**1. Introduction**

The female perspective on fertility after cancer treatment is, nowadays, an important issue. A 2012 study that analyses the information received from cancer survivors has found that there is a sex bias. Thus, men received more information regarding the influence of treatment on fertility 80% vs. 48%, and more men also received information about options to preserve fertility—68%, compared to 14% for women [1]. Statistics show that over 50% of men opted to cryopreserve sperm; only 2% of women undertook any means of fertility preservation [1]. The onco-fertility preservation necessity arises as 8% to 12% of all breast cancers occur before the age of 35 [2,3] and the tally rises to 15% for women 40 years old [4,5], in the backdrop of breast cancer being the most frequent of all cancers among women of childbearing age—affecting one-third of the young with cancer [6]. If we look at the breast cancer incidence in patients between the ages of 20 and 34 years, it stands at 1.9% of all newly diagnosed breast cancers and rises to 10.5% for breast cancers occurring in 35 and 44 year old women [7,8]. Breast cancer in young patients has special traits, characterized by specific oncogenic signaling pathways and associates a higher incidence of hormone receptor-negative, higher grade, and human EGF2 receptor-overexpressing tumors [9]. Advances in breast cancer early detection rates and treatment options have led to a five-year breast cancer survival rate of over 80% [10]. As greater survival rates are obtained, there is also a greater focus on achieving goals of motherhood and family completeness. Young Women's Breast Cancer Study concluded that 50% of women younger than 40 years have concerns about future fertility and pregnancy options, following chemotherapy and radiotherapy [11]. There is also a psychological burden upon cancer survivors as a result of the fertility concern, and there are wide-spread studies to attest to the rising awareness of such instances [12].

A 2012 review of fertility demographics in USA showed an increase in the number of women giving birth after 30 years of age, with a peak for white women at 35 years [13]. The infertility risk of a woman in her teens is 0.2%, which will rise to 2% by her twenties, and reach 20% in her early thirties, which thus acknowledges only the number and quality of oocytes—by the time that most women will consider getting pregnant, they are already 20% infertile [14], resembling an infertility pandemic in developed countries. Adding to this is the increased incidence of cancer in young women, which will increase the cost of treatment and frequently implies infertility. Up to 6% of fertile age women are cancer survivors, and the incidence of cancer increases from about 1 in 10,000 shortly after birth to about 1 in 300 by mid-forties [15]. Depending on the source, ovarian failure characterizes 6.3% up to 12% of women that are childhood cancer survivors [16] and up to 50% of the patients that receive oncologic treatment at 40 years old will suffer early ovarian failure [17]. Most studies take account of the abrupt onset of menopause five years from chemotherapy, as evidence of ovarian failure, underestimating subtler manifestations such as subfertility and diminished ovarian reserve.

Chemo-therapeutic agents, known for deleterious effects, include alkylating agents that are considered high risk, such as Cyclophosphamide, Mechlorethamine, Chlorambucil, Busulfan, and Melphalan, whose active metabolites form DNA crosslinks leading to its function and synthesis arrest [18]. They produce DNA double-strand breakage, followed by P63 mediated apoptosis. Platinum-based compounds, Cisplatin and Carboplatin covalently binds DNA and forms intra and interstrand bonds that produce DNA strands breakage during replication. DNA transcription, synthesis, and function are, thus, inhibited. They are considered intermediate risk, though there was no demonstrated specific toxicity upon human primordial follicles [15,19]. Antimetabolites include Methotrexate, 5-fluorouracil, and Cytarabine and inhibit DNS synthesis and ARN purines and Thymidylate synthesis, being considered of low risk. Vinca alkaloids, Vincristine and Vinblastine inhibit tubulin polymerization, producing microtubules disruption during mitosis with mitosis arrest in metaphase and consecutive cellular death [3,18]. It is labeled as low risk. Anthracyclines, Daunorubicin, Bleomycin, Adriamycin (doxorubicin) inhibit DNA synthesis and function and inhibit Topoisomerase II leading to DNA breakage. They also form free oxygen radicals that also affect DNA synthesis and function by DNA breakage. Doxorubicin determines DNA double-strand breakage and human primordial follicles P63 mediated apoptosis. Except for Doxorubicin, which is considered of medium risk, they are low risk [18].

#### **2. Current Fertility Preservation Methods**

The 2018 American Society of Clinical Oncology (ASCO) recommendations paint a clear image of current onco-fertility focusing and emerging possibilities. While patients will first be taken aback by a cancer diagnosis, it is recommended that, early in the therapeutic process, discussions be initiated with the fertile patients concerning the risk of diminished fertility induced by specific treatment and about the options available for fertility preservation. Oocytes and embryo freezing, due to technological progress, are considered the standard of care and are widely available. Considering the conflicting evidence concerning GnRH agonist use to protect ovarian reserve during oncologic treatment [19], ASCO recommends only using GnRH agonists in young patients that are not fit for other methods. Embryo cryo-preserving is routinely used to preserve embryos that were not used for fresh embryo transfer after IVF. Oocyte freezing may be an adequate option for patients currently without a male partner, for those who do not want to use donor sperm or have ethical or religious objections regarding embryo cryopreservation. As of 2012, ASCO no longer considers oocyte cryopreservation experimental. As multiple ovarian stimulation protocols are available, there is no longer a need to delay ovarian stimulation depending on the menstrual cycle, favoring oocyte retrieval. For estrogen receptor-positive breast cancer patients that may be at risk due to the elevated estrogen levels in classic stimulation

protocols, ASCO recommends aromatase inhibitors stimulation protocols as current studies do not show evidence of increased recurrence risk [20].

In this context of social pressure, and ultimately, financial stimulus, assisted reproduction technologies had a steep curve of evolution. In this process, IVF has greater visibility and striving for successful pregnancies produced more embryos. A limited number of the better-quality embryos are being selected for intrauterine embryo-transfer, while the remaining good quality embryos were frozen for cryopreservation. The resulting technological prowess led to increasing rates of embryo survival after freeze-thaw, and in turn, to a greater number of pregnancies, similar rates of pregnancies being obtained for fresh embryos and frozen ones. This highlighted the idea that all embryo-transfers should be realized with frozen embryos in a subsequent cycle; in favor of this judgment is the standing lower risk of ovarian hyperstimulation syndrome and a higher endometrial receptivity due to different gene expressions in stimulated versus unstimulated endometrium [21,22]. It seems that the cryo-preservation only strategy sustains a better chance for a viable pregnancy and lower abortion rates, and ovarian hyperstimulation has a 7% occurrence rate for fresh embryo pregnancies versus 1–3% for pregnancies with cryo-preserved embryos [23]. Frozen embryo pregnancies grew steadily, for example, from 28% in 2010 to 32% in 2011. Current trends contribute already by the discussed segmentation of IVF cycles, and pre-implantation genetic testing indicated the necessity by the advancing age of motherhood to the growing proportion of freeze-thaw cycles. Some countries like Switzerland, Finland, Holland, Iceland, and Sweden were fast to achieve 50% pregnancies from cryo-preserved embryos [24], mainly due to differences in embryo-transfer legislation and by the number limitation, which, in turn, increased the number of frozen embryos. As an answer to restrictive legislation like in Germany, Swiss, and Austria, or even interdiction as in Italy 2004–2009, oocyte-preserving techniques were fast emerging. Two types of cooling are used: slow freeze, which allows for cell dehydration, thus lowering ice formation and vitrification, creating a glass-like state by very fast cooling without ice formation. When cells are frozen very slow, excessive dehydration and shrinkage also lead to cellular death, and to avoid such an instance, cryo-protectants are typically used. Non-permeating cryo-protectants remain in the extracellular space, leading to the rising osmolality of the extracellular solution before freezing, and thus, preventing intracellular ice formation. The most used slow freezing cryoprotectant is a permeating agent, dimethyl sulfoxide—DMSO, and it was used as a cryo-protective agent for the first frozen-thaw human cleavage embryo transferred. DMSO displaces intracellular water; its cryo-protectant effect augments it with concentration but also increases cytotoxicity and clinical practice, as demonstrated by the toxic effect on patients [25]. The first successful mammalian embryo cryo-preservation was realized in 1972 by applying a cooling rate of ~1 ◦C/min up to −70 ◦C. This type of cooling is labeled equilibrium freezing, and the target is to maintain a sufficient intracellular dehydration rate to maintain intracellular water chemical potential in equilibrium with extracellular, partially frozen water. Mostly, ice formation occurs in the extracellular space with subsequent increases in cellular membrane osmotic pressure that accentuates after ice formation continues following nucleation due to the ice lattice excluding solvates that concentrate in the extracellular medium. Less permeable cell membranes rupture due to the abrupt rise in osmotic pressure if they cannot dehydrate fast enough. Intracellular ice formation may be lethal, and exposure to high concentrations of electrolytes may also cause cellular death [26]. Cryogenic injury is related to the cellular membrane permeability, and as a consequence, cells with higher membrane permeability have better survival rates at fast freezing rates, and cells with less permeable membranes need slower cooling. Vitrification was first used for mouse embryo cryopreservation, and became a viable alternative to conventional protocols, as it was shown to reduce cellular damage. During vitrification procedures, cells and tissues are exposed to cryo-protectants that dehydrate cells before commencing cooling. The most frequent method for embryo vitrification necessitates small specimen volumes to be exposed to super-fast cooling and warming rates. This technique was first used in human embryology by 1998 for cleavage stage embryos and then in 1999 for oocytes and pronuclear embryos. In the last 20 years, more vitrification methods were described, which involved a large array of cryo-preservers combinations such as Ethylene glycol (EG), DMSO, PROH

(1,2-propanediol) and sucrose, Ficoll (polysaccharide solution), Trehalose, with varying parameters of dilution and equilibrium, support and cooling systems, storage and warming devices [27]. In 2005, Kuwayama M. et al. considered that the usual embryo and oocyte vitrification implied using a 15% solution of DMSO, 15% Ethylene Glycol, and 0.5 M sucrose in a very small volume ≤1 μL [28]. The fast cooling in vitrification is achieved by immersing the specimen in liquid nitrogen, and from this, two techniques are realized: open and closed vitrification. The majority of embryos and oocytes are vitrified by direct exposure to liquid nitrogen in the open system, favored because of its fast cooling and warming rates, increasing the method's success [29]. The alternative is represented by the use of devices that mediate direct contact with liquid nitrogen-closed systems, presumably marred by lower cooling and thawing rates. It is currently considered that successful vitrification is closely dependent on successful cellular osmotic dehydration before cooling and on the warming rate than on the type and concentration of the cryo-preserving agent, so as to avoid water re-crystallization in the thawing cycle where very fast warming is a requisite. To highlight these conclusions, a recent study performed by a team that included Peter Mazur, one of the pioneers in the field, has demonstrated high survival rates after oocytes and embryos were vitrified without permeating cryoprotectants, and the thawing procedure was realized by ultra-fast warming by an infrared laser pulse [30]. Another study aimed to use a laser beam to dehydrate the blastocoel before vitrification and found substantial improvement in the clinical outcome by lowering the risk of ice recrystallization [31]. Vitrification is now considered the gold standard for oocyte and embryo cryopreservation [30]. Fast freezing protocols need a cell dehydration stage by cryo-protective agents to prevent ice formation, though the direct correlation between intracellular ice formation and cell death has yet to be well defined [26]. It seems that cell survival following cryo-preservation depends on the rate the cells are warmed during the thawing process, as cell damage does not occur during initial ice nucleation, but by another process during thawing and ice recrystallization [32] seems to be the main culprit. Among the first observations are the studies on organisms that naturally survive freezing and physiologically produce recrystallization inhibitors in large amounts [33]. Cryo-protectants based on carbohydrates represent an alternative to DMSO and have minimal or no toxicity; they act like glycerol, which is known to influence ice formation [34]. Ice recrystallization is inhibited by mono and poli-disaccharides, thus suggesting possible use in human cell cryopreservation. One study compared cellular viability after cryopreservation with mono and poli-disaccharides, and with the DMSO control. They found that the most powerful ice recrystallization inhibitors were 220 mM disaccharides solution, and the best viability was obtained with D-galactose 200 mM. It seems that the protective effect of D-galactose resides in its internalization, consequently lowering cellular osmotic stress [35]. Several studies used carbohydrates in the cryopreservation media, but did not strive to evaluate their efficiency, while in others, the success seemed dependent on the chemical structure or correlated carbohydrate efficiency with dehydration level of the milieu [36]. As recrystallization seems to be involved in cellular death derived from cryo-preservation [26,32], the intimate structural characteristic that is involved in recrystallization inhibition is not yet known. It is considered essential for the vitrification process's success to limit to the minimum the amount of vitrification specimen to obtain a high rate of cooling and warming, thus preventing ice formation, and in this respect, oocytes are suitable freeze due to the low surface volume ratio that makes the cell membrane difficult to traverse for water and cryo-protectants [37]. Further, mature oocyte vitrification in metaphase meiosis (MII) may disrupt and deregulate the meiosis spindle, increasing the risk of chromosomal aberrations [38]. Unlike oocytes, embryos are more tolerant of freezing because membrane characteristics change after fertilization, favoring dehydration during cryo-preservation [39]. Embryo cryo-conservation led to the development of numerous devices that facilitate cooling and warming procedures, such as Cryoloop, nylon loop, Hemi-straw system, electron microscope plate, glass capillary, and Cryotop. Open Pulled Straw (OPS) was the first device specifically conceived for ultrafast vitrification, which was introduced by Vajta in 1998, and is still considered to be one of the best devices. The Cryotop is considered to be one of the most efficient vitrification methods both for oocytes and embryos, providing high rates of survival for both humans and animal

models, and like other open cryo-preservation systems, directly contacts liquid nitrogen, increasing the risk of viral contamination [40]. Alternative methods were devised, such as micro-volume air-cooling (MVAC) that focused on preventing direct contact of the specimen with the liquid nitrogen [41].

There are patients for whom embryo cryo-preservation was not an option, and because oocyte cryo-preservation techniques had slower progress, ovarian tissue-slow-freeze represented the only fertility preservation method. Lately, oocyte cryo-preservation methods achieved good results, and the Practice Committee of the American Society for Reproductive Medicine (ASRM) reclassified oocyte cryopreservation technology as "nonexperimental" in 2013 [42]. While many oocyte preservation programs do not have long term data on oocyte preservation, especially for patients that received chemotherapy and radiotherapy treatment. Lastly, for increased chances of pregnancy, a few stimulation cycles could be necessary for oocyte preservation in light of evidence that using fresh oocytes only increases the chance of pregnancy by 5% [43].
