Radiol Oncol 2022; 56(1): 119-128. doi: 10.2478/raon-2021-0053
119
research article 
In vitro maturation of immature oocytes 
for fertility preservation in cancer patients 
compared to control patients with fertility 
problems in an in vitro fertilization program
Irma Virant-Klun1, Jure Bedenk2, Nina Jancar2
1 Clinical Research Centre, University Medical Centre Ljubljana, Ljubljana, Slovenia
2 Division of Obstetrics and Gynecology, University Medical Centre Ljubljana, Ljubljana, Slovenia 
Radiol Oncol 2022; 56(1): 119-128.
Received 5 August 2021
Accepted 25 November 2021
Correspondence to: Prof. Irma Virant-Klun, Ph.D., Clinical Research Centre, University Medical Centre Ljubljana, Zaloška cesta 2, 
SI 1000 Ljubljana, Slovenia. E-mail: irma.virant@kclj.si
Disclosure: No potential conflicts of interest were disclosed. 
This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Background. The aim of this study was to determine whether in vitro maturation (IVM) of immature oocytes after 
controlled hormonal stimulation of the ovaries could be important in cancer patients to improve their chances of 
conception in the future.
Patients and methods. After ovarian stimulation in cancer patients, the number of oocytes and their quality and 
maturity were compared to control patients with fertility problems in the in vitro fertilization (IVF) program. In both 
groups of patients, immature oocytes at the developmental stage of germinal vesicle were matured in vitro and 
the proportion of oocytes that matured in vitro was compared between groups. In a subset of women with fertility 
problems, intracytoplasmic sperm injection (ICSI) was performed on IVM oocytes to assess their ability to be fertilized 
and develop into an embryo compared to vivo matured oocytes in the same cycles and consider the procedure 
in cancer patients. 
Results. In patients with different cancers, the disease did not affect the number and quality of retrieved oocytes. 
In cancer patients, there was even a significantly lower proportion of immature oocytes than in patients with fertility 
problems (30.0% vs. 43.6%; P < 0.05). However, in patients with cancer, fewer oocytes per patient matured in vitro than 
in patients with fertility problems (1.39 ± 1.04 vs. 2.48 ± 1.83; P < 0.05). After ICSI, the proportions of fertilized oocytes 
and fertilized oocytes developing into an embryo did not differ between oocytes matured in vitro and in vivo in the 
same cycles.
Conclusions. Oocyte IVM is proving to be a reliable procedure for resolving immature oocytes after controlled 
ovarian stimulation in cancer patients.
Key words: cancer; fertility preservation; oocyte; in vitro maturation; vitrification 
Introduction
Many young women in the reproductive period 
of life who do not yet have children or would like 
to have another child suffer from cancer. Today, 
cancer therapies are successful, but unfortunately, 
they can negatively affect the ovarian function (in-
cluding oocyte quality) and fertility. At a median 
of 5.0 years from initial breast cancer diagnosis, 
49% patients after adjuvant chemotherapy with 
anthracyclines and taxanes and 11% after therapy 
with tamoxifen had become post- and peri-meno-
pausal.1 Decreased ovarian follicle reserve occurs 
in more than one-third of patients after breast can-
Radiol Oncol 2022; 56(1): 119-128.
Virant-Klun I et al. / In vitro maturation of immature oocytes for fertility preservation120
cer treatment resulting in permanent infertility.2 
In long-term female survivors of pediatric hema-
tologic malignancies 26.7% experienced premature 
ovarian insufficiency and face infertility after can-
cer treatment.3 The situation is similar with other 
cancers; cancer therapy is the cause of premature 
ovarian failure in 25% of women with this diagno-
sis.4 Therefore, it is very important to consider the 
preservation of female fertility before oncotherapy. 
An improvement in the survival rates of cancer pa-
tients and recent advances in assisted reproductive 
technologies have led to significant progress in fer-
tility preservation treatments. 
One option is vitrification and long-term storage 
of the patient's oocytes for later in vitro fertilization 
(IVF) with partner's sperm and transfer of embryos 
into the uterus. This program is established in many 
health care institutions around the world for a va-
riety of cancers, including breast cancer.5-12 Oocyte 
cryopreservation is an effective approach13-15, but it 
is still thought that further studies are needed in 
cancer patients to ensure the excellent outcomes 
obtained in women without cancer.16 After con-
trolled hormonal stimulation of the ovaries, in vitro 
maturation (IVM) of immature oocytes before vitri-
fication is recommended and not after vitrification/
devitrification procedure.17 Even if there are no dif-
ferences in survival rates between oocytes vitrified 
before or after IVM procedure, decreased matura-
tion rates of immature oocytes vitrified before IVM 
may be explained by underlying ultrastructural 
and biomolecular alterations.17 
Human oocyte cryopreservation may offer some 
advantages compared to embryo freezing in can-
cer patients and also eliminates some ethical, legal, 
and moral concerns of embryo freezing17, and is an 
option in young cancer patients who are single.9,18 
However, the chance of success depends primarily 
on the number of oocytes that have been vitrified in 
the patient15 and some breast cancer patients may 
have contraindications to exogenous gonadotropin 
administration for controlled ovarian stimulation.19 
Some recent data show that ovarian stimulation for 
oocyte vitrification does not modify disease-free 
survival and overall survival rates in patients with 
early breast cancer20 and the safety of pregnancy 
after an established diagnosis of breast cancer has 
been confirmed in numerous studies.21
In the case of vitrification and storage of oocytes, 
controlled hormonal stimulation of the ovaries is 
required to obtain oocytes. Despite careful hor-
monal stimulation of the ovaries, the significant 
proportion of oocytes obtained by ultrasound-
guided aspiration of ovarian follicles is immature 
as metaphase I (MI) oocytes or prophase I oocytes 
with germinal vesicle (GV). Immature MI oocytes 
mostly mature spontaneously in vitro and are vit-
rified, while immature GV oocytes do not mature 
spontaneously and are incapable of fertilization. 
Therefore, GV oocytes are not vitrified and stored 
in liquid nitrogen in clinical practice and are dis-
carded and lost to the patient. The important ques-
tion is whether the maturation of these oocytes in 
vitro makes sense. There is a lack of data regarding 
the outcome of in vitro matured oocytes cryopre-
served in cancer patients.22 Recently, the first birth 
achieved after fertility preservation using vitrifica-
tion of in vitro matured oocytes in a patient with 
breast cancer has been reported.23 
The purpose of this study was to investigate the 
effectiveness of maturation of immature GV oo-
cytes of cancer patients in laboratory conditions 
(in maturation medium and co-culture with cu-
mulus cells from mature oocytes of the same pa-
tients) compared to control women involved in the 
IVF program due to fertility problems. Because all 
oocytes of cancer patients are still frozen, we tried 
to elucidate the success of IVF procedure, actual-
ly intracytoplasmic sperm injection (ICSI), on the 
in vitro matured oocytes of patients with fertility 
problems as a model for cancer patients.
Patients and methods
This research was approved by the Slovenian 
National Medical Ethical Committee (No. 0120-
222/2016-2; KME 115/04/16). In this prospective 
research the immature (germinal vesicle-GV, pro-
phase I) oocytes of two groups of patients were 
included: i) 45 oocytes of 18 cancer patients with 
predominating breast cancer (Figure 1) and ii) 74 
oocytes of 21 healthy (non-cancer) patients (control) 
with fertility problems (partners of infertile men 
with impaired semen quality: oligozoospermia 
with less than 15 million spermatozoa/ml or tera-
tozoospermia with less than 4% morphologically 
normal spermatozoa according to the World Health 
Organization (WHO) Criteria 201024 who were in-
cluded in the IVF program. All patients were in the 
reproductive period of life, aged 18 to 43 years. 
Oocytes after controlled hormonal 
stimulation of the ovaries
In both groups of patients, both immature and 
mature oocytes were together retrieved after con-
trolled hormonal stimulation of the ovaries using 
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Virant-Klun I et al. / In vitro maturation of immature oocytes for fertility preservation 121
the same, antagonist protocol and ultrasound-
guided aspiration of ovarian follicles. In patients 
with fertility problems, the stimulation was started 
on day 2 of the menstrual cycle with 150 to 300 
I.U. of recombinant follicle-stimulating hormone 
(rFSH) daily. In cancer patients, the stimulation 
was initiated immediately after they have been 
sent to our department, no matter of the cycle 
phase. The ovarian stimulation was started with 
225 to 300 I.U. of rFSH. In breast cancer patients, 
an aromatase inhibitor – letrozole (2.5 mg every 
12 hours) was added to prevent estradiol rise and 
its possible detrimental effect on breast cancer. In 
all patients, the gonadotropin-releasing hormone 
(GnRH) antagonist was added, when dominant 
follicle measured 14 mm in a diameter. In patients 
with fertility problems, the oocyte maturation was 
triggered with choriogonadotropin alfa – Ovitrelle, 
when follicles measured 18 mm or more. If there 
were more than 15 follicles in both ovaries, the 
maturation triggering was performed with GnRH 
agonist. In majority of cancer patients, GnRH ag-
onist was used for oocyte maturation to prevent 
ovarian hyperstimulation (ovarian hyperstimula-
tion syndrome; OHSS), but some of them, if there 
were less than 10 follicles in both ovaries, were also 
treated by Ovitrelle. All follicles with a diameter 
of 16 mm or more were aspirated in all patients. 
A constant aspiration pressure of 180 mm Hg was 
used to aspirate the oocytes from the follicles. 
Mature oocytes with expressed polar body were 
immediately vitrified by soaking in a mixture of 
cryoprotectants, direct plunging into liquid nitro-
gen (-196oC), and stored in it, as described else-
where.25 
In vitro maturation of immature oocytes
Immature GV oocytes were exposed to the pro-
cedure of IVM in a seria of media of the IVM 
Maturation System (MediCult IVM®System, 
Origio/CooperSurgical, Denmark). 
For IVM, each GV oocyte was first exposed for 
two hours in the LAG Medium for conditioning 
and then for 24 to 28 hours to the maturation medi-
um of this system containing the reproductive hor-
mones: follicle stimulating hormone (FSH; 75 mIU/
ml) and human chorionic gonadotropin (HCG; 100 
mIU/ml) and in a co-culture with cumulus cells 
denuded from mature oocytes of the same patient, 
as described elsewhere (Figure 2).26 During incuba-
tion in these media, oocytes were cultured in a CO2-
incubator at 37oC and 6% CO2 in air. Oocytes were 
supposed to be mature (in a metaphase II) when 
they extruded a polar body. All oocytes of cancer 
patients and the majority of oocytes of women with 
fertility problems that matured in vitro were vitri-
fied and stored in liquid nitrogen for later clini-
cal use (IVF). Patients with ≥ 31% GV oocytes had 
increased oocyte immaturity. A subset of in vitro 
matured oocytes of women with fertility problems 
was fertilized in vitro. 
FIGURE 1. Types of disease in cancer patients included in this study. Breast cancer 
patients predominated. 
FIGURE 2. In vitro maturation of the oocyte in the maturation medium and 
in coculture with the cumulus cells of mature oocytes in the same patient at 
magnification 40 X (A) and magnification 100 X (B). 
CC = cumulus (granulosa) cells; O = oocyte; Red bar = A) 100 μm in B) 50 μm
A B
Radiol Oncol 2022; 56(1): 119-128.
Virant-Klun I et al. / In vitro maturation of immature oocytes for fertility preservation122
In vitro fertilization by ICSI 
In a subgroup of 17 in vitro matured oocytes from 
17 patients (1 oocyte per patient) with fertility 
problems (female partners of infertile men with 
oligozoospermia or teratozospermia) ICSI was per-
formed with partner's sperm one day later after 
oocyte and sperm retrieval from a couple. Oocytes 
were denuded by hyaluronidase to remove the 
cumulus cells and microinjection of one spermato-
zoon per oocyte was performed, as described else-
where.27 Only motile spermatozoa were used for 
ICSI. The next day, fertilization (presence of two 
pronuclei and extruded second polar body) was 
checked and embryo cleavage one day later. Good 
quality embryos were vitrified and stored in liquid 
nitrogen (-196oC) for later clinical use (transfer to 
the uterus). The rates of fertilization and embryo 
cleavage were compared between oocytes that ma-
tured in vitro and in vivo (in the ovaries; aspirated 
as mature) in the same patients after controlled 
ovarian hormonal stimulation.
Statistics
Both groups of patients, cancer and infertile pa-
tients, were compared in terms of the number of 
oocytes obtained after controlled hormonal stimu-
lation of their ovaries, the proportion of immature 
and degenerated oocytes, and the proportion of 
immature (GV) oocytes that matured in vitro. Due 
to the relatively small number of patients/oocytes 
included and the abnormal distribution of data, 
tested by Shapiro-Wilk normality test, non-para-
metric tests (Fisher's exact and Mann-Whitney U 
tests) were performed; statistical significance was 
set at P < 0.05. After ICSI, the fertilization and em-
bryo cleavage rates of oocytes that matured in vitro 
were compared with oocytes of the same patients 
that matured in vivo and were aspirated from the 
ovaries as mature oocytes in the same cycle of 
controlled ovarian hormonal stimulation using 
Fisher's exact and Wilcoxon tests; statistical signifi-
cance was set at P < 0.05. 
Results 
Cancer patients had different types of cancer, but 
breast cancer was predominant (Figure 1). The av-
erage age of cancer patients was 30.3 ± 6.3 years 
and of women with fertility problems 33.4 ± 5.0 
years. The two groups of women did not differ sig-
nificantly in their age. There was also no significant 
difference in the age of patients with breast cancer 
and patients with other cancers (32.0 ± 6.2 vs. 29.0 
± 6.0 years). 
Numbers, quality and immaturity of 
oocytes after controlled hormonal 
stimulation of the ovaries
After controlled hormonal stimulation of the ova-
ries, 198 oocytes were retrieved in cancer patients 
and 259 oocytes in infertile women. Cancer pa-
tients and patients with fertility problems did not 
differ significantly in the number of retrieved oo-
cytes (11.0 ± 9.0 oocytes/patient vs. 12.3 ± 9.2 oo-
cytes/patient), as revealed by Mann-Whitney U 
test. In cancer patients, the proportion of immature 
GV and MI oocytes was significantly lower than in 
patients with fertility problems (30.0% vs. 43.6%; P 
< 0.05), as revealed by Fisher's exact test (Table 1). 
The groups did not differ significantly in the pro-
portion of GV oocytes (23.0% vs. 28.6%) (Table 1); 
among patients with immature oocytes, 50% of 
cancer patients and 48.0% of patients with fertility 
problems had increased proportion (≥ 31%) of GV 
oocytes with a germinal vesicle, which did not dif-
fer significantly. There was also no difference in the 
proportion of degenerated oocytes between cancer 
patients and patients with fertility problems (8.6 vs. 
6.5%) (Table 1).
If we considered the type of cancer, we found 
that there was no significant difference in the 
number of all immature (MI and GV) oocytes in 
patients with breast cancer compared to patients 
with fertility problems or patients with other can-
cers (4.17 ± 3.25 vs. 5.29 ± 3.76 and 2.92 ± 2.47 im-
mature oocytes/patient, respectively), as revealed 
by Mann-Whitney U test. There was also no sta-
tistically significant difference in the proportion of 
all immature (GV and MI) oocytes in breast cancer 
patients compared to patients with other cancers 
or patients with fertility problems (36.0% vs. 27.0% 
and 43.6%), as found using the Fisher's exact test. 
Nevertheless, there was a tendency for a signifi-
cantly higher proportion of immature, GV oocytes 
in breast cancer patients compared to patients 
with other cancers (31.43% vs. 18.75%; P = 0.0531, 
Fisher's exact test).
In vitro matured oocytes
Forty-five GV oocytes in cancer patients and 74 
GV oocytes in patients with fertility problems un-
derwent IVM procedure (Table 2); the proportion 
of oocytes (15.5% in cancer patients and 12.2% in 
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Virant-Klun I et al. / In vitro maturation of immature oocytes for fertility preservation 123
women with fertility problems) degenerated just 
before or during conditioning in LAG medium. 
We found that a lower proportion of oocytes ma-
tured in vitro in cancer patients compared to pa-
tients with fertility problems (66.0 vs. 80.0%), how-
ever, the difference was not statistically significant, 
as revealed by Fisher's exact test. In spite of that, 
number of oocytes that matured in vitro per patient 
was significantly lower in cancer patients than in 
patients with fertility problems (1.39 ± 1.04 vs. 2.48 
± 1.83 oocytes/patient; P < 0.05, Mann-Whitney U 
test), as shown in Table 2. 
In cancer patients, there was also a lower pro-
portion of oocytes that matured in vitro in patients 
with breast cancer than in patients with other can-
cers and patients with fertility problems (54.5% vs. 
81.2% and 80.0%) (Table 2). The difference between 
patients with breast cancer and women with fertil-
ity problems tended to be statistically significant 
(P = 0.0862; Fisher's exact test) and was probably 
not significant due to the relatively low number of 
oocytes included. 
Overall, 198 oocytes were retrieved in cancer pa-
tients, of which 139 were mature. Following IVM, 
the number of total mature oocytes increased to 164 
(13.0% increase in mature oocyte yield). In patients 
with fertility problems, 259 oocytes were retrieved, 
of which 146 were mature. After IVM, the number 
of total mature oocytes increased to 198, which 
means 20.1% increase in mature oocyte yield. Thus, 
there was no significant difference in the yield of 
mature oocytes after IVM between cancer patients 
and patients with fertility problems. 15.5% (7/45) 
GV oocytes in cancer patients and 12.2% (9/74) GV 
oocytes in patients with fertility problems degener-
ated before in vitro maturation procedure.
Results of ICSI of in vitro matured 
oocytes in patients with fertility 
problems
In vitro fertilization of 49 oocytes in 17 patients with 
fertility problems (average age 34.3 ± 4.4 years) was 
performed by ICSI with partner's semen (in 2 men 
oligozoospermia and 15 men teratozoospermia). 
After performing this method, 27 (55.1%) oocytes 
were fertilized (expressing two pronuclei and two 
polar bodies) and 23 (85.2%) fertilized oocytes (zy-
gotes) further developed into an embryo, as shown 
in Table 3; four zygotes did not cleave further. 
Good quality embryos were vitrified and stored 
in liquid nitrogen for future clinical use in patients 
(transfer into the uterus). 
TABLE 1. Differences in the number, quality and immaturity of oocytes after 
controlled hormonal stimulation of the ovaries in cancer patients compared to 
patients with fertility problems
Cancer 
patients 
(n = 18)
Patients with 
fertility problems 
(n = 21)
Age (years) 30.3 ± 6.3 33.4 ± 5.0
Number of retrieved oocytes 198 259
Oocytes per patient 11.0 ± 9.0 12.3 ± 9.2
Number of degenerated oocytes 17 (8.6%) 17 (6.5%)
Number of immature (MI + GV) oocytes 59 (30.0%)* 113 (43.6%) *
Number of immature GV oocytes 45 (23.0%) 74 (28.6%)
* = statistically significant difference (P = 0.0064) revealed by Fisher's exact test; significance was 
set at P < 0.05; GV = germinal vesicle; MI = metaphase I (oocyte meiosis)
TABLE 2. Numbers and percentages of oocytes that matured in vitro in patients with 
different cancers compared to patients with fertility problems 
Number of oocytes 
that underwent in 
vitro maturation
Number of oocytes that 
matured in vitro
All cancer patients
(n = 18) 38 / 45
25 
(1.39 ± 1.04 per patient)*
(66.0%)
Patients with breast cancer 
(n = 8) 22 
12 
(54.5%)
Patients with other cancers 
(n = 10) 16 
13 
(81.2%)
Patients with fertility problems 
(n = 21) 65 / 74
52 
(2.48 ± 1.83 per patient)* 
(80.0%)
* = statistically significant difference (P < 0.05; Mann-Whitney U test) 
After ICSI, the fertilization and cleavage rates 
of 49 oocytes that matured in vitro were compared 
with 121 oocytes of the same patients that matured 
in vivo and were aspirated from their ovaries as 
mature oocytes (metaphase II [MII] oocyte meiosis) 
in the same cycle of controlled ovarian hormonal 
stimulation. Of the 121 oocytes obtained as mature 
oocytes, 69 oocytes were fertilized, representing a 
fertilization rate of 57.0%. Sixty-one fertilized oo-
cytes further developed into an embryo (Table 3). 
Fisher's exact test revealed no statistical differ-
ences in the proportions of fertilized oocytes, non-
cleaved zygotes, and embryos obtained by ICSI on 
in vitro and in vivo matured oocytes (Table 3). 
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In patients with an increased proportion of im-
mature (GV) oocytes (≥ 31%), there was a tendency 
for a lower proportion of fertilized oocytes and 
a higher proportion of non-cleaved zygotes, but 
the differences were not statistically significant 
(Table 4).
Discussion
The results of this research show that cancer and 
control healthy patients with fertility problems 
did not differ in the number and quality of oocytes 
after controlled hormonal stimulation of their ova-
ries, which is positive. In cancer patients, there was 
even a significantly lower proportion of immature 
oocytes than in patients with fertility problems. 
However, in patients with cancer, fewer oocytes 
per patient matured in vitro than in patients with 
fertility problems (1.39 ± 1.04 vs. 2.48 ± 1.83, P < 
0.05). Following ICSI of oocytes in patients with 
fertility problems, the fertilization and embryo 
cleavage rates were approximately the same in oo-
cytes that matured in vitro and in vivo in the same 
patients, in the same cycles of controlled hormonal 
stimulation of the ovaries. This is also to be expect-
ed in cancer patients.
The proportion of mature, MII oocytes in the 
patients with fertility problems included in this 
research was relatively low (56.4%) compared to 
the internationally accepted reference value of 70-
80%28, because we included mainly patients with 
a higher proportion of immature oocytes which 
did not reflect the average condition; in cancer pa-
tients, the proportion of mature oocytes was higher 
(70%) and within the reference value.28 The num-
ber and quality of oocytes in cancer patients did 
not differ between different cancers and from con-
trol patients with fertility problems. The same has 
been found in other studies for different types of 
cancer such as breast cancer, lymphoma, gliomas 
and other cancers.29,30 For breast cancer, the results 
of various studies are otherwise contradictory. In a 
study by Malacarne et al., as in our study, the aver-
age number of oocytes obtained per breast cancer 
patient after ovarian stimulation did not differ sig-
nificantly from healthy control women including 
oocyte donors, women with fertility preservation 
for non-medical reasons, and female partners of in-
fertile men in an IVF program31; it was concluded 
that patients with breast cancer undergoing con-
trolled ovarian hormonal stimulation for fertility 
preservation can expect the ovarian response pre-
dicted for their age. The results obtained by differ-
ent studies do not support the notion of a negative 
impact of the breast cancer gene 1/2 (BRCA1/2) 
mutation on the ovarian response of women with 
breast cancer.31-33 Nevertheless, the results of some 
other studies suggest the reduced number and ma-
turity of oocytes obtained for cryostorage in pa-
tients with breast cancer34,which may be attributed 
to the higher grade of cancer35 or different expres-
sion of hormonal receptors.36 
There is little data in the literature on how dif-
ferent cancers affect the oocyte IVM in cancer pa-
tients. The oocyte IVM rate in breast cancer patients 
was found to be approximately 53.2 to 64.2% in the 
TABLE 3. Non-significant differences in fertilized oocytes, non-cleaved zygotes, and 
cleavage embryos obtained by intracytoplasmic sperm injection (ICSI) on in vitro 
matured and in vivo matured oocytes of patients with fertility problems (in the same 
cycles)
ICSI cycles 
(n = 17)
In vitro matured 
oocytes
In vivo matured 
oocytes*
Number of microinjected oocytes 49 121
Fertilized oocytes 27 (55.1%)
69
(57.0%)
Non-cleaved zygotes 4 (15.0%)
8 
(11.6%) 
Cleavage embryos 23 (85.2%)
61
(88.4%)
* = non-significant differences, as revealed by Fisher's exact test
TABLE 4. Non-significant differences in results of intracytoplasmic sperm injection 
(ICSI) cycles (fertilized oocytes, non-cleaved zygotes, cleavage embryos) on in 
vitro matured oocytes of patients with fertility problems regarding the number of 
immature (germinal vesicle [GV]) oocytes
ICSI cycles (n = 17) ≤ 30% GV oocytes ≥ 31% GV oocytes
Female age (years) 34.4 ± 3.0 34.3 ± 5.6 
Number of microinjected oocytes 16 33 
Fertilized oocytes 11 (69.0%) 16 (48.5%)
Non-cleaved zygotes 0 (0%) 4 (25.0%)
Cleavage embryos 11 (100%) 12 (75%)
Non-significant differences, as revealed by Fisher's exact test
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Virant-Klun I et al. / In vitro maturation of immature oocytes for fertility preservation 125
study of Shalom Paz et al.37, which is very similar to 
our study (54.5%), or slightly higher – 62.0%, 66.0% 
or 66.7% in some other studies.22,32 In this study, 
the proportion of immature oocytes matured in 
vitro tended to be lower in breast cancer patients 
(54.5%) than in patients with fertility problems 
(80.0%), while this was not observed in patients 
with other cancers (81.2%). Although, the differ-
ence was not significant, possibly due to relatively 
low number of patients and oocytes included. In a 
study conducted by Liu et al., 811 genes were iden-
tified that were expressed differently in malignant 
breast tissue compared to healthy breast tissue38; 
among the up-regulated genes was also a group of 
genes involved in the cell cycle and progesterone-
mediated oocyte maturation. For cancer patients, 
Cohen et al. found that the mean oocyte maturation 
rate in stimulated IVF cycles was 38%39, which is 
significantly lower than in our study (66.0%; 54.5% 
in breast cancer and 81.0% in other cancers). In our 
study, IVM of oocytes in coculture with cumulus 
cells from mature oocytes in the same patients may 
have been beneficial, at least in part providing an 
ovarian niche.26 Also Chatroudi et al. found that cu-
mulus cell supplementation in IVM culture media 
enhances the viability of human embryos (blas-
tocysts) after IVF.40 The rates of in vitro matured 
oocytes in cancer patients and patients with fertil-
ity problems in our study were very similar to the 
published rates of IVM of immature oocytes in the 
usual IVF program, where 65.0%, 68.7%, 68.9% and 
69.7% maturation rates were obtained in different 
maturation media.41-43 Oktay et al. reported the 45% 
increase in mature oocyte yield after IVM of imma-
ture oocytes after controlled hormonal stimulation 
of the ovaries in breast cancer patients and a high 
fertilization rate of these oocytes.44 Moreover, an 
IVM of oocytes retrieved without hormonal stim-
ulation of the ovaries was considered for fertility 
preservation in breast cancer patients to avoid the 
ovarian stimulation, shorten the time to oocyte re-
trieval, and not to increase both the serum estradiol 
level and delay in cancer treatment.45-47 
It should be noted that our study was limited to 
a relatively small number of patients involved and 
a small number of oocytes. In cancer patients and 
patients with fertility problems, we tried to per-
form as comparable controlled hormonal stimula-
tion of the ovaries as possible using a GnRH an-
tagonist. Nevertheless, we also had to take certain 
safety precautions in cancer patients. In these pa-
tients, the ovarian hormonal stimulation was initi-
ated immediately after they have been sent to our 
department, no matter of the cycle phase to be fast 
and prevent further progression of disease. The oo-
cyte maturation in cancer patients was initiated by 
GnRH analogue to prevent hyperstimulation, but 
more patients with less than 10 follicles were also 
treated with Ovitrelle similar to patients with fer-
tility problems. Thus, in most patients, oocyte mat-
uration was triggered by Ovitrelle. If we used ex-
actly the same method of hormonal ovarian stimu-
lation and triggering oocyte maturation in cancer 
patients and patients with fertility problems, there 
might be more immature oocytes in cancer pa-
tients, but this was not possible for cancer-related 
safety reasons. 
In addition, for safety reasons, breast cancer 
patients were also treated with an aromatase in-
hibitor, letrozole, to prevent an increase in estra-
diol and worsening of the disease. Thus, based on 
our own experience and literature48,49, we believe 
that both random start of hormonal stimulation in 
cancer patients and use of aromatase inhibitor in 
patients with breast cancer do not affect the num-
ber, maturity and in vitro maturation of oocytes ob-
tained in these patients. 
Letrozole treatment may also increase the in-
traovarian androgen levels, which have a negative 
impact on granulosa cells (apoptosis) in the late an-
tral and pre-ovulatory follicles.50 In this research, 
granulosa (cumulus) cells were used in co-culture 
for oocyte maturation, which may lower the matu-
ration rate. However, we performed in vitro matu-
ration of oocytes with cumulus cells of mature oo-
cytes because our previous work showed that co-
culture with cumulus cells does not affect the pro-
portion of in vitro matured oocytes, but improves 
the molecular status of oocytes (gene expression 
profile) compared to oocytes matured in vivo.26
In patients with fertility problems, we deter-
mined the FSH, LH and AMH levels in early fol-
licular phase of the cycle as well as the number 
of antral follicles. For cancer patients, we have no 
such data. Only informative ovarian scan with an-
tral follicle estimation was performed at the begin-
ning of ovarian stimulation.
In our study, approximately the same propor-
tion of oocytes were fertilized and further cleaved 
into an embryo after ICSI of in vitro and in vivo ma-
tured oocytes. Some studies have shown poorer 
embryo development and live birth rates with in 
vitro matured oocytes51,52, which could be linked to 
structural and morphologic differences in human 
oocytes after IVM53 due to suboptimal maturation 
medium and lack of ovarian niche. It needs to be 
point out that in our study, oocyte IVM was per-
formed in coculture with cumulus cells from ma-
Radiol Oncol 2022; 56(1): 119-128.
Virant-Klun I et al. / In vitro maturation of immature oocytes for fertility preservation126
ture oocytes of the same patients thus providing a 
degree of ovarian niche. In spite of a lower rate of 
good-quality embryos and different developmen-
tal dynamics of embryos, pregnancy rates as well 
as live births did not necessarily differ after oocyte 
IVM, as found by Roesner et al.54 Moreover, in a 
matched setting between IVM and IVF babies born 
from women with polycystic ovaries, no significant 
increased risk associated with IVM has been iden-
tified in 2-year-old singletons born after IVM and 
after a mean follow-up up to 7.5 years.55,56 In gen-
eral, more studies are urgently required to improve 
IVM –vitrification method to successfully preserve 
oocytes collected from cancer patients.57,58
Conclusions
We may conclude that ‘rescue’ of immature oo-
cytes with IVM is a useful strategy to improve the 
mature oocyte yield of fertility preservation cycles 
in cancer patients. Immature oocytes retrieved dur-
ing oocyte and also embryo cryopreservation cy-
cles in cancer patients should not be discarded in 
order to improve the future potential of pregnancy 
in these patients. Their immature oocytes can ma-
ture in vitro comparable to healthy controls. After 
ICSI, approximately the same proportion of in vitro 
matured oocytes could be fertilized and developed 
into an embryo as in oocytes matured in vivo. 
Acknowledgments 
We would like to thank all colleagues, gynecolo-
gists, embryologists, and nurses at the Department 
of Gynecology and Obstetrics, Reproductive Unit 
(IVF), who in any way helped in this work. We also 
thank all the patients who kindly donated imma-
ture oocytes for this research. Last but not least, we 
would also like to thank our institution, University 
Medical Centre Ljubljana, which funded this re-
search as part of a tertiary research project. 
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