Abstract
Survival rates after cancer have increased significantly in recent decades; however, these treatments also have drawbacks, and patients (or parents in the case of children) must be informed of the long-term side effects of oncological treatments and the possible options for preserving the fertility of these patients. It is important to set out clearly the possible risks of developing ovarian failure or azoospermia with oncological treatments. These will depend on the age of the patients and on the type, dose and duration of chemotherapy, and on the field, dose and duration of radiotherapy.
Oocyte Vitrification as an Option for Fertility Preservation
Every year many women worldwide are diagnosed with cancer. More than 90% of cancer patients undergo invasive cancer therapy, such as chemo- and radiotherapy [1]. Most chemotherapy regimens include the alkylating agent cyclophosphamide, which is known to cause a significant loss in ovarian follicle reserve, and may result in infertility and early menopause. The irreversible destruction of germ cells after using both radio- and chemotherapy is due to a direct apoptotic effect on oocytes [2]. On the other hand, advances in oncological treatments and better screening programs have significantly improved the life expectancy thus increasing the population of young cancer survivors. Therefore, protection against iatrogenic infertility caused by cancer therapies is considered indispensable to allow patients to have a chance to conceive in the future and to have their own genetic offspring.
Fertility preservation (FP) is not limited only to cancer patients but in any situation in which ovarian function is compromised, as other non-oncological diseases or different situations related to ovarian surgery. In these cases, an intervention to safeguard gametes for future use is required to uphold fertility potential. Severe systemic autoimmune diseases requiring therapy potentially harmful to their ovaries as cyclophosphamide for refractory rheumatoid arthritis [3], for severe manifestations of systemic lupus erythematosus, such as proliferative nephritis, affection of the central nervous system, pneumonitis or severe thrombocytopenia [4] or in other diseases as Wegener’s granulomatosis [5]. Patients needing bone marrow transplantation are also associated with high risk of ovarian failure as high doses of chemo- and radiotherapy are applied to destroy the preexisting bone marrow [6].
Repetitive conservative surgery on the ovaries can also lead to premature ovarian failure by diminishing ovarian reserve with almost half pregnancy rate after primary surgery [7]. Endometriosis, as one of the most frequent pathologies in gynecologic surgery, can also result in a considerable loss of ovarian reserve [8].
Once this technique became available for medical indications, its use for nonmedical reasons was not long in coming. There is a vast population increasingly using FP, which includes women who wish to postpone motherhood and childbirth for a variety of reasons. These motivations are frequently grouped as “social reasons” or “elective fertility preservation (EFP)” where the main reason is age-related fertility decline [9]. In today’s society, many women are entirely dedicated to their careers and delay pregnancy beyond young childbearing years, so that women are often forced to choose between motherhood and their professional aspirations and financial security [10]. As a consequence, most women visit reproductive clinics usually over their mid-30s. This leads to repeated failed cycles at advanced maternal age or engagement to egg donation programs. Additionally, lack of partner is also a common reason for delaying pregnancy and occurs mainly in developed countries, where significantly low birth rates are reported [10].
Oocyte vitrification as an FP method is undoubtedly one of the greatest advances made in assisted reproduction in recent years, providing the opportunity to offer an option to these women. Thanks to vitrification oocytes can now be harvested and safely stored to attempt pregnancy in the future when diseases are cured or when circumstances become more favorable [11]. Currently, the options suggested for FP also include embryo cryopreservation and ovarian tissue cryopreservation. However, the need for a male partner or donor sperm, in addition to ethical and religious concerns, means that embryo cryopreservation would not be the most suitable option for all single women. Ovarian tissue cryopreservation is the technique of choice in prepubertal patients [12, 13] and for women with hormone-dependent diseases [14]. Recent publications suggest that ovarian cortex cryopreservation and transplantation is an effective method for preserving fertility offering real possibilities for future maternity [15]. Despite the achievements provided by this technology, it is still labeled as “experimental” but hopefully the evidence available will soon help to remove it, so it can be openly introduced to the clinical practice [16]. Therefore, oocyte vitrification is becoming the gold standard technique for FP in adult patients, and it is the best option for all women to maintain their reproductive autonomy [17].
Brief Outline of the Basic Principles and Methodology of Oocyte Vitrification
Following the first report of a pregnancy using a frozen thawed oocyte in 1986 [18], many efforts were carried out to overcome all the drawbacks posed by the method which are mainly related to the architecture of the oocyte itself and to the high cryosensitivity associated to this cell. The intracellular ice formation related to the large volume of the cell, chilling injury, and osmotic damage are the main causes of high oocyte sensitivity to cryopreservation. The introduction of vitrification allowed to overcome all these drawbacks and led to cryopreserve oocytes successfully.
Vitrification is defined as the glass-like solidification of an aqueous solution at low temperature. This process is facilitated by applying high cooling rates and increasing viscosity using high concentration of cryoprotectant agents (CPAs) [19]. These agents either permeate the cell membrane or create an osmotic gradient allowing to reduce water content and, as a result, avoiding ice crystal formation. However, CPAs can be toxic at high concentration; thereby protocols have been modified in order to minimize damage. In this way, by significantly reducing the volume of the sample, the cooling rate is increased and the concentration of CPAs can be reduced while maintaining successful vitrification.
The methodology used for oocyte vitrification may differ significantly between laboratories, especially when distinct devices and solutions are used. Since the first vitrification solution was developed [20], several CPAs have been proposed as being effective. At the moment, a mixture of penetrating CPAs, such as ethylene glycol and dimethylsulfoxide, in combination with a non-penetrating CPA, such as sucrose or trehalose is the most commonly used approach [21].
The basic procedure is simple and quick [22], although it requires experienced hands. Oocytes are gradually exposed to hypertonic solutions of CPAs and osmotic agents in order to remove water from cells and allow the penetration of permeable CPAs. Once the oocytes are loaded on/in a suitable container specifically designed for vitrification, they are immediately submerged into liquid nitrogen. Traditionally, the devices that use minimum volumes are directly plunged into liquid nitrogen to allow direct contact with samples, and are commonly known as open systems. Conversely, closed systems avoid direct contact between samples and liquid nitrogen thanks to their hermetic sealing prior to vitrification. The premise of the vitrification approach is that the higher the cooling rate, the greater the likelihood of true vitrification being achieved. However, this is not the whole truth. A high warming rate is also required to succeed as demonstrated [23]. Nowadays, consolidated oocyte vitrification programs exist in assisted reproductive technology, which have led to an increasing number of healthy newborns [24].
Oocyte Vitrification in Oncological Patients
Gonadotoxicity in cancer patients depends on various factors, such as age, ovarian reserve, type of chemotherapy, especially while using alkylating agents, type of cancer, and cumulative doses received [25, 26]. Many young women recover their ovarian function and reproductive capability once chemotherapy is completed, especially with low-dose and low-gonadotoxic chemotherapy [27]. However, options to preserve fertility should be consider from the moment of diagnosis due to egg quality may be suboptimal thus lowering the chances of pregnancy. Oocyte vitrification is currently being offered to cancer patients, however, there have been some matters of concern.
One of the main worries is the need of multiple follicular stimulation which may cause delay in the beginning of chemotherapy. However, controlled ovarian stimulation (COS) can initiate in the luteal phase, randomly, or irrespectively of the menstrual cycle date when patients come for their first consultation [28, 29]. The strategy’s efficacy has been previously evidenced by similar IVF and clinical outcomes compared with the conventional start of COS in the follicular phase [28, 29, 30, 31]. In a recent study we did not observe impaired ovarian response between cancer patients stimulated either at luteal or random start and EFP patients who were stimulated following the conventional start at the follicular phase [32]. Another point of concern is whether the stimulation with gonadotropins would affect the evolution of breast cancer, which is the most common malignancy in reproductive age and the most frequent diagnosis of people undergoing any FP option [11, 27]. High estradiol levels are the main objection for patients with hormone-dependent tumors, as in vitro cellular proliferation has been demonstrated when acute exposure to estrogens occurs, and would contribute to the activation of pro-oncogenes in breast cancer [33, 34]. The protocol described by Oktay et al. [34] prevents the potential risk associated with high estradiol levels. This combines aromatase inhibitor letrozole with gonadotrophins as it supports low estradiol levels during stimulation. This protocol is considered safe and is very useful for those patients affected by hormone sensitive tumors as it significantly lowers estradiol levels compared with other stimulation protocols without letrozole [36].
Evidence for cancer patients who have returned to use their stored oocytes once they have overcome the disease is still scarce although interesting data is starting to emerge [32]. One study reported the birth of twins after the combined use of ovarian tissue cryopreservation and vitrified oocytes harvested after tissue grafting [37]. Another publication by our group reported the birth of a healthy baby boy in a patient diagnosed with non-Hodgkin’s lymphoma [11]. Additionally, few other case reports have been published that report the live births achieved by FP patients affected by different cancer types [38, 39, 40]. Our last publication compiles the outcome achieved by 80 cancer patients who returned to attempt pregnancy after FP [31]. In this study we showed IVF results after warming of the stored oocytes and compared the outcome with that achieved by EFP patients and reported a total of 25 healthy babies born from cancer patients.
Oocyte Vitrification for EFP: General Aspects and Clinical Outcomes
The demand for oocyte vitrification due to social reasons has increased in recent years and evidence about the outcomes of IVF cycles in this population is starting to grow. A study published by our group provided data on the outcomes achieved by 137 women who chose EFP and returned to attempt pregnancy [40]. As expected, the vast majority of women who came to vitrify their oocytes were single (75.6%) and professionals with a high level of education (72.8%), which coincide with the essential motivations described for this population. Most of these women decided EFP at advanced age (63%) when aged 37–40 years, a non-negligible 16.2% were aged ≥40 upon vitrification, and the small minority were aged <30 years. It is striking that the age of the women who underwent EFP matched that of those who traditionally came to IVF clinics to treat infertility problems. This is indeed a contradiction because the fundamentals of FP consist in preserving fertility and not preserving infertility.
As predicted, age at vitrification and number of oocytes were identified as factors strongly related to success. In young woman aged ≤35 years, a large number of oocytes were retrieved and finally vitrified, and the survival and clinical outcomes were the equivalent to those achieved in our egg-banking program for ovum donation, with the highest success rates for the youngest group of women (≤29 years). Otherwise, predictably fewer oocytes and worse outcomes were obtained as age increased, which resembled the results of the infertile population of a similar age. Similarly, the effect of female age was observed when data were analyzed according to the reason for FP.
That way, IVF outcome with vitrified oocytes is, as it is for fresh oocytes, strongly dependent on maternal age at the time of vitrification as the number of mature oocytes is age-related and post-warming survival rates diminish with increasing age. The increase in chromosomal abnormalities with maternal age correlates strongly with diminished embryo viability, and it is well known that almost 80% of oocytes are already aneuploid by the age of 40. These observations allow us to assume that older women will require more vitrified oocytes to achieve a live birth compared to younger women, which would need at least 8–10 MII vitrified oocytes to achieve a reasonable success rate.
Another simultaneously published study, which included 128 autologous IVF cycles with vitrified oocytes of which 32 came from EFP patients, reached similar conclusions [42]. These authors considered that the clearest threshold between better and worse outcomes was <38 years versus ≥38 years. Accordingly, the clinical pregnancy rate for patients aged <38 years at the time of oocyte cryopreservation was 60.2%, compared to one of 43.9% for those aged ≥38 years at the time of oocyte cryopreservation. Their recommendations were to cryopreserve 15–20 MII oocytes for the women aged <38 years, which allows them roughly a 70–80% chance of at least one live birth, and 25–30 MII oocytes for the women aged 38–40 years, which allows them roughly a 65–75% chance of at least one live birth. Obviously, when considering 38 years instead of 36 years as the proposed cutting point, cryopreserving more oocytes is recommended.
Factors Related to IVF Outcomes in Elective and Oncological FP
Due to the increasing interest in FP, it is crucial for reproductive medicine providers to be aware of the success rates and limiting factors of oocyte vitrification in order to provide patients with proper counselling. Recently, we published a study intended to explore the scope and functioning of FP in our practice [32]. We also aimed to determine the possible impact of the underlying malignant disease on the IVF outcome in the cancer patients who had their oocytes vitrified for FP (Onco-FP group) by a comparison with the results achieved by women who underwent EFP (EFP group). This report comprised the largest series to date, with more than 6,000 women performing over 8,000 FP cycles, of whom ~700 had returned to attempt pregnancy, and with 162 and 25 healthy babies born in the EFP group and the Onco-FP group, respectively.
When attempting to identify different factors related to success in the two different study populations, special attention was drawn to age as it is recognized as one of the strongest confounders in assisted reproduction. Notably in our study, 70% of women in the EFP group were older than 35 years and 15% were aged ≥40 years by the time of vitrification (Figure 20.1). This distribution was the opposite in the Onco-FP group, where 70% of the patients were younger than 35 years. In fact, this could explain the statistically fewer oocytes recovered and vitrified per cycle that we observed in the EFP group.
It was relevant that in the present study we did not observe any impaired ovarian response in cancer patients, which agrees with recent publications [43–46]. Nonetheless, no consensus on this issue exists. A meta-analysis published in 2012 concluded that we should expect fewer oocytes retrieved after COS for FP in cancer patients compared with healthy age-matched patients [47]. Even so, it is worth noticing that this meta-analysis included studies with differing ovarian stimulation protocols that used milder stimulation in cancer patients and different inclusion criteria [44], which may introduce a bias.
Doubtlessly, when analyzing the ovarian response it would be interesting to consider the ovarian stimulation protocol. In our study, the most widely used protocol in EFP was the antagonist, while the antagonist + letrozole was employed in the majority of cancer patients. The reasons for using different COS protocols in this study clearly obey the presence of two very different populations. We observed more retrieved and vitrified oocytes when the antagonist protocol was administered to both EFP and Onco-FP patients (Table 20.1). This was most probably because the antagonist protocol was used in the vast majority of our patients, especially in the EFP group. Besides, the agonist protocol was used in very few cases, which means that the comparison is not very accurate.
Table 20.1 Number of oocytes retrieved and vitrified per cycle according to controlled ovarian stimulation protocol and FP group
| A. Nº Retrieved oocytes/cycle | ||
|---|---|---|
| Type of COS protocol | Elective FP | Onco-FP |
| Antagonist | 10.5 ± 7.3a* | 13.4 ± 9.5a* |
| Agonist | 8.8 ± 5.9b | 8.3 ± 6.2b |
| Antagonist + Letrozole | NA | 11.2 ± 8.1a |
| B. Nº Vitrified oocytes/cycle | ||
|---|---|---|
| Type of COS protocol | Elective FP | Onco-FP |
| Antagonist | 8.1 ± 5.8a* | 10.3 ± 7.5a* |
| Agonist | 6.5 ± 4.8b | 5.8 ± 4.6b |
| Antagonist + Letrozole | NA | 8.6± 6.6c |
*P<0.05.
Different superscripts for comparisons intra FP group indicate statistical differences (P<0.05).
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