12.0|
|Denuded oocytes||59||5.1||13.6b||27.1b||40.7a, b||20.3|
|Denuded oocytes||30||10.0||10.0c||76.7a, b||86.7b||3.3|
MI; Metaphase II, COC; Cumulus oocyte complex, Opp; Opposite ovaries, Vit-trans; Vitrified-transplanted ovaries GV; Germinal vesicle, GVBD; Germinal vesicle breakdown and 7w-fresh; Fresh ovaries removed from 7-week-old normal mice. Percentages with same letters in each column are significantly different (p<0.05).
The number of denuded oocytes that reached
GVBD or MII stage in the Vit-trans group (70%)
was significantly higher than the Opp group
(40.7%). In each group there were a few numbers
of degenerated oocytes (
In order to assess the capacity of mature oocytes
after vitrification and transplantation, the matured
oocytes obtained from Opp and 7w-fresh groups
were fertilized and cultured
There was only a significant difference in fertilization
rate and formation of 2PN observed
between the Opp (42.9%) and 7w-fresh groups
(73.1%). The Vit-trans oocytes had a mediocre
rate of IVF (56.5%) between Opp and 7w-fresh
Comparison of in vitro fertilization rate between groups
Vit-trans; Vitrified, transplanted ovaries, 7w-fresh; Fresh ovaries removed from 7-week-old normal mice and 2PN; Two pronuclear. Percentages with same letters in each column are significantly different (p<0.05).
*;The percentage of ova with male and female pronucleus.
Cleavage stages were observed until the blastocyst stage, which was approximately 96 hours after fertilization. The results are summarized in table 3.
From a total of 13 zygotes obtained from Vittrans
group IVF, 12 zygotes were cultured and
5 embryos (41.7%) reached the 8-cell stage. No
additional progress was visualized in this group
by 96 hours post-observation. There were no
significant differences between Vit-trans, Opp
and 7w-fresh groups on the development of
embryos to the 8-cell stage after 72 hours of
|Experimental Groups||Total||24 hours||48 hours||72 hours||96 hours|
|2-cell (%)||2-cell (%)||4-cell (%)||4-cell (%)||8-cell (%)||8-cell (%)||Morula (%)||Blastocyst (%)|
Vit-trans; Vitrified, transplanted ovaries and7 week-fresh; Fresh ovaries removed from 7-week-old normal mice. Percentages with same letters in each column are significantly different (p<0.05).
The major challenge of ovarian tissue transplantation
is still ischemia. Attempts have been
made to overcome ischemia-related damage
that exists at transplantation by treatment with
GnRH and antioxidant agents in addition to
microvascular anastomosis (
In this research, we evaluated the IVM, IVF
and IVD rates of oocytes following cryopreservation
of whole ovaries on cryotops and
subsequent autotransplantation. During ovarian
dissection and oocyte retrieval, there was
an elevated rate of denuded oocytes in the
Vit-trans and Opp groups compared to the 7wfresh
group. The IVM rates of COCs retrieved
from Vit-trans and 7w-fresh groups were the
same, as was the IVF rate and developmental
competence of oocytes that reached the 8-cell
stage. The freezing protocol used during this
research contained EG. According to Salehnia
et al. the vitrification of whole mouse ovaries using EG was useful and had no harmful effects
on follicle morphology (
During this research the whole ovary was
transplanted to the gluteal muscle. We have
previously demonstrated that the gluteal muscle
is suitable site for ovarian transplantation
The lower percentage of GVBD and MII
oocytes, the higher percentage of degenerated
oocytes and lower IVF rates in the Opp group
in contrast to other groups might result from
the adverse effect of FSH and LH up regulation
during two weeks of vitrification and the few
days of transplantation followed by hormone
therapy in the absence of other ovary. Although
the fertilization rate in the Vit-trans group was
lower than 7w-fresh group, this difference was
not significant. It has been reported that the
presence of granolusa cells around the oocytes
caused a higher IVF rate (
Despite lower retrieval of COCs from Vittrans
ovaries, it seems that for
IVD of generated embryos from Vit-trans
ovaries was observed. The combination of cryopreservation
(cryotop) and subsequent transplantation
on gluteal muscle have been shown
to preserve ovarian function. Research is ongoing
for ovarian cryopreservation, transplantation