Intro
There is a concerning increase in cancer diagnoses
according to the Iranian Cancer Society. Today, due
to medical advances, many cancers are treatable with
timely diagnosis and follow up. The patient can re-
turn to a normal life after radiotherapy, chemothera-
py, or surgical tumor excision. Therefore, many can-
cers are no longer considered incurable. Although, in
many cases, chemotherapy and radiotherapy aim to
save lives, premature ovarian failure and reduction of
follicular reserve is undeniable. By taking into con-
sideration the probable infertility of cancer patients,
preservation of their reproductive ability prior to
onset of cancer treatment is crucial ( 1 , 2 ). Different
methods of assisted reproductive techniques that in-
clude oocyte, embryo and ovarian tissue cryopreser-
vation have helped these patients. The use of these
techniques in single or married, as well as young
and older women differ. Hence, the most appropriate
technique is selected according to the patients’ cir-
cumstances ( 1 - 7 ). In cases where adequate time exists
for ovarian stimulation, embryo cryopreservation is
considered the gold standard and an acceptable clini-
cal technique. However, if embryo cryopreservation
is not an option due to the absence of a sexual partner,
unwillingness to use donor sperm, or for any other
reason, the oocytes can be frozen ( 6 ). Ovarian tissue
cryopreservation is another technique that has a long
history of use, but with a new purpose. Limitations
of oocyte cryopreservation exist, such as the impos-
sibility of stimulating ovaries in patients with hyper-
stimulation syndrome. Under these circumstances,
ovarian tissue cryopreservation is more accepted and
approved ( 2 , 5 ). In this technique numerous follicles
at different stages of maturity are preserved without
delays to cancer treatment. In addition, for single or
young girls this is the best choice to preserve their
reproductive ability ( 3 , 4 ).
The Royan Human Ovarian Tissue Bank was es-
tablished in 2010 with the intent to provide fertility
preservation services to cancer patients eligible for
preservation of reproductive ability. We have estab-
lished the maximum age for inclusion in the Tissue
Bank as 35 years. Cases of malignancy where tu-
mors have metastasized to the ovarian tissue are not
accepted for cryopreservation. In other cases there is no exclusion for acceptance. Patients undergo an ini-
tial consultation that determines individual factors of
age, marital status, physical and mental conditions,
cancer type, its progression stage and grade, level of
previous treatments, earlier infertility treatment and
prognosis after treatment. After the initial consulta-
tion, the best fertility preservation technique is se-
lected. A contract is signed between the Ovarian Tis-
sue Bank and the patient after the consultation. This
contract includes patients’ rights and sample mainte-
nance insurance, as well as informing patients about
the use of her own sample after treatment, which is
approved by the Royan Ethical Committee.
The procedure for ovarian tissue cryopreserva-
tion at the Royan Human Ovarian Tissue Bank is as
follows. An ovarian tissue sample is removed from
the patient by laparoscopy, laparotomy, unilateral
or bilateral oophorectomy according to the patient’s
condition. The sample is transferred to the Ovarian
Bank in the shortest possible time (approximately
1 hour) in Medium 199+Heppes (HTCM, Gibco,
Paisley, UK)+20% human serum albumin (HAS,
Biotest, Germany) as transfer medium at 4°C and
on ice. In the laboratory, initially, the transferred
tissue is washed in HTCM+20% HSA medium,
after which the medullary part is removed. Next,
the cortical part is thinned and 10×5×1 mm strips
are obtained from the thin cortex. These steps are
all performed on a cool pad. Finally, the stripes are
vitrified in a two-step process, equilibration and
vitrification. In the first step (equilibration), each
strip is washed in equilibrium medium composed
of HTCM, ethylene glycol (EG, Sigma, St. Louis,
MO, USA), Dimethyl sulphoxide (DMSO, Sigma,
USA, each 7.5%) and 20% HSA for 15 minutes at
4°C. In the second step (vitrification), each strip
is washed in 15% HTCM, 15% DSMO and 15%
EG, 0.25 M sucrose, and 20% HSA for 10 minutes
at 4°C. The extra medium is completely removed
from the strips, after which they are directly trans-
ferred into liquid nitrogen. Of note, we randomly
fix one strip before cryopreservation for histologi-
cal evaluation (H&E staining and Semi thin). For
tissue evaluation, one vitrified strip is warmed and
assessed histologically. Warming is performed in
4 steps in descending concentrations (1, 0.5, 0.25,
and 0.125) of sucrose. The base medium is com-
prised of HTCM+20% HSA. The histological as-
sessment markers considered for tissue evaluation
include total integrity, follicular population, oo-
cyte degeneration, vacuolization and granulation
of the nucleus, oolemma and ooplasm conditions,
zona pellucida situation (in secondary or preantral
follicle), coherence and connectivity of granulosa
cells ( Fig .1 ).
Primordial (short arrows), primary (long arrows) and preantral (black arrow) follicles in A., B., C. Control, D., E. and F. Vitrified hu-
man ovarian tissues. Hematoxylin and eosin (H&E) staining (magnification: ×20, ×50 μm).
Functionality of the entire ovarian tissue is con-
sidered by the presence or absence of the corpus
luteum or corpus albicans in tissue. Finally, the in-
formation is kept and filed in a histology descrip-
tion form.