**3. New Techniques of Fertility Preservation**

Ovarian tissue cryo-preservation may be an alternative to oocyte preservation, because for some patients' ovarian tissue cryo-preservation, it may eliminate the need for the oncologic treatment delay needed for stimulation cycles for oocyte retrieval. There is hope that ovarian tissue transplant reinstates not only fertility, but also endocrine function. The slow freeze was compared with vitrification methods for the ovarian tissue [44] and some authors consider slow freezing a better method due to greater primordial follicle density and viability, less apoptotic cells and better morpho-functional aspects [44], while others did not find a significant difference in regards to these characteristics [45]. Shi et al. [46] showed that vitrification produces less DNA fragmentation regarding primordial follicles, and vitrification also produces superior results for the granulosa and stromal cells ultra-structure after vitrification. The large variety and the array of conflicting results stem from lack of standardization of the cryo-preservation protocols, the large spread of cryoprotective agents concentration, the variety of experimental animals, the varying implanting sites of the ovarian tissue, the different duration of observation protocols, and last but not least, the varying methods of success measurement. Clinical benefits of ovarian transplantation may be disputed, but, for sure, it benefits fundamental science on ovarian function, primordial follicle activation and development arrest as provided by studies such as those of Winkler et al., and more recently, Silber et al. and Hayashi et al. [47,48]. ASCO acknowledges that ovarian tissue cryopreservation and transplantation does not need ovarian stimulation and may be immediately performed. The added benefit resides in that it does not need sexual maturity, being the only method fit for children in the scope of fertility preservation and being able to restore the global ovarian function. As of 2018, ASCO considers ovarian tissue preservation still experimental, but keeps it open for evidence that can change this status [20]. Improving techniques led to an estimated 35%–40% live birth [49], since the first alive human baby, obtained as a result of ovarian cortex auto-transplant, was reported in 2004, and there were more than 100 live births worldwide; however, global reach of the procedure remains low, limiting further progress [49]. There are growing numbers of studies trying to discern how the best results can be obtained, by ovarian strip vitrification, or slow freezing, whole ovary versus ovarian strip, or the best place to insert the implant as one study concluded that implant location could significantly affect the results [50]. One group [51] compared whole ovary vitrification vs. slow freezing and concluded that the efficacy of whole ovaries cryopreservation by vitrification was higher than those by conventional freezing and rapid freezing, and that conventional freezing of ovarian cortical strips was more effective than cryopreservation of whole ovaries, independent of the way of whole ovary cryopreservation. Most of the live births after ovarian tissue cryopreservation, so far, have been achieved by slow controlled freezing, and only two teams achieved live births from ovarian tissue vitrification [52,53], while others still use the slow freezing method. Laboratories are striving to find the optimal concentration of cryo-protectants for the best results in ovarian tissue cryopreservation by vitrification. One of the pioneers of ovarian tissue vitrification, Silber S [53], reported that he and his team have used only ovarian tissue vitrification since 2008, after 11 years of slow freezing use. Vitrification may overcome the negative effects of freezing by inhibiting ice crystal formation, and also, vitrification has advantages related to its relatively lower cost, and does not require sophisticated freezing machines or ultra-specialized laboratory staff.

During the antenatal period, human ovaries lose to follicular atresia 80% of its germinal cells to roughly 500,000–1,000,000 at birth [54] and reaching puberty with only 300,000 to 500,000 oocytes; of these, just 400–500 will be selected for ovulation in the following 30–40 years and the rest will be extinguished. The 1% selected for ovulation is subjected to an FSH dependent process that leads to a dominant follicle that produces ovulation during the same cycle. It is considered that follicular activation in humans takes place in a wave pattern, taking effect even during pregnancy or contraceptive medication [54,55]. Follicular recruitment varies with age, from more than 1400 at the beginning of the third decade of life to less than 30 towards the end of the fifth decade and follicular destruction takes place in great numbers before or after follicular recruitment dependent or not on the menstrual cycle, with a decreasing rate during the lifetime, with more follicular loss occurring in young women [56]. The remaining ovarian follicle cohort thus declines in number throughout the lifetime, leading to reduced fertility in the fourth decade, irregular menses by the middle of the fifth decade, and menopause at around 50 years of age. This process remains in a mysterious equilibrium of reproductive aging and organismal aging as it is one of the most precocious aging phenomena in women, but new studies are bringing fresh hypotheses on how ovarian follicles are being activated. Some authors realized that following auto-transplant of ovary tissue after a very marked spike in AMH, the values stabilized at low levels. It has been described as a 'burn out effect', and there is still debate around the implicated mechanisms. One study concluded that the increased number of growing follicles versus resting follicles might be due to the downregulation of PTEN gene expression and subsequent augmentation of follicular recruitment [57]. A Japanese team led by Kawamura [58] recently proposed a premature ovarian insufficiency treatment by Hippo signaling dysregulation, realized by fragmenting ovarian tissue followed by Akt application and autografting. The serine/threonine kinase Akt (protein kinase B or PKB) has become a major focus of attention because of its critical role in regulating diverse cellular functions, including metabolism, growth, proliferation, survival, transcription and protein synthesis, while Hippo signaling is a conserved pathway regulating organ size by cell proliferation, apoptosis, and stem cell activity. It is thought that the disruption of the Hippo pathway contributes to cancer development. Other authors consider that follicle activation and 'burn-out' have an important contribution to post-implantation follicular depletion affecting ovarian tissue grafts [59]. SonerCelik et al. [60] research team's findings regarding ovarian cryo-preservation and auto-transplantation demonstrate that expression of inhibitor proteins that control primordial follicle reserve decreases in cryopreserved ovaries after transplantation. The observation is consistent with the ovarian activity rush observed by others [61,62], and thus they debate the recommendation of follicular activation prior (in vitro activation IVA) to transplantation [58,63]. The longevity of the transplanted ovarian tissue varies widely and may depend on the age of the woman at cryo-preservation [64], some blaming the revascularization rate after transplantation as an important issue [65]; however, long functioning viability of more than ten years has been reported [66]. This success has promoted ovary cryo-preservation for potential use in severe genetic conditions with a risk of primary ovarian insufficiency like sickle cell anemia, thalassemia, Turner syndrome, and galactosemia [67]. A study performed mathematical modeling after losing 50% of the ovarian reserve after mono ovariectomy and concluded that the maintenance of ovarian function suggests an extra-ovarian, probably an age-dependent regulation agent of reproductive decline [68]. A review of cases comprising women with unilateral oophorectomy concluded that the menopause age was lowered by only 1.8 years [69]. By the same logic, it has been suggested that extracting ovarian strips early in life would not substantially affect menopause age, but by cryo-preservation, the ovarian tissue would later be re-implanted, thus conveniently delaying menopause [70]. A proposed mechanism for this ovarian function refers to the downregulation of follicular activation as the follicle pool is diminishing [71]. Another method described for fertility augmentation refers to autologous stem cell ovarian transplantation with, seemingly, relative success [72]. Tilly et al. have long provoked the fertility dogma that women dispose from birth of a fixed,

limited number of follicles, by affirming the existence of oogonial stem cells that can be activated, thus jumpstarting fertility [73]. There are reports that sphingosine-1-phosphate has cytoprotective functions in human ovaries, two studies showing that S1P reduces primordial follicle loss in human ovarian tissue xenografted in mice and exposed to cyclophosphamide as an in-vivo model of chemotherapy-induced ovarian damage [74,75]. Another study suggested the use of Sphingosine-1-phosphate to reduce the follicular atresia occurring during the freeze-thawing procedure [76].

As an alternative to ovarian tissue transplants, probing the hypotheses that limited success with ovarian tissue strips is due to the limited and late graft vascularization, there were animal and human trials with whole ovary transplants with limited success [77]. Some limitations were linked to the sheer mass of the ovary (sometimes animal trial included bovine ovaries) posing difficulties in the freezing process, being slow or exhibiting vitrification even after cannulation of the main vessels with cryo-protectants and likely experiencing ice recrystallization in the thawing [78]. Another obstacle is presented by the reperfusion lesions for the prevention, for which some authors tried edaravone, as it is supposed to relieve oxidative stress [79]. An observed supplemental difficulty results from the extensive dissection needed for extraction, but mostly for the ovarian implant, especially venous anastomosis due to the thin venous walls even for experienced teams like M Brännström's that is pioneering uterus transplantation [80].

Owing to the dispute that entangles the use of GnRH agonists [19], alternatives are looked for with the scope to protect ovarian function during chemotherapy. One example of such a protecting agent is represented by Sphingosine-1-phosphate [74,75], others examined co-administration of imatinib, (a 2-phenyl amino pyrimidine derivative that inhibits activity of the tyrosine kinase domains of c-Abl, c-Kit) and platelet-derived growth factor receptor, as they have been reported to attenuate follicle depletion in mice caused by cytotoxic treatments [81], although other studies failed to evidence the protective effect [82]. Anti-Müllerian hormone (AMH) represents another example of hopeful agents to be used in fertility preservation. AMH is part of the transforming growth factor (TGF)-beta family, with a central role in the control of sexual differentiation and follicular genesis, and while serum AMH levels have long been used in reproductive medicine as an indicator of ovarian reserve, it is now investigated as a protective agent [83].

The advent of new technologies brought new fields of research, and while ovarian strip auto-transplant harbors hope for both fertility preservation after oncologic therapy and menopause delay, studies searching for artificial support or even fully artificial ovaries are in full stride. A team is proposing a bioprosthetic ovary that was assembled using 3D printed microporous scaffolds in order to restore ovarian function [84]. Additionally, this is not a singular example since the oncofertility field is evolving; meanwhile, eager bioengineers have sought to create artificial ovaries with biomaterials and isolated follicles [85].

### **4. Conclusions**

Societal pressures pushed forward the long and successful experience with ART due to the increasing age of childbearing, which, in turn, exposed women intending to procreate to a higher risk of malign conditions. This is coupled with the better chance and longer disease-free survival by novel chemotherapy schemes that produce a growing population of women trying to conceive at increasing ages, often after oncologic treatment. Oncofertility studies crosslink with female aging studies and general fertility. There are promising technologies from oogonial stem cells activation, artificial scaffold bioprosthetic ovaries, and proactive ovarian tissue extraction for future use that could push female fertility farther away into what is now senescence.

**Author Contributions:** Conceptualization, C.B.C. and D.C.R.; methodology, C.B.C., D.C.R. and A.P.; validation, R.-C.P.; investigation, R.-C.P.; writing—original draft, C.B.C. and D.C.R.; writing—review & editing, R.-C.P., D.C.R. and A.P.; visualization, A.P.; supervision, C.B.C. and A.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflict of interest.
