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

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 (including 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-menopausal.¹ Decreased ovarian follicle reserve occurs in more than one-third of patients after breast cancer 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 cancer treatment.³ The situation is similar with other cancers; cancer therapy is the cause of premature ovarian failure in 25% of women with this diagnosis.⁴ Therefore, it is very important to consider the preservation of female fertility before oncotherapy. An improvement in the survival rates of cancer patients and recent advances in assisted reproductive technologies have led to significant progress in fertility 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 variety of cancers, including breast cancer.^{5, 6, 7, 8, 9, 10, 11, 12} Oocyte cryopreservation is an effective approach^{5, 6, 7, 8, 9, 10, 11, 12}, 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 controlled hormonal stimulation of the ovaries, in vitro maturation (IVM) of immature oocytes before vitrification is recommended and not after vitrification/ devitrification procedure.¹⁷ Even if there are no differences in survival rates between oocytes vitrified before or after IVM procedure, decreased maturation 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 cancer 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 hormonal 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 vitrified, 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 discarded and lost to the patient. The important question 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 cryopreserved in cancer patients.²² Recently, the first birth achieved after fertility preservation using vitrification 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 oocytes of cancer patients in laboratory conditions (in maturation medium and co-culture with cumulus cells from mature oocytes of the same patients) 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, actually intracytoplasmic sperm injection (ICSI), on the in vitro matured oocytes of patients with fertility problems as a model for cancer patients.

This research was approved by the Slovenian National Medical Ethical Committee (No. 0120222/2016-2; KME 115/04/16). In this prospective research the immature (germinal vesicle-GV, prophase 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 teratozoospermia with less than 4% morphologically normal spermatozoa according to the World Health Organization (WHO) Criteria 2010²⁴ who were included in the IVF program. All patients were in the reproductive period of life, aged 18 to 43 years.

Figure 1

In vitro maturation of immature oocytes

Immature GV oocytes were exposed to the procedure 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 medium of this system containing the reproductive hormones: 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 incubation in these media, oocytes were cultured in a CO₂incubator at 37^oC 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 vitrified and stored in liquid nitrogen for later clinical 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 2

Cancer patients had different types of cancer, but breast cancer was predominant (Figure 1). The average 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 significantly 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).

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 ovaries, 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 oocytes 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 expected 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 7080%²⁸, because we included mainly patients with a higher proportion of immature oocytes which did not reflect the average condition; in cancer patients, the proportion of mature oocytes was higher (70%) and within the reference value.²⁸ The number and quality of oocytes in cancer patients did not differ between different cancers and from control 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 average number of oocytes obtained per breast cancer patient after ovarian stimulation did not differ significantly from healthy control women including oocyte donors, women with fertility preservation for non-medical reasons, and female partners of infertile men in an IVF program³¹; it was concluded that patients with breast cancer undergoing controlled ovarian hormonal stimulation for fertility preservation can expect the ovarian response predicted for their age. The results obtained by different 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, 32, 33} Nevertheless, the results of some other studies suggest the reduced number and maturity of oocytes obtained for cryostorage in patients with breast cancer³⁴,which may be attributed to the higher grade of cancer³⁵ or different expression of hormonal receptors.³⁶

There is little data in the literature on how different cancers affect the oocyte IVM in cancer patients. The oocyte IVM rate in breast cancer patients was found to be approximately 53.2 to 64.2% in the 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 difference 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 identified 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 progesteronemediated 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 cumulus cell supplementation in IVM culture media enhances the viability of human embryos (blastocysts) after IVF.⁴⁰ The rates of in vitro matured oocytes in cancer patients and patients with fertility 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, 42, 43} Oktay et al. reported the 45% increase in mature oocyte yield after IVM of immature 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 stimulation of the ovaries was considered for fertility preservation in breast cancer patients to avoid the ovarian stimulation, shorten the time to oocyte retrieval, and not to increase both the serum estradiol level and delay in cancer treatment.^{45, 46, 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 perform as comparable controlled hormonal stimulation of the ovaries as possible using a GnRH antagonist. Nevertheless, we also had to take certain safety precautions in cancer patients. In these patients, the ovarian hormonal stimulation was initiated 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 oocyte 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 fertility problems. Thus, in most patients, oocyte maturation was triggered by Ovitrelle. If we used exactly the same method of hormonal ovarian stimulation and triggering oocyte maturation in cancer patients and patients with fertility problems, there might be more immature oocytes in cancer patients, but this was not possible for cancer-related safety reasons.

In addition, for safety reasons, breast cancer patients were also treated with an aromatase inhibitor, letrozole, to prevent an increase in estradiol and worsening of the disease. Thus, based on our own experience and literature^48,49, 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 number, maturity and in vitro maturation of oocytes obtained in these patients.

Letrozole treatment may also increase the intraovarian androgen levels, which have a negative impact on granulosa cells (apoptosis) in the late antral and pre-ovulatory follicles.⁵⁰ In this research, granulosa (cumulus) cells were used in co-culture for oocyte maturation, which may lower the maturation rate. However, we performed in vitro maturation of oocytes with cumulus cells of mature oocytes because our previous work showed that coculture with cumulus cells does not affect the proportion 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 determined the FSH, LH and AMH levels in early follicular 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 antral follicle estimation was performed at the beginning of ovarian stimulation.

In our study, approximately the same proportion of oocytes were fertilized and further cleaved into an embryo after ICSI of in vitro and in vivo matured oocytes. Some studies have shown poorer embryo development and live birth rates with in vitro matured oocytes^51,52, 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 performed in coculture with cumulus cells from mature oocytes of the same patients thus providing a degree of ovarian niche. In spite of a lower rate of good-quality embryos and different developmental 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 identified in 2-year-old singletons born after IVM and after a mean follow-up up to 7.5 years.^55,56 In general, more studies are urgently required to improve IVM –vitrification method to successfully preserve oocytes collected from cancer patients.^57,58

We may conclude that ‘rescue’ of immature oocytes with IVM is a useful strategy to improve the mature oocyte yield of fertility preservation cycles in cancer patients. Immature oocytes retrieved during oocyte and also embryo cryopreservation cycles in cancer patients should not be discarded in order to improve the future potential of pregnancy in these patients. Their immature oocytes can mature 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.